**Part 1**

**Animal Models and Techniques** 

**1** 

**Ischemic Neurodegeneration in** 

**Stroke-Prone Spontaneously** 

**Prevention with Antioxidants** 

*College of Bioresource Sciences, Nihon University (NUBS),* 

Stroke involves cerebral infarction and hemorrhaging and is associated with very high mortality. Previous reports have indicated that ischemic stimulation such as the reoxygenation that occurs after hypoxia produces a large quantity of reactive oxygen species (ROS) that strongly induces neuronal death *in vivo* and *in vitro* (Negishi et al., 2001). Indeed, this is considered to be the factor that most strongly induces cell death in cerebral ischemia. In recent years, apoptosis has been suggested to be the mechanism responsible for ischemic

Stroke-prone spontaneously hypertensive rats (SHRSP) are widely used as a model of human stroke (Yamori et al., 1974). In this model, blood pressure is elevated as age increases, as is found in humans; and the rats eventually die of stroke. One feature of this model is that strokes develop spontaneously following severe hypertension (more than 150 mmHg). Therefore, in SHRSP, because strokes develop after the onset of elevated blood pressure, elevated blood pressure is considered to be the most critical factor for stroke induction. However, interestingly, the neuronal cells of this model exhibit a great vulnerability compared with normal control WKY/Izm rats during the reoxygenation conditions following hypoxia (Tagami et al., 1998; Yamagata et al., 2010c). In addition to the influence of blood pressure in SHRSP/Izm rats, the neuronal vulnerability of this model strongly contributes to stroke development. SHRSP/Izm rats are susceptible to apoptosis under conditions of hypoxia and reoxygenation (H/R) (Tagami et al., 1998). The expression of antioxidant enzymes in SHRSP/Izm rats is attenuated in comparison with that in WKY/Izm rats. We highlight that this attenuation of antioxidant enzymes is related to the vulnerability of neuronal cells (Yamagata et al., 2000b). Furthermore, an altered susceptibility to apoptosis was detected in the astrocytes of SHRSP/Izm rats compared with

neuronal death in animal stroke models (Tagami et al., 1998).

those of WKY/Izm rats (Yamagata et al., 2010a).

**1. Introduction** 

**Hypertensive Rats and Its** 

*Laboratory of Molecular Health Science of Food, Department of Food Bioscience and Biotechnology,* 

**Such as Polyphenols** 

Kazuo Yamagata

 *Japan* 

### **Ischemic Neurodegeneration in Stroke-Prone Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols**

Kazuo Yamagata

*Laboratory of Molecular Health Science of Food, Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University (NUBS), Japan* 

### **1. Introduction**

Stroke involves cerebral infarction and hemorrhaging and is associated with very high mortality. Previous reports have indicated that ischemic stimulation such as the reoxygenation that occurs after hypoxia produces a large quantity of reactive oxygen species (ROS) that strongly induces neuronal death *in vivo* and *in vitro* (Negishi et al., 2001). Indeed, this is considered to be the factor that most strongly induces cell death in cerebral ischemia. In recent years, apoptosis has been suggested to be the mechanism responsible for ischemic neuronal death in animal stroke models (Tagami et al., 1998).

Stroke-prone spontaneously hypertensive rats (SHRSP) are widely used as a model of human stroke (Yamori et al., 1974). In this model, blood pressure is elevated as age increases, as is found in humans; and the rats eventually die of stroke. One feature of this model is that strokes develop spontaneously following severe hypertension (more than 150 mmHg). Therefore, in SHRSP, because strokes develop after the onset of elevated blood pressure, elevated blood pressure is considered to be the most critical factor for stroke induction. However, interestingly, the neuronal cells of this model exhibit a great vulnerability compared with normal control WKY/Izm rats during the reoxygenation conditions following hypoxia (Tagami et al., 1998; Yamagata et al., 2010c). In addition to the influence of blood pressure in SHRSP/Izm rats, the neuronal vulnerability of this model strongly contributes to stroke development. SHRSP/Izm rats are susceptible to apoptosis under conditions of hypoxia and reoxygenation (H/R) (Tagami et al., 1998). The expression of antioxidant enzymes in SHRSP/Izm rats is attenuated in comparison with that in WKY/Izm rats. We highlight that this attenuation of antioxidant enzymes is related to the vulnerability of neuronal cells (Yamagata et al., 2000b). Furthermore, an altered susceptibility to apoptosis was detected in the astrocytes of SHRSP/Izm rats compared with those of WKY/Izm rats (Yamagata et al., 2010a).

Ischemic Neurodegeneration in Stroke-Prone

development (Fig. 4).

SHRSP/Izm rats.

Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols 5

apoptotic. About 41% of the WKY/Izm neurons died 1.5 hours after reoxygenation (necrosis = 12%, apoptosis = 29%). On the other hand, 78% of SHRSP/Izm neurons died (necrosis = 15%, apoptosis = 63%). Following three hours of reoxygenation, 99% of cells from both strains had died. In SHRSP/Izm rat neurons, fragmentation of DNA was strongly induced by 36 hours of hypoxia and reoxygenative stimulation for three hours (Tagami et al., 1998). The H/R induced apoptosis of neuronal cells in SHRSP/Izm rats (Yamagata et al., 2010c). The neuronal cells of SHRSP/Izm rats were strongly induced into apoptosis with 3 or 5 hours of reoxygenation following hypoxia. When DNA fragmentation was examined using a TUNEL method, few of the SHRSP/Izm rat neurons displayed DNA fragmentation when incubated under normal oxygen concentrations (data not shown). However, after 3 hours of reoxygenation following 36 hours of hypoxia, marked DNA fragmentation was seen. At the same time, many lipid droplets were detected in the cells (Tagami et al., 1998). We classified the apoptotic levels in H/R conditions via a morphologic analysis of neuronal death (Tagami et al., 1998, 1999). We demonstrated the criteria for neuronal apoptosis in the SHRSP/Izm rats in Table 1 and Figure 1. Neuronal axons and dendrites are lost in the early stages of apoptosis, and many lipid droplets are seen in the neuronal cell body (A, initial stage of apoptosis). Furthermore, cells shrink as apoptosis advances (B, second stage of apoptosis; C, third stage of apoptosis). The neuronal cell membrane is lost in the advanced stage of apoptosis, and the nucleus disappears (D). Figure 2 is considered to show the second stage of apoptosis (Tagami et al., 1998: Yamagata et al., 2010c). These processes eventually lead to cell death. From these results, it is suggested that the neuronal weakness of SHRSP/Izm rats is associated with stroke

Fig. 1. Our criteria to determine apoptosis and necrosis in neurons during H/R in

A. initial stage, B, second stage, C. third stage and D necrosis (Tagami et al., 1998)

Epidemiologic study indicated the possibility of preventing stroke using antioxidants such as dietary polyphenols (Vita, 2005). Polyphenols are substances produced by plants via photosynthesis, and their structures contain many hydroxyl groups (–OH). Polyphenols are found in vegetables, fruit, and processed products. They are also found abundantly in red wine, tea, soybeans, and coffee. The preventive effects of polyphenols include the inhibition of blood pressure elevation, cholesterol-lowering activity, hypoglycemic activity, antioxidant activity, and antimutagen activity (Sies et al., 2010). The effects of polyphenols differ between substances, but most are capable of "antioxidation". It is considered that the antioxidative effects of polyphenols are advantageous in their roles as defensive substances that protect plant components from oxidation. Polyphenols are found in trace amounts in our diet and have been demonstrated to prevent degenerative diseases such as cancer and cardiovascular disease (Manach et al., 2004). This review describes the vulnerability of neuronal cells and susceptibility of astrocytes in SHRSP in stroke conditions. Furthermore, we describe the prophylactic effects of apigenin, epigallocatechin-3-gallate (EGCG), and resveratrol on endothelial cells as well as their stroke preventive effects.

### **2. Susceptibility of neuronal cells and astrocytes of SHRSP/Izm rats during cerebral ischemia**

The reoxygenation after cerebral ischemia rapidly generates a large quantity of ROS. The following chain of events leads to neuronal cell injury (Love, 1999). Free radicals are generated early in the period of the reperfusion and cause neuronal damage (Bolli, 1991). Cerebral ischemia–reperfusion induced neuronal cell death is usually apoptotic (Rothstein et al., 1994). Here, we describe alteration in neuronal cells and astrocytes related to apoptosis in SHRSP/Izm rats during H/R.

### **2.1 Neuronal vulnerability of SHRSP during stroke and oxidative stress**

Neuronal death because of cerebral ischemic stress strongly induces apoptosis (Rothstein et al., 1994). Reports indicate that the production of hydroxyl radicals is strongly induced in SHRSP/Izm rats during H/R (Negishi et al., 2001). SHRSP/Izm and WKY/Izm rats produce hydroxyl radicals in their hippocampi when subjected to reoxygenation after 20 minutes of hypoxia. However, SHRSP/Izm rats display significantly increased hydroxyl production when compared with normal WKY/Izm control rats (Tagami et al., 1998). In SHRSP/Izm rats the production of hydroxyl radicals is strongly induced during H/R (Negishi et al., 2001). The increased levels of hydroxyl radicals produced by SHRSP/Izm rats may induce neuronal injury. These findings suggest that capturing the hydroxyl radicals produced during H/R, in which the level of antioxidant substances is decreased, would be beneficial for preventing neuronal injury (Yamagata et al., 2010c).

### **2.2 The neuronal cells of SHRSP/Izm rats strongly induce apoptosis during H/R**

Neuronal cells are easily damaged during H/R. We examined neuronal cells during hypoxia using SHRSP/Izm and WKY/Izm rats. After 24 hours of hypoxia, neuronal cell death was not observed in WKY/Izm or SHRSP/Izm rats. However, after 36 hours of hypoxia, neuronal cell death increased in SHRSP/Izm rats. This was not observed in WKY/Izm rats. The findings of a morphologic examination of SHRSP/Izm rats indicated that most neuronal cell death was

Epidemiologic study indicated the possibility of preventing stroke using antioxidants such as dietary polyphenols (Vita, 2005). Polyphenols are substances produced by plants via photosynthesis, and their structures contain many hydroxyl groups (–OH). Polyphenols are found in vegetables, fruit, and processed products. They are also found abundantly in red wine, tea, soybeans, and coffee. The preventive effects of polyphenols include the inhibition of blood pressure elevation, cholesterol-lowering activity, hypoglycemic activity, antioxidant activity, and antimutagen activity (Sies et al., 2010). The effects of polyphenols differ between substances, but most are capable of "antioxidation". It is considered that the antioxidative effects of polyphenols are advantageous in their roles as defensive substances that protect plant components from oxidation. Polyphenols are found in trace amounts in our diet and have been demonstrated to prevent degenerative diseases such as cancer and cardiovascular disease (Manach et al., 2004). This review describes the vulnerability of neuronal cells and susceptibility of astrocytes in SHRSP in stroke conditions. Furthermore, we describe the prophylactic effects of apigenin, epigallocatechin-3-gallate (EGCG), and resveratrol on

**2. Susceptibility of neuronal cells and astrocytes of SHRSP/Izm rats during** 

**2.1 Neuronal vulnerability of SHRSP during stroke and oxidative stress** 

The reoxygenation after cerebral ischemia rapidly generates a large quantity of ROS. The following chain of events leads to neuronal cell injury (Love, 1999). Free radicals are generated early in the period of the reperfusion and cause neuronal damage (Bolli, 1991). Cerebral ischemia–reperfusion induced neuronal cell death is usually apoptotic (Rothstein et al., 1994). Here, we describe alteration in neuronal cells and astrocytes related to apoptosis

Neuronal death because of cerebral ischemic stress strongly induces apoptosis (Rothstein et al., 1994). Reports indicate that the production of hydroxyl radicals is strongly induced in SHRSP/Izm rats during H/R (Negishi et al., 2001). SHRSP/Izm and WKY/Izm rats produce hydroxyl radicals in their hippocampi when subjected to reoxygenation after 20 minutes of hypoxia. However, SHRSP/Izm rats display significantly increased hydroxyl production when compared with normal WKY/Izm control rats (Tagami et al., 1998). In SHRSP/Izm rats the production of hydroxyl radicals is strongly induced during H/R (Negishi et al., 2001). The increased levels of hydroxyl radicals produced by SHRSP/Izm rats may induce neuronal injury. These findings suggest that capturing the hydroxyl radicals produced during H/R, in which the level of antioxidant substances is decreased, would be beneficial

**2.2 The neuronal cells of SHRSP/Izm rats strongly induce apoptosis during H/R**  Neuronal cells are easily damaged during H/R. We examined neuronal cells during hypoxia using SHRSP/Izm and WKY/Izm rats. After 24 hours of hypoxia, neuronal cell death was not observed in WKY/Izm or SHRSP/Izm rats. However, after 36 hours of hypoxia, neuronal cell death increased in SHRSP/Izm rats. This was not observed in WKY/Izm rats. The findings of a morphologic examination of SHRSP/Izm rats indicated that most neuronal cell death was

endothelial cells as well as their stroke preventive effects.

for preventing neuronal injury (Yamagata et al., 2010c).

**cerebral ischemia** 

in SHRSP/Izm rats during H/R.

apoptotic. About 41% of the WKY/Izm neurons died 1.5 hours after reoxygenation (necrosis = 12%, apoptosis = 29%). On the other hand, 78% of SHRSP/Izm neurons died (necrosis = 15%, apoptosis = 63%). Following three hours of reoxygenation, 99% of cells from both strains had died. In SHRSP/Izm rat neurons, fragmentation of DNA was strongly induced by 36 hours of hypoxia and reoxygenative stimulation for three hours (Tagami et al., 1998). The H/R induced apoptosis of neuronal cells in SHRSP/Izm rats (Yamagata et al., 2010c). The neuronal cells of SHRSP/Izm rats were strongly induced into apoptosis with 3 or 5 hours of reoxygenation following hypoxia. When DNA fragmentation was examined using a TUNEL method, few of the SHRSP/Izm rat neurons displayed DNA fragmentation when incubated under normal oxygen concentrations (data not shown). However, after 3 hours of reoxygenation following 36 hours of hypoxia, marked DNA fragmentation was seen. At the same time, many lipid droplets were detected in the cells (Tagami et al., 1998). We classified the apoptotic levels in H/R conditions via a morphologic analysis of neuronal death (Tagami et al., 1998, 1999). We demonstrated the criteria for neuronal apoptosis in the SHRSP/Izm rats in Table 1 and Figure 1. Neuronal axons and dendrites are lost in the early stages of apoptosis, and many lipid droplets are seen in the neuronal cell body (A, initial stage of apoptosis). Furthermore, cells shrink as apoptosis advances (B, second stage of apoptosis; C, third stage of apoptosis). The neuronal cell membrane is lost in the advanced stage of apoptosis, and the nucleus disappears (D). Figure 2 is considered to show the second stage of apoptosis (Tagami et al., 1998: Yamagata et al., 2010c). These processes eventually lead to cell death. From these results, it is suggested that the neuronal weakness of SHRSP/Izm rats is associated with stroke development (Fig. 4).

Fig. 1. Our criteria to determine apoptosis and necrosis in neurons during H/R in SHRSP/Izm rats.

A. initial stage, B, second stage, C. third stage and D necrosis (Tagami et al., 1998)

Ischemic Neurodegeneration in Stroke-Prone

**during H/R** 

their vulnerability.

**2.4 Characteristics of SHRSP/Izm rat astrocytes during stroke** 

The functions of the astrocytes regulate outbreaks of cerebropathy (Chen & Swanson, 2003). In brain lesions, reactive astrocyte numbers increase and promote the development of stroke (Pekny & Nilsson, 2005). This characteristic of the astrocytes of SHRSP/Izm rats may be related to brain disease (Chen & Swanson, 2003). We separated astrocytes from the brain of fetal SHRSP/Izm rats and cultured them. We compared the proliferation of astrocytes from WKY/Izm with SHRSP/Izm rats under various culture conditions (Yamagata et al., 1995). The astrocytes isolated from fetuses are not influenced by blood pressure. We examined the characteristics of astrocytes from SHRSP/Izm rats in environments that were not influenced by blood pressure. We found that the growth of astrocytes from SHRSP/Izm rats was increased in comparison with those from WKY/Izm rats (Yamagata et al., 1995). We suggest that the numbers of astrocytes of the SHRSP/Izm rats are increased and that this strongly leads to the gliosis following damage. In the rat brain transient cerebral ischemia model, epidermal growth factor (EGF) receptor is related to mechanism of astrocyte reactivity. The details are not known, but astrocyte numbers of SHRSP/Izm rats may increase by cell division through EGF stimulation during the appearance of cerebral blood vessel pathogenesis. This proliferation of astrocytes is enhanced by vascular smooth muscle cells in SHRSP/Izm rats (Yamori et al., 1981). In fibrinoid necrosis degeneration by hypertension, the barrier function of endothelial cells diminishes and blood plasma components leak out of the circulation (Johansson, 1999). In SHRSP/Izm rats, there is denaturation of smooth muscle cells of the media, necrosis with

Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols 7

The apoptosis in neuronal cells of SHRSP/Izm is strongly induced by reperfusion after ischemia (Tagami et al., 1998). Simultaneously, oxidative stress can induce antioxidant enzymes in neuronal cells. Antioxidant enzymes can prevent the apoptosis caused by oxidation stress. Furthermore, the Bcl2 gene is an oncogene related to human lymphoma and is able to inhibit the apoptosis induced by neurodegeneration stimuli (Akhtar et al, 2004). We highlight that the Bcl2 gene expression in SHRSP/Izm rat neuronal cells is significantly attenuated after 30 minutes of reoxygenation following hypoxia in comparison with that in WKY/Izm rats (Yamagata et al., 2000b). The decrease in the expression of Bcl2 leads to release of the cytochrome C from mitochondria. Thereafter, caspase activity increases and can strongly induce apoptosis. In SHRSP/Izm rat neurons, gene expression of thioredoxin II (Txn2) and mitochondrial cytochrome c oxidase III (CO III) decreased in a fashion similar to Bcl2 30 minutes after reoxygenation following hypoxia (Yamagata et al., 2000b). Txn2 provides protection against ROS via its SH group. In addition, these proteins have many functions that contribute to intracellular signal transduction. Namely, CO III is associated with energy metabolism in mitochondria. It transfers electrons from the reduced form of cytochrome C to molecular oxygen. Vitamin E and CO III are present in mitochondria where they protect the cell from injury by free radicals (Yang & Korsmeyer, 1996). Attenuation of Bcl2 and CO III gene expression in SHRSP/Izm rat neuronal cells may reduce energy metabolism and redox control during posthypoxic reoxygenation. The decrease of viability in SHRSP/Izm rat neurons, unlike that in WKY/Izm rat neurons, may be associated with

**2.3 Gene expression of Bcl2 and thioredoxin II in neuronal cells of SHRSP/Izm rats** 

Fig. 2. Second stage of apoptosis in neurons during H/R in SHRSP/Izm rats. N: nucleus


Cited references (Tagami et al., 1998, 1999).

Table 1. The morphological criteria for neuronal apoptosis in the SHRSP/Izm rats.

Fig. 2. Second stage of apoptosis in neurons during H/R in SHRSP/Izm rats. N: nucleus

1 Initial stage The cells lose their axons and dendrites,

2 Second stage The cells become round, small, and electron-

3 Advanced stage The cells lose their cytoplasm and cell

4 Final stage The cells become electron-lucent,

Table 1. The morphological criteria for neuronal apoptosis in the SHRSP/Izm rats.

Features of morphological

prominent invagination

become small and dark before disappearing)

and numerous lipid droplets appear in the cell bodies, although cell organelles remain intact

dense, and their nuclei demonstrate

membrane, and their nuclei become small and dark before disappearing

organelles decrease in number, and nuclei contain abnormal clusters of chromatin (the cells lose their cytoplasm and cell membrane, and their nuclei

Stage Criteria of neuronal death

Cited references (Tagami et al., 1998, 1999).

### **2.3 Gene expression of Bcl2 and thioredoxin II in neuronal cells of SHRSP/Izm rats during H/R**

The apoptosis in neuronal cells of SHRSP/Izm is strongly induced by reperfusion after ischemia (Tagami et al., 1998). Simultaneously, oxidative stress can induce antioxidant enzymes in neuronal cells. Antioxidant enzymes can prevent the apoptosis caused by oxidation stress. Furthermore, the Bcl2 gene is an oncogene related to human lymphoma and is able to inhibit the apoptosis induced by neurodegeneration stimuli (Akhtar et al, 2004). We highlight that the Bcl2 gene expression in SHRSP/Izm rat neuronal cells is significantly attenuated after 30 minutes of reoxygenation following hypoxia in comparison with that in WKY/Izm rats (Yamagata et al., 2000b). The decrease in the expression of Bcl2 leads to release of the cytochrome C from mitochondria. Thereafter, caspase activity increases and can strongly induce apoptosis. In SHRSP/Izm rat neurons, gene expression of thioredoxin II (Txn2) and mitochondrial cytochrome c oxidase III (CO III) decreased in a fashion similar to Bcl2 30 minutes after reoxygenation following hypoxia (Yamagata et al., 2000b). Txn2 provides protection against ROS via its SH group. In addition, these proteins have many functions that contribute to intracellular signal transduction. Namely, CO III is associated with energy metabolism in mitochondria. It transfers electrons from the reduced form of cytochrome C to molecular oxygen. Vitamin E and CO III are present in mitochondria where they protect the cell from injury by free radicals (Yang & Korsmeyer, 1996). Attenuation of Bcl2 and CO III gene expression in SHRSP/Izm rat neuronal cells may reduce energy metabolism and redox control during posthypoxic reoxygenation. The decrease of viability in SHRSP/Izm rat neurons, unlike that in WKY/Izm rat neurons, may be associated with their vulnerability.

### **2.4 Characteristics of SHRSP/Izm rat astrocytes during stroke**

The functions of the astrocytes regulate outbreaks of cerebropathy (Chen & Swanson, 2003). In brain lesions, reactive astrocyte numbers increase and promote the development of stroke (Pekny & Nilsson, 2005). This characteristic of the astrocytes of SHRSP/Izm rats may be related to brain disease (Chen & Swanson, 2003). We separated astrocytes from the brain of fetal SHRSP/Izm rats and cultured them. We compared the proliferation of astrocytes from WKY/Izm with SHRSP/Izm rats under various culture conditions (Yamagata et al., 1995). The astrocytes isolated from fetuses are not influenced by blood pressure. We examined the characteristics of astrocytes from SHRSP/Izm rats in environments that were not influenced by blood pressure. We found that the growth of astrocytes from SHRSP/Izm rats was increased in comparison with those from WKY/Izm rats (Yamagata et al., 1995). We suggest that the numbers of astrocytes of the SHRSP/Izm rats are increased and that this strongly leads to the gliosis following damage. In the rat brain transient cerebral ischemia model, epidermal growth factor (EGF) receptor is related to mechanism of astrocyte reactivity. The details are not known, but astrocyte numbers of SHRSP/Izm rats may increase by cell division through EGF stimulation during the appearance of cerebral blood vessel pathogenesis. This proliferation of astrocytes is enhanced by vascular smooth muscle cells in SHRSP/Izm rats (Yamori et al., 1981). In fibrinoid necrosis degeneration by hypertension, the barrier function of endothelial cells diminishes and blood plasma components leak out of the circulation (Johansson, 1999). In SHRSP/Izm rats, there is denaturation of smooth muscle cells of the media, necrosis with

Ischemic Neurodegeneration in Stroke-Prone

**nutrition** 

Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols 9

Fig. 4. Alteration of astrocytes and neuronal apoptosis by H/R stimulation.

as antioxidant nutrients can be used to reduce the risk of stroke.

**3.1 Possible role of polyphenols against cerebral ischemia injury** 

**3. Endothelial dysfunction and importance of stroke prevention through** 

The risk of stroke increases with the presence of arteriosclerosis in cerebral blood vessel endothelial cells. Here, we describe preventive action for endothelial cell disorders by food components. The secretion of cytokines by initial lesions strongly activates endothelial cells, vascular smooth muscle cells, and blood cells. For example, endothelial cells are strongly influenced by the effects of inflammatory cytokines such as tumor necrosis factor alpha (TNF-) and interleukin beta (IL-1) (Kofler et al., 2005). As a consequence, monocytic adhesion to endothelial cells is induced, which promotes various arteriosclerotic processes. Among these, the oxidative stress produced during the early period of the disorder triggers arteriosclerosis. When the various arteriosclerotic reactions begin simultaneously, they are very difficult to inhibit. Therefore, it is best to inhibit ROS production in the early stages of the disorder in order to avoid arteriosclerosis (Kondo et al., 2009). Indeed, the effects of nutritional components with antioxidant activity on the redox regulation of ROS in stroke conditions have been reported previously. It is considered to be possible to inhibit blood vessel disorders in the early stages and that the inhibition of ROS production using polyphenols prevents the development of arteriosclerosis (Manach et al., 2004). Therefore, it is very likely that arteriosclerosis prevention via the consumption of appropriate foods such

Cerebral ischemia induces the rapid production of a large quantity of ROS and induces cell injury through self-perpetuating reactions. Free radicals are produced within several minutes of reoxygenation after cerebral ischemia and induce brain cell injury (Bolli, 1991). Cerebral ischemia elevates the intracellular level of calcium ions and activates calcium-

a rise in blood pressure, and destruction of the blood–brain barrier (BBB) in perforating branch arteries (Tagami et al.,1987). We have indicated the possibility that attenuated endothelial barrier functions might be induced by comparing the astrocytic potency of SHRS/Izm rats (Yamagata et al., 1997b).

Glutamate is released as a neurotransmitter by nerve terminals and activates astrocytes. Furthermore, glutamate uptake via a glutamate transporter in the cell membrane is mediated by astrocytes. Glutamate produces lactate in astrocytes and the lactate produced by astrocytes is supplied as an energy source to neuronal cells (Pellerin & Magistretti 1994). Concurrently, the lactate supplied by astrocytes is important for the recovery of the neuronal cells after ischemia (Schurr et al., 1997; Dringen et al., 1995). We demonstrated that there is decreased lactate produced in cultured astrocytes from SHRSP/Izm rats when compared with that from WKY/Izm rat astrocytes during hypoxia (Yamagata et al., 2000a). The decreased lactate production by SHRSP/Izm rat astrocytes may cause neuronal cell death through reduced energy supply.

Furthermore, we examined characteristics of SHRSP/Izm rat astrocytes during stroke. In H/R, the expression levels of intercellular adhesion molecule-1 (ICAM1), monocyte chemotactic protein-1 (MCP1), and vascular cell adhesion molecule-1 (VCAM1) in astrocytes from SHRSP/Izm rats were increased in comparison with that in astrocytes from WKY/Izm rats (Yamagata et al., 2010a). In addition, production of glial cell linederived neurotrophic factor (GDNF) by adenosine, H2O2, glutamate, sphingosine-1 phosphate (S1P) was decreased during H/R in astrocytes from SHRSP/Izm rats in comparison with that from astrocytes from WKY/Izm rats (Yamagata et al., 2002; 2003; 2007a) (Fig. 3). Moreover, production of l-serine by nitric oxide (NO) stimulation decreased in SHRSP/Izm rats in comparison with that in WKY/Izm rats (Yamagata et al., 2006). Not all of the differences seen in SHRSP/Izm rats compared with WKY/Izm rats may be related to the generation of neuronal dysfunction in SHRSP/Izm rats. However, decreased astrocytic lactate and GDNF production may worsen energy conditions and nutrition status of SHRSP/Izm rat neurons (Yamagata et al., 2008). We suggest that attenuation of astrocyte functions accelerates neuronal cell death during stroke and may participate in its appearance (Fig. 4).

Fig. 3. Expression of GDNF in SHRSP/Izm rats by H/R stimulation. H/R: hypoxia and reoxygenation: S1P: sphingosine-1-phosphate, GDNF: glial cell line derived neurotrophic factor

a rise in blood pressure, and destruction of the blood–brain barrier (BBB) in perforating branch arteries (Tagami et al.,1987). We have indicated the possibility that attenuated endothelial barrier functions might be induced by comparing the astrocytic potency of

Glutamate is released as a neurotransmitter by nerve terminals and activates astrocytes. Furthermore, glutamate uptake via a glutamate transporter in the cell membrane is mediated by astrocytes. Glutamate produces lactate in astrocytes and the lactate produced by astrocytes is supplied as an energy source to neuronal cells (Pellerin & Magistretti 1994). Concurrently, the lactate supplied by astrocytes is important for the recovery of the neuronal cells after ischemia (Schurr et al., 1997; Dringen et al., 1995). We demonstrated that there is decreased lactate produced in cultured astrocytes from SHRSP/Izm rats when compared with that from WKY/Izm rat astrocytes during hypoxia (Yamagata et al., 2000a). The decreased lactate production by SHRSP/Izm rat astrocytes may cause neuronal cell

Furthermore, we examined characteristics of SHRSP/Izm rat astrocytes during stroke. In H/R, the expression levels of intercellular adhesion molecule-1 (ICAM1), monocyte chemotactic protein-1 (MCP1), and vascular cell adhesion molecule-1 (VCAM1) in astrocytes from SHRSP/Izm rats were increased in comparison with that in astrocytes from WKY/Izm rats (Yamagata et al., 2010a). In addition, production of glial cell linederived neurotrophic factor (GDNF) by adenosine, H2O2, glutamate, sphingosine-1 phosphate (S1P) was decreased during H/R in astrocytes from SHRSP/Izm rats in comparison with that from astrocytes from WKY/Izm rats (Yamagata et al., 2002; 2003; 2007a) (Fig. 3). Moreover, production of l-serine by nitric oxide (NO) stimulation decreased in SHRSP/Izm rats in comparison with that in WKY/Izm rats (Yamagata et al., 2006). Not all of the differences seen in SHRSP/Izm rats compared with WKY/Izm rats may be related to the generation of neuronal dysfunction in SHRSP/Izm rats. However, decreased astrocytic lactate and GDNF production may worsen energy conditions and nutrition status of SHRSP/Izm rat neurons (Yamagata et al., 2008). We suggest that attenuation of astrocyte functions accelerates neuronal cell death during stroke and may

SHRS/Izm rats (Yamagata et al., 1997b).

death through reduced energy supply.

participate in its appearance (Fig. 4).

Fig. 3. Expression of GDNF in SHRSP/Izm rats by H/R stimulation. H/R: hypoxia and reoxygenation: S1P: sphingosine-1-phosphate,

GDNF: glial cell line derived neurotrophic factor

Fig. 4. Alteration of astrocytes and neuronal apoptosis by H/R stimulation.

### **3. Endothelial dysfunction and importance of stroke prevention through nutrition**

The risk of stroke increases with the presence of arteriosclerosis in cerebral blood vessel endothelial cells. Here, we describe preventive action for endothelial cell disorders by food components. The secretion of cytokines by initial lesions strongly activates endothelial cells, vascular smooth muscle cells, and blood cells. For example, endothelial cells are strongly influenced by the effects of inflammatory cytokines such as tumor necrosis factor alpha (TNF-) and interleukin beta (IL-1) (Kofler et al., 2005). As a consequence, monocytic adhesion to endothelial cells is induced, which promotes various arteriosclerotic processes. Among these, the oxidative stress produced during the early period of the disorder triggers arteriosclerosis. When the various arteriosclerotic reactions begin simultaneously, they are very difficult to inhibit. Therefore, it is best to inhibit ROS production in the early stages of the disorder in order to avoid arteriosclerosis (Kondo et al., 2009). Indeed, the effects of nutritional components with antioxidant activity on the redox regulation of ROS in stroke conditions have been reported previously. It is considered to be possible to inhibit blood vessel disorders in the early stages and that the inhibition of ROS production using polyphenols prevents the development of arteriosclerosis (Manach et al., 2004). Therefore, it is very likely that arteriosclerosis prevention via the consumption of appropriate foods such as antioxidant nutrients can be used to reduce the risk of stroke.

### **3.1 Possible role of polyphenols against cerebral ischemia injury**

Cerebral ischemia induces the rapid production of a large quantity of ROS and induces cell injury through self-perpetuating reactions. Free radicals are produced within several minutes of reoxygenation after cerebral ischemia and induce brain cell injury (Bolli, 1991). Cerebral ischemia elevates the intracellular level of calcium ions and activates calcium-

Ischemic Neurodegeneration in Stroke-Prone

Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols 11

Inhibiton of VEGF Osada

Inhibition of superoxide anion-mediated impairment Ma

Induction of calcium dependent activation of the NO Chen

Inhibition of TNFα-induced LOX-1 expression Yamagata

Inhibiton of MCP-1 expression Hong et al., (2007)

Inhibiton of TNFα-induced MCP-1 production Ahn et al., (2008)

Decrease of caveolin-1 expression Li et al., (2009) Decrease of endothelin-1 expression and secretion Reiter et al., (2010) Induction of the NO Auger et al., (2010)

Protection against the oxidative stress by the NO Jin et al., (2009)

(authors and issue)

Zhang et al (2000)

(2006)

et al., (2004)

et al., (2008)

et al., (2010)

et al., (2011)

et al., (2000)

Chae et al., (2007)

Zheng et al., (2009)

Lee et al., (2010)

Ou et al., (2010)

Wang et al., (2010)

Yang et al., (2010)

Neuhaus et al., (2004)

Potenza et al., (2007)

Yamagata et al., (2010b)

Zou and Chiou

Lee et al., (2007)

Navarro-Nunez et al., (2008)

Polyphenol(s) Effects on pathological condition(s) Ref

Inhibiton of laser-induced choroidal

Inhibition of COX-2 expression and adhesion of

Inhibition of platelet adhesion and thrombus

Inhibition of high glucose and TNFα-induced

EGCG Increase of the prostacyclin production Mizugaki

Inhibition of the vascular-endothelial growth factorinduced intracellular signaling and mitogenesis

Inhibits the angiotensin II-induced adhesion molecule

Improves endothelial function and insulin sensitivity,

Protection against linoleic-acid-induced endothelial

Protects against oxidized LDL-induced endothelial dysfunction by inhibiting LOX-1-mediated signaling

Decreases thrombin/paclitaxel-induced endothelial

inhibiton of angiotensin II-induced endothelial barrier

reduces blood pressure, and protects against

Protection of against oxidized LDL-induced

adhesion molecule expression

myocardial I/R injury in SHR

endothelial dysfunction

tissue factor expression

dysfunction

Apigenin Endothelium-dependent vasorelaxant and antiproliferative effect

neovascularization

monocytes

formation

expression

cell activation

dependent proteases. Moreover, these reactions activate xanthine dehydrogenase (XDH) and produce xanthine oxidase (XOD) (Thompson-Gorman & Zweier, 1990). It is considered that the superoxide anion radicals produced via this pathway cause neuronal death. However, the consumption of polyphenol-rich foods, such as fruits and vegetables, is beneficial for preventing vascular disorders (Manach et al., 2004). Epidemiological studies have indicated that an inverse correlation exists between polyphenolic consumption and the risk of having to undergo a cardiovascular procedure (Arts & Hollman, 2005). Polyphenols induce the production of vasodilatory factors such as NO (Auger et al., 2010) and prostacyclin (PGI2) (Mizugaki et al., 2000) and inhibit the synthesis of endothelin-1, which induces vasoconstriction in endothelial cells (Reiter et al., 2010). On the other hand, the polyphenols present in the skin of grapes and in wine inhibit the proliferation and migration of smooth muscle cells (Lee et al., 2009). Polyphenols may eliminate the active oxygen produced by reoxygenation after cerebral ischemia via their antioxidative effects.

### **3.2 Vasorelaxant effects of polyphenols on endothelial cells**

Epidemiological analysis has suggested that polyphenols have protective effects against heart disease. The polyphenols that protect against heart disease are found in foods including cocoa, wine, grape pips, berries, tea, tomatoes, soybeans, and pomegranates (Chong et al., 2010). The mechanisms by which polyphenols reduce the risk of heart disease are associated with the prevention of endothelial cell disorders. Endothelial cell disorders strongly induce arteriosclerosis, which subsequently progresses to heart disease and stroke. Therefore, the prevention of endothelial cell disorders by polyphenols is effective in preventing heart disease and stroke. Table 2 shows the effects of the typical polyphenols apigenin, EGCG, and resveratrol on endothelial cells. Jin et al. (2009) demonstrated that apigenin (0.5 – 72.0 M) enhanced concentration-dependent relaxation in aortas. Apigenin action is mediated by weakening the oxidative stress and by NO reduction. On the other hand, it has been shown that stimulation of expression of endothelial NOS (eNOS) by apigenin occurs through phosphatidylinositol 3-kinase/Akt (PI3K/Akt) for Ca2+ dependence (Chen et al., 2010). Moreover, the blockade of adhesion of monocytes and cyclooxygenase (COX)-2 expression in endothelial cells by apigenin has been reported (Lee et al., 2007). We have shown that apigenin strongly inhibits high glucose- and TNF--induced VCAM1 expression and the adhesion of U937 in human endothelial cells (Yamagata et al., 2010b). These effects of apigenin are caused by the inhibition of Iionkinase (IKK) and IKK/IKKi. From these findings, we suggested that the mechanism by which apigenin inhibits the expression of adhesion molecules and the adhesion of monocytic U937 to endothelial cells involves nuclear factor kappa beta (NF-). From the structure and inhibitory activity profiles of dietary flavonoids, it was recognized that the double bond found in the C-ring of flavonoids and the third hydroxyl group (A-ring) are required for the inhibition of VCAM1 gene expression (Yamagata et al., 2010b). Apigenin may inhibit monocytic adhesion caused by superoxide anions as well as block reductions in NO activity. From these reports, it is considered that apigenin reduces the levels of ROS, promotes NO activity, and inhibits cell adhesion. Moreover, apigenin strongly inhibited the TNF- stimulated expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (Yamagata et al., 2011) and the double bond of the C ring of apigenin is essential for this action (Fig. 5). As shown in Figure. 5, the inhibition of LOX-1 expression by apigenin requires a flavone frame, a double bond in the C-ring, and the absence of a third hydroxyl group in the Band C-rings, which are not found in naringenin (not active) (Yamagata et al., 2011).

dependent proteases. Moreover, these reactions activate xanthine dehydrogenase (XDH) and produce xanthine oxidase (XOD) (Thompson-Gorman & Zweier, 1990). It is considered that the superoxide anion radicals produced via this pathway cause neuronal death. However, the consumption of polyphenol-rich foods, such as fruits and vegetables, is beneficial for preventing vascular disorders (Manach et al., 2004). Epidemiological studies have indicated that an inverse correlation exists between polyphenolic consumption and the risk of having to undergo a cardiovascular procedure (Arts & Hollman, 2005). Polyphenols induce the production of vasodilatory factors such as NO (Auger et al., 2010) and prostacyclin (PGI2) (Mizugaki et al., 2000) and inhibit the synthesis of endothelin-1, which induces vasoconstriction in endothelial cells (Reiter et al., 2010). On the other hand, the polyphenols present in the skin of grapes and in wine inhibit the proliferation and migration of smooth muscle cells (Lee et al., 2009). Polyphenols may eliminate the active oxygen

produced by reoxygenation after cerebral ischemia via their antioxidative effects.

and C-rings, which are not found in naringenin (not active) (Yamagata et al., 2011).

Epidemiological analysis has suggested that polyphenols have protective effects against heart disease. The polyphenols that protect against heart disease are found in foods including cocoa, wine, grape pips, berries, tea, tomatoes, soybeans, and pomegranates (Chong et al., 2010). The mechanisms by which polyphenols reduce the risk of heart disease are associated with the prevention of endothelial cell disorders. Endothelial cell disorders strongly induce arteriosclerosis, which subsequently progresses to heart disease and stroke. Therefore, the prevention of endothelial cell disorders by polyphenols is effective in preventing heart disease and stroke. Table 2 shows the effects of the typical polyphenols apigenin, EGCG, and resveratrol on endothelial cells. Jin et al. (2009) demonstrated that apigenin (0.5 – 72.0 M) enhanced concentration-dependent relaxation in aortas. Apigenin action is mediated by weakening the oxidative stress and by NO reduction. On the other hand, it has been shown that stimulation of expression of endothelial NOS (eNOS) by apigenin occurs through phosphatidylinositol 3-kinase/Akt (PI3K/Akt) for Ca2+ dependence (Chen et al., 2010). Moreover, the blockade of adhesion of monocytes and cyclooxygenase (COX)-2 expression in endothelial cells by apigenin has been reported (Lee et al., 2007). We have shown that apigenin strongly inhibits high glucose- and TNF--induced VCAM1 expression and the adhesion of U937 in human endothelial cells (Yamagata et al., 2010b). These effects of apigenin are caused by the inhibition of Iionkinase (IKK) and IKK/IKKi. From these findings, we suggested that the mechanism by which apigenin inhibits the expression of adhesion molecules and the adhesion of monocytic U937 to endothelial cells involves nuclear factor kappa beta (NF-). From the structure and inhibitory activity profiles of dietary flavonoids, it was recognized that the double bond found in the C-ring of flavonoids and the third hydroxyl group (A-ring) are required for the inhibition of VCAM1 gene expression (Yamagata et al., 2010b). Apigenin may inhibit monocytic adhesion caused by superoxide anions as well as block reductions in NO activity. From these reports, it is considered that apigenin reduces the levels of ROS, promotes NO activity, and inhibits cell adhesion. Moreover, apigenin strongly inhibited the TNF- stimulated expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (Yamagata et al., 2011) and the double bond of the C ring of apigenin is essential for this action (Fig. 5). As shown in Figure. 5, the inhibition of LOX-1 expression by apigenin requires a flavone frame, a double bond in the C-ring, and the absence of a third hydroxyl group in the B-

**3.2 Vasorelaxant effects of polyphenols on endothelial cells** 


Ischemic Neurodegeneration in Stroke-Prone

preventive effects for vascular disorders.

Fig. 5. Structures and LOX-1 inhibitory activities of apigenins.

gene expressions).

○: active (indicates that the compound dose-dependently inhibited TNFα-induced LOX-1

Many studies have demonstrated that ischemic heart disease is decreased by wine intake, and in particular, it has been shown that the antioxidative effects of the polyphenols found in red wine are important for cardioprotection. It was shown that this cardioprotective effect is caused by the actions of resveratrol. It has been confirmed that resveratrol displays various pharmacologic actions such as antioxidant activity in humans

Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols 13

(EGCG), and the content order of these compounds is as follows: EGCG>EGC>ECG>EC. Catechins are also responsible for the bitter taste of green tea. Catechins account for around 13%–30% of the dry weight of tea leaves (Wolfram, 2007). EGCG suppresses the expression of adhesion molecules such as MCP1 (Ahn et al., 2008; Chae et al., 2007; Hong et al., 2007) and expression of endothelin-1 (Reiter et al., 2010). Like apigenin, EGCG inhibits the expression of monocyte adhesion molecules in endothelial cells stimulated with TNF- (Ahn et al., 2008; Zheng et al., 2010) and it has been reported that EGCG inhibits the TNF- induced expression of activator protein-1 in endothelial cells and increased the expression of HO-1. These findings suggest that EGCG inhibits the expression of activator protein-1 and increases the expression of HO-1, both of which aid endothelial protection. Furthermore, a least one study demonstrated that EGCG downregulated the endothelial cell activation induced by linoleic acid via caveolin-1 (Zheng et al., 2009). Six hours of linoleate exposure induced the expression of caveolin-1 and COX-2 in caveolae. However, pretreatment with EGCG inhibited the expression of caveolin-1 and COX-2 induced by linoleic acid. Exposure to linoleic acid also increased the levels of several kinases (p38 MAPK, extracellular signal regulated kinase 1/2 8ERK1/2), and amino kinase terminal (Akt). According to these findings, EGCG activates several enzymes in endothelial cell caveolae and may have many


COX; cyclooxygenase, GPx1; glutathione peroxidase 1, I/R; ischemia/reperfusion, LDL; low density lipoprotein, LOX-1; lectin-like oxidized low-density lipoprotein receptor-1, MCP-1; monocyte chemotactic protein-1, NO; nitric oxide, Nox4; NADPH oxidase subunit, PAF; platelet-activating factor, SOD1; superoxide dismutase 1, SHR, spontaneously hypertensive rats, TNF; tumor necrosis factor, VEGF; vascular endothelial growth factor,

Table 2. Studies on the protective effects of apigenin, EGCG and resveratrol in endothelial cells

EGCG is a catechin that is found in green tea. The catechins found in tea include epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate

Inhibition of VEGF-induced angiogenesis Lin

Inhibition of MCP-1 synthesis and secretion Cullen et al., (2007)

Brakenhielm et al., (2001)

Shigematsu et al., (2003)

et al., (2003)

Brito et al., (2006)

Csiszar et al., (2006)

Chow et al (2007)

Juric et al., (2007)

Klinge et al., (2008)

Spanier et al (2009)

Schilder et al., (2009)

et al., (2009)

et al., (2010)

Lin et al., (2010)

Chang et al., (2011)

Xu et al., (2009)

Resveratrol Inhibition of angiogenesis, tumor growth, and wound healing

PAF, or oxidants.

endothelial cell death

diastolic impairment

cellular senescence

GPx1 and Nox4

LOX-1 signaling

barrier

VEGF; vascular endothelial growth factor,

cells

estrogen receptor alpha

endothelial dysfunction

the gene expression of SOD1,

inhibition of NF-kappaB

Prevention of superoxide-dependent inflammatory responses induced by I/R,

Protection against peroxynitrite-mediated

Attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages.

Prevention of concentric hypertrophy and

Induction of NO production by increasing

Reduces oxidative stress by modulating

Prevention of hyperglycemia-induced

Decrease of oxidized LDL-evoked

Protecton of oxidized LDL-induced

breakage of the blood-brain

Induction of NADPH oxidases 1 and 4 mediate

Decrease of mitochondrial oxidative stress Ungvari

Protecton of H 2O2-induced oxidative stress Kao

COX; cyclooxygenase, GPx1; glutathione peroxidase 1, I/R; ischemia/reperfusion, LDL; low density lipoprotein, LOX-1; lectin-like oxidized low-density lipoprotein receptor-1, MCP-1; monocyte

chemotactic protein-1, NO; nitric oxide, Nox4; NADPH oxidase subunit, PAF; platelet-activating factor, SOD1; superoxide dismutase 1, SHR, spontaneously hypertensive rats, TNF; tumor necrosis factor,

Table 2. Studies on the protective effects of apigenin, EGCG and resveratrol in endothelial

EGCG is a catechin that is found in green tea. The catechins found in tea include epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate

Attenuation of TNF alpha-induced activation;

(EGCG), and the content order of these compounds is as follows: EGCG>EGC>ECG>EC. Catechins are also responsible for the bitter taste of green tea. Catechins account for around 13%–30% of the dry weight of tea leaves (Wolfram, 2007). EGCG suppresses the expression of adhesion molecules such as MCP1 (Ahn et al., 2008; Chae et al., 2007; Hong et al., 2007) and expression of endothelin-1 (Reiter et al., 2010). Like apigenin, EGCG inhibits the expression of monocyte adhesion molecules in endothelial cells stimulated with TNF- (Ahn et al., 2008; Zheng et al., 2010) and it has been reported that EGCG inhibits the TNF- induced expression of activator protein-1 in endothelial cells and increased the expression of HO-1. These findings suggest that EGCG inhibits the expression of activator protein-1 and increases the expression of HO-1, both of which aid endothelial protection. Furthermore, a least one study demonstrated that EGCG downregulated the endothelial cell activation induced by linoleic acid via caveolin-1 (Zheng et al., 2009). Six hours of linoleate exposure induced the expression of caveolin-1 and COX-2 in caveolae. However, pretreatment with EGCG inhibited the expression of caveolin-1 and COX-2 induced by linoleic acid. Exposure to linoleic acid also increased the levels of several kinases (p38 MAPK, extracellular signal regulated kinase 1/2 8ERK1/2), and amino kinase terminal (Akt). According to these findings, EGCG activates several enzymes in endothelial cell caveolae and may have many preventive effects for vascular disorders.

Fig. 5. Structures and LOX-1 inhibitory activities of apigenins. ○: active (indicates that the compound dose-dependently inhibited TNFα-induced LOX-1 gene expressions).

Many studies have demonstrated that ischemic heart disease is decreased by wine intake, and in particular, it has been shown that the antioxidative effects of the polyphenols found in red wine are important for cardioprotection. It was shown that this cardioprotective effect is caused by the actions of resveratrol. It has been confirmed that resveratrol displays various pharmacologic actions such as antioxidant activity in humans

Ischemic Neurodegeneration in Stroke-Prone

dehydrogenase, XOD; xanthine oxidase.

J 15 (10): 1798-1800.

cells. Life Sci 82 (17-18): 964-968.

Am J Clin Nutr 81 (Suppl 1): 317S-325S.

Keywords; Endothelial cells, Ischemic stroke, Polyphenol, SHRSP.

**5. Conclusion** 

**6. Abbreviations** 

**7. References** 

Spontaneously Hypertensive Rats and Its Prevention with Antioxidants Such as Polyphenols 15

Endothelial cell dysfunction causes arteriosclerosis and promotes neuronal demise after stroke. Enhanced neuronal sensitivity to oxidative stress contributes to the neuronal death observed in SHRSP/Izm rats. Also, enhanced oxidative stress after hypoxia-reoxygenation is important in ischemic stroke. Polyphenols reduce oxidation stress and have a protective effect on endothelial and neuronal cells. Antioxidant nutrients such as polyphenols may prevent or

BBB; blood–brain barrier, CO III; cytochrome c oxidase III, COX; cyclooxygenase, CVD; cardiovascular disease, EC; epicatechin, ECG; epicatechin gallate, EGC; epigallocatechin, EGCG; epigallocatechin-3-gallate, EGF; epidermal growth factor, eNOS; endothelial NOS, - GCS; gamma glutamylcystenyl synthase, GDNF; glial cell line-derived neurotrophic factor, GSH: glutathione, HO-1; hemoxigenase-1, H/R; hypoxia and reoxygenation, ICAM1; intercellular adhesion molecule-1, IL-1; interleukin beta, IKK; IIKKkinase, LDL; lowdensity lipoprotein, LOX-1; lectin-like oxidized low-density lipoprotein receptor-1, MCP1; monocyte chemotactic protein-1, NO; nitric oxide, NF-; nuclear factor kappa beta, oxLDL; oxidized LDL, PGI2; prostacyclin, PI3K/Akt; phosphatidylinositol 3-kinase/Akt, ROS; reactive oxygen species, SHRSP/Izm; spontaneously hypertensive rats/Izm, S1P; sphingosine-1-phosphate, TNF-; tumor necrosis factor alpha, TRX; thioredoxin, VCAM1; vascular cell adhesion molecule-1, WKY/Izm; Wistar Kyoto rat/Izm, XDH; xanthine

Ahn, H.Y., Xu, Y. & Davidge, S.T. (2008). Epigallocatechin-3-O-gallate inhibits TNFalpha-

Akhtar, R.S., Ness, J.M. & Roth, K.A. (2004). Bcl-2 family regulation of neuronal development and neurodegeneration. Biochim Biophys Acta 1644(2-3): 189-203. Arts, I.C., & Hollman, P.C. (2005). Polyphenols and disease risk in epidemiologic studies.

Auger, C., Kim, J.H., Chabert, P., Chaabi, M., Anselm, E., Lanciaux, X., Lobstein, A. &

Bråkenhielm, E., Cao, R. & Cao, Y. (2001). Suppression of angiogenesis, tumor growth, and

Brito, P.M., Mariano, A., Almeida, L.M. & Dinis, T.C. (2006). Resveratrol affords protection

hydroxyl moieties. Biochem Biophys Res Commun 393 (1): 162-167. Bolli, R. (1991). Oxygen-derived free radicals and myocardial reperfusion injury: an

overview. Cardiovasc Drugs Ther (Suppl 2): 249-268.

glutathione. Chem Biol Interact 164 (3): 157-166.

induced monocyte chemotactic protein-1 production from vascular endothelial

Schini-Kerth, V.B. (2010). The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on

wound healing by resveratrol, a natural compound in red wine and grapes. FASEB

against peroxynitrite-mediated endothelial cell death: A role for intracellular

reduce endothelial dysfunction and neuronal cell injury during cerebral ischemia.

(Brito et al., 2006; Chow et al., 2007; Spanier et al., 2009; Ungvari et al., 2009). Resveratrol is considered to decrease circulating low-density lipoprotein (LDL) cholesterol levels and thereby reduce the risk of cardiovascular disease (CVD) (Ramprasath & Jones, 2010). Resveratrol inhibits atherosclerosis and improves the function of endothelial cells in animal models. There have been many studies of resveratrol actions, which have shown that it has various effects on endothelial cells, as shown in Table 2. The effects of resveratrol and red wine on endothelial cells were investigated using experimental hypercholesterolemic rabbits (Zou et al., 2003). It was found that hypercholesterolemic rabbits displayed significant improvements in the functions of their endothelial cells after the administration of resveratrol (3 mg/kg/day), red wine (4 ml/kg/day), or nonalcoholic red wine (4 ml/kg/day) for 12 weeks. Moreover, they demonstrated decreased levels of plasma endothelin-1 and NO, which are increased by hypercholesterolemia. On the other hand, it was also shown that resveratrol protects against injury to the BBB caused by oxidized LDL (oxLDL) (Lin et al., 2010). It is considered that the mechanism behind these effects of resveratrol involves amelioration of the effects of oxLDL on the expression of occludin and ZO-1, which aids the stability of tight junctions. Resveratrol regulates the expression of tight junction proteins as a means of protecting against the disruption of the BBB induced by oxLDL. In a rat postischemic reoxygenation model, resveratrol decreased ROS generation (Shigematsu et al., 2003), and the effect of resveratrol on cerebral infarction was also examined in a rat middle cerebral artery occlusion (MCA) model (Sinha et al., 2002). In addition, after MCA and 2 hours of reperfusion, the rats were evaluated for motor disorders, malondialdehyde (MDA), reduced glutathione, and infarct volume. After MCA, increases in the frequency of functional motility disorders and the levels of MDA and reduced glutathione were observed. On the other hand, the administration of resveratrol prevented these increases and significantly decreased the infarct volume. These findings indicate that resveratrol inhibits the organ injuries produced by ischemia–reperfusion. The other polyphenols found in wine are not known to have this effect. Correspondingly, resveratrol prevents myocardial infarction by reducing peroxide levels. It is suggested that this effect can be attributed to the antioxidative effects of resveratrol (Dudley et al., 2008).

### **4. Preventive effects of antioxidant drugs and polyphenols for SHRSP rat neurons during stroke**

We indicated that high dose vitamin E induced neutral gamma glutamylcystenyl synthase (-GCS), GSH levels, and strongly prevented neuronal death (Yamagata et al., 2009). Furthermore, we have shown that ebselen, a seleno–organic antioxidant (Yamagata et al., 2008), amlodipine, and carvedilol (Yamagata et al., 2004) prevented neuronal cell death in SHRSP/Izm rats. Another study demonstrated that the expression of VCAM1 by TNF in astrocytes isolated from SHRSP/Izm rats was increased compared with that in those from WKY/Izm rats. However, apigenin strongly attenuated TNF--induced VCAM1 mRNA and protein expression and suppressed the adhesion of U937 cells and SHRSP/Izm astrocytes (Yamagata et al., 2010a). It is suggested that apigenin regulates adhesion molecule expression in reactive astrocytes during ischemia and prevents neuronal death.

### **5. Conclusion**

14 Advances in the Preclinical Study of Ischemic Stroke

(Brito et al., 2006; Chow et al., 2007; Spanier et al., 2009; Ungvari et al., 2009). Resveratrol is considered to decrease circulating low-density lipoprotein (LDL) cholesterol levels and thereby reduce the risk of cardiovascular disease (CVD) (Ramprasath & Jones, 2010). Resveratrol inhibits atherosclerosis and improves the function of endothelial cells in animal models. There have been many studies of resveratrol actions, which have shown that it has various effects on endothelial cells, as shown in Table 2. The effects of resveratrol and red wine on endothelial cells were investigated using experimental hypercholesterolemic rabbits (Zou et al., 2003). It was found that hypercholesterolemic rabbits displayed significant improvements in the functions of their endothelial cells after the administration of resveratrol (3 mg/kg/day), red wine (4 ml/kg/day), or nonalcoholic red wine (4 ml/kg/day) for 12 weeks. Moreover, they demonstrated decreased levels of plasma endothelin-1 and NO, which are increased by hypercholesterolemia. On the other hand, it was also shown that resveratrol protects against injury to the BBB caused by oxidized LDL (oxLDL) (Lin et al., 2010). It is considered that the mechanism behind these effects of resveratrol involves amelioration of the effects of oxLDL on the expression of occludin and ZO-1, which aids the stability of tight junctions. Resveratrol regulates the expression of tight junction proteins as a means of protecting against the disruption of the BBB induced by oxLDL. In a rat postischemic reoxygenation model, resveratrol decreased ROS generation (Shigematsu et al., 2003), and the effect of resveratrol on cerebral infarction was also examined in a rat middle cerebral artery occlusion (MCA) model (Sinha et al., 2002). In addition, after MCA and 2 hours of reperfusion, the rats were evaluated for motor disorders, malondialdehyde (MDA), reduced glutathione, and infarct volume. After MCA, increases in the frequency of functional motility disorders and the levels of MDA and reduced glutathione were observed. On the other hand, the administration of resveratrol prevented these increases and significantly decreased the infarct volume. These findings indicate that resveratrol inhibits the organ injuries produced by ischemia–reperfusion. The other polyphenols found in wine are not known to have this effect. Correspondingly, resveratrol prevents myocardial infarction by reducing peroxide levels. It is suggested that this effect can be

attributed to the antioxidative effects of resveratrol (Dudley et al., 2008).

**neurons during stroke** 

neuronal death.

**4. Preventive effects of antioxidant drugs and polyphenols for SHRSP rat** 

We indicated that high dose vitamin E induced neutral gamma glutamylcystenyl synthase (-GCS), GSH levels, and strongly prevented neuronal death (Yamagata et al., 2009). Furthermore, we have shown that ebselen, a seleno–organic antioxidant (Yamagata et al., 2008), amlodipine, and carvedilol (Yamagata et al., 2004) prevented neuronal cell death in SHRSP/Izm rats. Another study demonstrated that the expression of VCAM1 by TNF in astrocytes isolated from SHRSP/Izm rats was increased compared with that in those from WKY/Izm rats. However, apigenin strongly attenuated TNF--induced VCAM1 mRNA and protein expression and suppressed the adhesion of U937 cells and SHRSP/Izm astrocytes (Yamagata et al., 2010a). It is suggested that apigenin regulates adhesion molecule expression in reactive astrocytes during ischemia and prevents Endothelial cell dysfunction causes arteriosclerosis and promotes neuronal demise after stroke. Enhanced neuronal sensitivity to oxidative stress contributes to the neuronal death observed in SHRSP/Izm rats. Also, enhanced oxidative stress after hypoxia-reoxygenation is important in ischemic stroke. Polyphenols reduce oxidation stress and have a protective effect on endothelial and neuronal cells. Antioxidant nutrients such as polyphenols may prevent or reduce endothelial dysfunction and neuronal cell injury during cerebral ischemia.

### **6. Abbreviations**

BBB; blood–brain barrier, CO III; cytochrome c oxidase III, COX; cyclooxygenase, CVD; cardiovascular disease, EC; epicatechin, ECG; epicatechin gallate, EGC; epigallocatechin, EGCG; epigallocatechin-3-gallate, EGF; epidermal growth factor, eNOS; endothelial NOS, - GCS; gamma glutamylcystenyl synthase, GDNF; glial cell line-derived neurotrophic factor, GSH: glutathione, HO-1; hemoxigenase-1, H/R; hypoxia and reoxygenation, ICAM1; intercellular adhesion molecule-1, IL-1; interleukin beta, IKK; IIKKkinase, LDL; lowdensity lipoprotein, LOX-1; lectin-like oxidized low-density lipoprotein receptor-1, MCP1; monocyte chemotactic protein-1, NO; nitric oxide, NF-; nuclear factor kappa beta, oxLDL; oxidized LDL, PGI2; prostacyclin, PI3K/Akt; phosphatidylinositol 3-kinase/Akt, ROS; reactive oxygen species, SHRSP/Izm; spontaneously hypertensive rats/Izm, S1P; sphingosine-1-phosphate, TNF-; tumor necrosis factor alpha, TRX; thioredoxin, VCAM1; vascular cell adhesion molecule-1, WKY/Izm; Wistar Kyoto rat/Izm, XDH; xanthine dehydrogenase, XOD; xanthine oxidase.

Keywords; Endothelial cells, Ischemic stroke, Polyphenol, SHRSP.

### **7. References**


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**2** 

*Germany* 

**Frameless Stereotaxy in Sheep –** 

**for Translational Stroke Research** 

Donald Lobsien5, Björn Nitzsche1 and Johannes Boltze1,2

*3Institute of Neuroscience, Technical University Munich, Munich, 4Institute of Analytical Chemistry, University of Leipzig, Leipzig, 5Department of Neuroradiology, University of Leipzig, Leipzig,* 

*2Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig,* 

The continuous pathologic reduction of cerebral blood flow is mainly caused by thromboembolic occlusion of a brain-supplying artery or cerebral blood vessel disruption. These events, representing the most important causes for ischemic or hemorrhagic strokes respectively, lead to an acute breakdown of neuronal function, secondary brain damage by numerous mechanisms and the loss of cerebral tissue. In industrialized nations, stroke accounts for every third case of death. Cerebral stroke furthermore represents the most frequent reason for permanent disability in adulthood (Kolominsky-Rabas et al., 2006) and is therefore considered to be one of the most dreaded diseases from a clinical, socio-

Intravenous thrombolysis by tissue plasminogen activator (tPA) is currently the only FDAapproved, effective and potentially curative treatment (Blinzler et al., 2011) for ischemic stroke. However, this approach is restricted to a narrow time window of 4.5 hours (Hacke et al., 2008). The approach is further limited by a sharply increasing number needed to treat (Hacke et al., 2008; Lansberg et al., 2009) and a significant risk for fatal adverse events (Shobha et al., 2011) at later stages of this time window. As a result, more than 95% of all stroke patients do not significantly benefit from systemic thrombolytic treatment (Barber et al., 2001). Alternatively, endovascular thrombolysis under thorough radiological surveillance can be applied in specialized centers, extending the therapeutic time window to

**1. Introduction** 

economic and individual, patient-related perspective.

**1.1 Current state of the art clinical stroke treatment and diagnosis** 

up to 8.0 hours under optimal conditions (Natarajan et al., 2009).

Antje Dreyer1,2, Albrecht Stroh3, Claudia Pösel1, Matthias Findeisen4, Teresa von Geymüller1,2,

*1Fraunhofer Institute for Cell Therapy and Immunology,* 

*Department of Cell Therapy, Leipzig,* 

**Neurosurgical and Imaging Techniques** 


## **Frameless Stereotaxy in Sheep – Neurosurgical and Imaging Techniques for Translational Stroke Research**

Antje Dreyer1,2, Albrecht Stroh3, Claudia Pösel1, Matthias Findeisen4, Teresa von Geymüller1,2, Donald Lobsien5, Björn Nitzsche1 and Johannes Boltze1,2 *1Fraunhofer Institute for Cell Therapy and Immunology, Department of Cell Therapy, Leipzig, 2Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig, 3Institute of Neuroscience, Technical University Munich, Munich, 4Institute of Analytical Chemistry, University of Leipzig, Leipzig, 5Department of Neuroradiology, University of Leipzig, Leipzig, Germany* 

### **1. Introduction**

20 Advances in the Preclinical Study of Ischemic Stroke

Yamagata, K., Tagami, M., Nara, Y., Fujino, H., Kubota, A., Numano, F., Kato, T. & Yamori

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Zheng, Y., Lim, E.J., Wang, L., Smart, E.J., Toborek, M. & Hennig, B. (2009). Role of caveolin-

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expression is heme oxygenase-1 dependent. Metabolism 59 (10): 1528-1535. Zou, Y., & Chiou, G.C. (2006). Apigenin inhibits laser-induced choroidal neovascularization and regulates endothelial cell function. J Ocul Pharmacol Ther 22 (6): 425-430. Zou, J.G., Wang, Z.R., Huang, Y.Z., Cao, K.J. & Wu, J.M. (2003). Effect of red wine and wine

expression in human endothelial cells. J Nutr Biochem 21 (2): 116-124. Yamagata, K., Tagami, M., & Yamori, Y. (2010c). Neuronal vulnerability of stroke-prone

(9-10): 686-691.

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their expression. Stroke Res Treat pii:386389. 1-11.

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and cell death. Blood 88 (2): 386-401.

of apigenin. Gen Pharmacol 35 (6): 341-347.

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Suppl (7): 121s-123s.

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Y. (1997b). Faulty induction of blood-brain barrier functions by astrocytes isolated from stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 24

production and induces neuronal vulnerability in stroke-prone spontaneously

GSH levels, and inhibits neuronal death in stroke-prone spontaneously hypertensive

Stroke status evoked adhesion molecule genetic alterations in astrocytes isolated from stroke-prone spontaneously hypertensive rats and the apigenin inhibition of

Inhibits high glucose and tumor necrosis factor alpha-induced adhesion molecule

spontaneously hypertensive rats to ischemia and its prevention with antioxidants

necrosis factor alpha plus high glucose-induced LOX-1 expression in human

evidence obtained by successive selective breeding of stroke-prone and-resistant

Mechanisms of structural vascular changes in genetic hypertension: analyses on cultured vascular smooth muscle cells from spontaneously hypertensive rats. 61

Yun, Y.P. (2000). Endothelium-dependent vasorelaxant and antiproliferative effects

1 in EGCG-mediated protection against linoleic-acid-induced endothelial cell

against tumor necrosis factor-α-induced monocyte chemoattractant protein-1

polyphenol resveratrol on endothelial function in hypercholesterolemic rabbits. Int

The continuous pathologic reduction of cerebral blood flow is mainly caused by thromboembolic occlusion of a brain-supplying artery or cerebral blood vessel disruption. These events, representing the most important causes for ischemic or hemorrhagic strokes respectively, lead to an acute breakdown of neuronal function, secondary brain damage by numerous mechanisms and the loss of cerebral tissue. In industrialized nations, stroke accounts for every third case of death. Cerebral stroke furthermore represents the most frequent reason for permanent disability in adulthood (Kolominsky-Rabas et al., 2006) and is therefore considered to be one of the most dreaded diseases from a clinical, socioeconomic and individual, patient-related perspective.

### **1.1 Current state of the art clinical stroke treatment and diagnosis**

Intravenous thrombolysis by tissue plasminogen activator (tPA) is currently the only FDAapproved, effective and potentially curative treatment (Blinzler et al., 2011) for ischemic stroke. However, this approach is restricted to a narrow time window of 4.5 hours (Hacke et al., 2008). The approach is further limited by a sharply increasing number needed to treat (Hacke et al., 2008; Lansberg et al., 2009) and a significant risk for fatal adverse events (Shobha et al., 2011) at later stages of this time window. As a result, more than 95% of all stroke patients do not significantly benefit from systemic thrombolytic treatment (Barber et al., 2001). Alternatively, endovascular thrombolysis under thorough radiological surveillance can be applied in specialized centers, extending the therapeutic time window to up to 8.0 hours under optimal conditions (Natarajan et al., 2009).

Frameless Stereotaxy in Sheep –

due to minimal mortality rates.

Neurosurgical and Imaging Techniques for Translational Stroke Research 23

and porcine (Imai et al., 2006) models for which middle cerebral artery occlusion (MCAO) techniques have been described. For anatomical reasons, most large animal models require enucleation and show high mortality rates (The STAIR Participants, 1999), rendering their

Early models of cerebral hemorrhage were reported more than 45 years ago (Klintworth, 1965), using the application of mechanical force (e.g. balloon inflation) to induce cerebral hemorrhages. More contemporary models are often based on atlas-guided stereotaxic injections (Bullock et al., 1984) of autologous blood or bacterial collagenase into cerebral tissue, predominantly the rodent striatum (MacLellan et al., 2010). MR-guided application techniques have recently been reported in primates (Zhu et al., 2011) but are rarely used,

To compensate the above mentioned common limitations of large animal models an ovine model of ischemic stroke using permanent MCAO was developed by our group (Boltze et al. 2008). This model avoids enucleation and allows for long term observation of subjects

Briefly, the method can be described as follows. A transcranial access is performed between the left eye and ear. Animals should be placed on the right side to avoid ventilation insufficiency due to a gaseous rumen edema during surgery. After superficial shaving and disinfection, the temporal muscle is incised at the Linea temporalis and temporally elevated from the parietal skull bone. Then, a trepanation of about 1 x 1 cm is performed right behind the orbital rim. After careful incision of the dura mater, the MCA is permanently electrocoagulated by a bipolar forceps. Occlusion of either one or two MCA branches or the entire cortical vessel allows a detailed control of lesion size and functional deficits (Boltze et al., 2008). The drill hole may be sealed by sterile bone cement after suturing of the dura. However, leaving the craniotomy open (only covered by the temporal muscle) avoids pathophysiological increase of intracerebral pressure in early post-stroke phases, significantly reducing post-stroke mortality. After refixation of the temporal muscle at the Linea temporalis and suturing the skin wound, the animals can be taken back to the stable and are allowed to recover. Adequate post stroke analgetic and antibiotic treatment has to be ensured. For any details regarding animal medication, behavioral phenotyping, advanced imaging and the surgical procedure itself, please refer to Boltze et al. (2008). Species-appropriate housing can be realized with comparatively low efforts and over extended time periods. The price per sheep is relatively low and the species is broadly available. A special feature of the sheep stroke model is the control of lesion size and subsequent behavioral deficits by occlusion of the cortical MCA or a defined number of its branches. A protocol for testing and quantification of neurological functions is available to assess the impact of the MCAO modality and a potential therapeutic procedure. Moreover, the model is eligible for detailed MR, CT and positron emission tomography (PET) studies

applicability for long term safety and efficacy trials difficult.

**1.4 The ovine stroke model and its use for preclinical research** 

presumable due to ethical and financial restrictions.

as well as the assessment of autologous cell therapies.

**2. Stereotaxy and cell tracking for stroke-related applications** 

Stereotaxic concepts were developed as minimally invasive surgical approaches which use three-dimensional Cartesian or polar coordinate systems to localize small targets inside the

Cerebral ischemia needs to be discriminated from hemorrhagic stroke by means of magnetic resonance imaging (MRI) or computer tomography (CT) prior to the start of therapy, as inducing thrombolysis after hemorrhagic strokes is fatal. The mentioned imaging modalities are also used to monitor disease progression and the beneficial, or eventually detrimental, impact of any therapeutic intervention.

Even though intracerebral hemorrhages are treated by multimodal strategies including neurosurgical interventions and diligent monitoring of blood pressure (Flower & Smith, 2011), the event is still associated with high morbidity and mortality rates (Rymer, 2011), leaving about 80% of patients dead or disabled. Repeated patient surveys by MRI or CT is therefore pivotal for early detection of complications after hemorrhagic stroke. In summary, imaging procedures are of utmost clinical importance for diagnosis, treatment and onward care after ischemic and hemorrhagic strokes.

### **1.2 Current recommendations for preclinical stroke research**

Preclinical and translational stroke research aims to overcome the aforementioned therapeutic and prognostic limitations by the development of novel treatment strategies. The application of stem cell based therapies is currently among the most promising approaches in the field (Burns & Steinberg, 2011) and the first clinical trials have already been initiated (Sahota & Savitz, 2011). However, despite thorough research, the development of novel stroke therapies is so far characterized by continuous setbacks and the general failure to translate promising findings from animal models into effective clinical treatment paradigms (Del Zoppo, 1995). In particular this holds true for the development of neuroprotective therapies (O'Collins et al., 2006).

The inability to translate preclinical findings into clinical therapies has been a matter of debate for more than 15 years. International expert committees like the "Stroke Treatment Academic and Industry Roundtable" (STAIR) and "Stem Cell Therapies as an Emerging Paradigm in Stroke" (STEPS) consortia were formed to define, discuss and publish recommendations for adequate preclinical stroke research (The STAIR Participants, 1999; The STEPS Participants, 2009). Current guidelines for state of the art assessment of novel stroke treatment strategies comprise the design and application of relevant animal models (Fisher et al., 2009), the definition of the optimal route of administration for any therapeutic agent (Savitz et al., 2011; The STAIR Participants, 1999), as well as the choice of relevant imaging protocols to monitor therapeutic safety and efficacy (Savitz et al., 2011).

### **1.3 The role of large animal models in translational stroke research**

Cerebral ischemia is mainly modeled by transient or permanent occlusion of the middle cerebral artery (MCA; Howells et al., 2010). Rodent models are widely available and represent experimental key systems for preclinical stroke research. These models offer many advantages such as well established methodology including surgical techniques, imaging procedures, histological techniques and protocols for molecular biology. Further, the availability of genetically modified strains and excellent tools to assess functional outcome favor the use of rodent stroke models.

However, the impact of a particular therapy in the gyrencephalic brain can only be assessed in large animals. Therefore, the use of a second, predominantly large animal species has been recommended by the STAIR and STEPS committees. Existing large animal models include rabbit (Amiridze et al., 2009), canine (Kang et al., 2007), feline (Garcia et al., 1977),

Cerebral ischemia needs to be discriminated from hemorrhagic stroke by means of magnetic resonance imaging (MRI) or computer tomography (CT) prior to the start of therapy, as inducing thrombolysis after hemorrhagic strokes is fatal. The mentioned imaging modalities are also used to monitor disease progression and the beneficial, or eventually detrimental,

Even though intracerebral hemorrhages are treated by multimodal strategies including neurosurgical interventions and diligent monitoring of blood pressure (Flower & Smith, 2011), the event is still associated with high morbidity and mortality rates (Rymer, 2011), leaving about 80% of patients dead or disabled. Repeated patient surveys by MRI or CT is therefore pivotal for early detection of complications after hemorrhagic stroke. In summary, imaging procedures are of utmost clinical importance for diagnosis, treatment and onward

Preclinical and translational stroke research aims to overcome the aforementioned therapeutic and prognostic limitations by the development of novel treatment strategies. The application of stem cell based therapies is currently among the most promising approaches in the field (Burns & Steinberg, 2011) and the first clinical trials have already been initiated (Sahota & Savitz, 2011). However, despite thorough research, the development of novel stroke therapies is so far characterized by continuous setbacks and the general failure to translate promising findings from animal models into effective clinical treatment paradigms (Del Zoppo, 1995). In particular this holds true for the development of

The inability to translate preclinical findings into clinical therapies has been a matter of debate for more than 15 years. International expert committees like the "Stroke Treatment Academic and Industry Roundtable" (STAIR) and "Stem Cell Therapies as an Emerging Paradigm in Stroke" (STEPS) consortia were formed to define, discuss and publish recommendations for adequate preclinical stroke research (The STAIR Participants, 1999; The STEPS Participants, 2009). Current guidelines for state of the art assessment of novel stroke treatment strategies comprise the design and application of relevant animal models (Fisher et al., 2009), the definition of the optimal route of administration for any therapeutic agent (Savitz et al., 2011; The STAIR Participants, 1999), as well as the choice of relevant

Cerebral ischemia is mainly modeled by transient or permanent occlusion of the middle cerebral artery (MCA; Howells et al., 2010). Rodent models are widely available and represent experimental key systems for preclinical stroke research. These models offer many advantages such as well established methodology including surgical techniques, imaging procedures, histological techniques and protocols for molecular biology. Further, the availability of genetically modified strains and excellent tools to assess functional outcome

However, the impact of a particular therapy in the gyrencephalic brain can only be assessed in large animals. Therefore, the use of a second, predominantly large animal species has been recommended by the STAIR and STEPS committees. Existing large animal models include rabbit (Amiridze et al., 2009), canine (Kang et al., 2007), feline (Garcia et al., 1977),

imaging protocols to monitor therapeutic safety and efficacy (Savitz et al., 2011).

**1.3 The role of large animal models in translational stroke research** 

impact of any therapeutic intervention.

care after ischemic and hemorrhagic strokes.

neuroprotective therapies (O'Collins et al., 2006).

favor the use of rodent stroke models.

**1.2 Current recommendations for preclinical stroke research** 

and porcine (Imai et al., 2006) models for which middle cerebral artery occlusion (MCAO) techniques have been described. For anatomical reasons, most large animal models require enucleation and show high mortality rates (The STAIR Participants, 1999), rendering their applicability for long term safety and efficacy trials difficult.

Early models of cerebral hemorrhage were reported more than 45 years ago (Klintworth, 1965), using the application of mechanical force (e.g. balloon inflation) to induce cerebral hemorrhages. More contemporary models are often based on atlas-guided stereotaxic injections (Bullock et al., 1984) of autologous blood or bacterial collagenase into cerebral tissue, predominantly the rodent striatum (MacLellan et al., 2010). MR-guided application techniques have recently been reported in primates (Zhu et al., 2011) but are rarely used, presumable due to ethical and financial restrictions.

### **1.4 The ovine stroke model and its use for preclinical research**

To compensate the above mentioned common limitations of large animal models an ovine model of ischemic stroke using permanent MCAO was developed by our group (Boltze et al. 2008). This model avoids enucleation and allows for long term observation of subjects due to minimal mortality rates.

Briefly, the method can be described as follows. A transcranial access is performed between the left eye and ear. Animals should be placed on the right side to avoid ventilation insufficiency due to a gaseous rumen edema during surgery. After superficial shaving and disinfection, the temporal muscle is incised at the Linea temporalis and temporally elevated from the parietal skull bone. Then, a trepanation of about 1 x 1 cm is performed right behind the orbital rim. After careful incision of the dura mater, the MCA is permanently electrocoagulated by a bipolar forceps. Occlusion of either one or two MCA branches or the entire cortical vessel allows a detailed control of lesion size and functional deficits (Boltze et al., 2008). The drill hole may be sealed by sterile bone cement after suturing of the dura. However, leaving the craniotomy open (only covered by the temporal muscle) avoids pathophysiological increase of intracerebral pressure in early post-stroke phases, significantly reducing post-stroke mortality. After refixation of the temporal muscle at the Linea temporalis and suturing the skin wound, the animals can be taken back to the stable and are allowed to recover. Adequate post stroke analgetic and antibiotic treatment has to be ensured. For any details regarding animal medication, behavioral phenotyping, advanced imaging and the surgical procedure itself, please refer to Boltze et al. (2008).

Species-appropriate housing can be realized with comparatively low efforts and over extended time periods. The price per sheep is relatively low and the species is broadly available. A special feature of the sheep stroke model is the control of lesion size and subsequent behavioral deficits by occlusion of the cortical MCA or a defined number of its branches. A protocol for testing and quantification of neurological functions is available to assess the impact of the MCAO modality and a potential therapeutic procedure. Moreover, the model is eligible for detailed MR, CT and positron emission tomography (PET) studies as well as the assessment of autologous cell therapies.

### **2. Stereotaxy and cell tracking for stroke-related applications**

Stereotaxic concepts were developed as minimally invasive surgical approaches which use three-dimensional Cartesian or polar coordinate systems to localize small targets inside the

Frameless Stereotaxy in Sheep –

quality (Boltze et al., 2008).

performance of the sterotaxic injections.

**3.2.1 Fiducial marker positioning and imaging** 

Fig. 1. Maxillary splint with fiducial marker

heads)

intervention.

Neurosurgical and Imaging Techniques for Translational Stroke Research 25

as well as thorough medical inspections and blood screening, medication and vaccination ensure a significant reduction of postoperative complications and thereby enhance study

Anesthesia is performed as described elsewhere (Boltze et al., 2008). Animals should be intubated after induction of anesthesia and placed in a prone ("sphinx") position during surgery and imaging. Vital parameters (electrocardiogram, oxygen saturation, blood pressure, rectal body temperature) should be continuously monitored during any surgical

The neuronavigation device, BrainSightTM, is a frameless system that allows for MRI data set based planning of surgical approaches as well as for surveillance and precision control of the surgical intervention with an optical position sensor (Frey et al., 2004). An individual 3D-reconstruction of the head, especially the brain, is required for the precise planning and

The MR-compatible fiducial markers are attached to a maxillary splint (Fig. 1a). The use of the splint is different from neurosurgical approaches in human medicine, where fiducial markers can be fixed directly to cranial bones. This is not recommended in animals due to safety and welfare issues, especially when the animal is awake between MRI data set acquisition and surgery. The maxillary splint consists of a mouthpiece (Fig. 1a, 1) and two angled arms (Fig. 1a/b, 2) that hold the fiducial markers (Fig. 1a/b, 4). The mouthpiece is inserted carefully, avoiding damage to or constriction of the tracheal tube. It can be adapted to the individual shapes of the maxillary molars and the hard palate by using thermoplastic, which cures within a few minutes. The fiducial markers are usually placed in the area between the cheeks and the ears. The maxillary splint has to be adapted to each individual sheep to ensure maximum precision. The splint has to be inserted for imaging and surgery.

a) maxillary splint with mouthpiece (1), angeled arms (2), bar spacer to the skin (3) and fiducial marker (4); b) sheep before surgery, the maxillary splint is inserted and fixed with a bandage. The angeled arms (2) support the fiducial markers (4); c) 3D-MRI reconstruction of the skin illustrates the position of fiducial makers between cheek and ear (black arrow

**3.2 Frameless stereotaxy in sheep – preparation and data acquisition** 

body. The approach can be used for both diagnostic and therapeutic applications, as it allows the placement or the removal of a specimen from a certain location within the body with highest precision and minimal damage to the surrounding tissue. Stereotaxy is of particular importance in neurosurgery where the technique is routinely used for diagnosis and treatment of intracranial tumors (Willems et al., 2006), as well as for the application of deep brain stimulation electrodes in Parkinson's disease (Starr et al., 1998) and neuropathic pain (Stadler et al., 2011). The fibrinolytic evacuation of intracranial hemorrhages by a stereotaxic apporach has also been reported (Samadani & Rohde, 2009). Moreover, stereotaxic stem cell injections into the human brain are used in phase I and II clinical trials, as the local administration of therapeutic compounds close to the lesion is considered to be advantageous.

Albeit these concepts may have been strongly perpetuated towards clinical application clinical trials during the last years; the first reported results unfortunately resemble the translational failures that were known from past efforts. This holds true for experimental treatments in the field of stroke (Kondziolka et al., 2005) and Parkinson's disease (Gross et al., 2011) although these concepts were positively evaluated in preceding rodent studies. This emphasizes the relevance of large animal models as an important translational milestone. Whereas simplified stereotaxic devices based on brain atlases are widely available for rodents, the accuracy and complexity of human stereotaxic devices can currently only be modeled in primates. However, this complexity, including the individual, "lesion-specific" application of substances or the induction of phenotypically varying intacerebral hematomas may be critically needed in translational research to mimic the more heterogenic patient populations enrolled in clinical trials.

We expanded the sheep model to fill this methodological gap and to provide an additional large animal model for translational research in cell transplantation after ischemic stroke and intracerebral hemorrhages. Our model allows precise, MR-guided implantation of magnetically labeled, bone-marrow derived mesenchymal stem cells (stroke treatment) and autologous blood samples into the ovine brain (hemorrhage induction). The technique was developed using the BrainsightTM neuronavigation system (Rogue Research Inc., Quebec, Canada) and several modifications were applied to adapt the system to the ovine skull anatomy. Cell tracking can be performed reliably using clinical MR scanners with adequate resolution and sensitivity.

This chapter describes the methodology of image-guided frameless stereotaxic surgery in sheep with special emphasis on (i) the application of an autologous therapeutic cell population (e.g., the mesenchymal stem cells), (ii) the previous labeling and subsequent imaging protocols for MR-based cell tracking in sheep and (iii) the MR-guided induction of cerebral hemorrhage in the species.

### **3. Technical description of surgery**

### **3.1 General information about the species and handling requirements**

The neurosurgical approach for stereotaxy in sheep requires hornless subjects for easy accessibility of cranial structures. Merino sheep may be of advantage as many hornless strains can be found in this widely available race. Adult merino sheep weigh approximately 80 kilograms (ewe) to 130 kilograms (rams) and have a wither height of about 0.9 meter. This body size allows for relatively easy handling. Frequent and early contact to humans facilitates familiarization and improves the handling. Species appropriate housing, feeding

body. The approach can be used for both diagnostic and therapeutic applications, as it allows the placement or the removal of a specimen from a certain location within the body with highest precision and minimal damage to the surrounding tissue. Stereotaxy is of particular importance in neurosurgery where the technique is routinely used for diagnosis and treatment of intracranial tumors (Willems et al., 2006), as well as for the application of deep brain stimulation electrodes in Parkinson's disease (Starr et al., 1998) and neuropathic pain (Stadler et al., 2011). The fibrinolytic evacuation of intracranial hemorrhages by a stereotaxic apporach has also been reported (Samadani & Rohde, 2009). Moreover, stereotaxic stem cell injections into the human brain are used in phase I and II clinical trials, as the local administration of therapeutic compounds close to the lesion is considered to be

Albeit these concepts may have been strongly perpetuated towards clinical application clinical trials during the last years; the first reported results unfortunately resemble the translational failures that were known from past efforts. This holds true for experimental treatments in the field of stroke (Kondziolka et al., 2005) and Parkinson's disease (Gross et al., 2011) although these concepts were positively evaluated in preceding rodent studies. This emphasizes the relevance of large animal models as an important translational milestone. Whereas simplified stereotaxic devices based on brain atlases are widely available for rodents, the accuracy and complexity of human stereotaxic devices can currently only be modeled in primates. However, this complexity, including the individual, "lesion-specific" application of substances or the induction of phenotypically varying intacerebral hematomas may be critically needed in translational research to mimic the more

We expanded the sheep model to fill this methodological gap and to provide an additional large animal model for translational research in cell transplantation after ischemic stroke and intracerebral hemorrhages. Our model allows precise, MR-guided implantation of magnetically labeled, bone-marrow derived mesenchymal stem cells (stroke treatment) and autologous blood samples into the ovine brain (hemorrhage induction). The technique was developed using the BrainsightTM neuronavigation system (Rogue Research Inc., Quebec, Canada) and several modifications were applied to adapt the system to the ovine skull anatomy. Cell tracking can be performed reliably using clinical MR scanners with adequate

This chapter describes the methodology of image-guided frameless stereotaxic surgery in sheep with special emphasis on (i) the application of an autologous therapeutic cell population (e.g., the mesenchymal stem cells), (ii) the previous labeling and subsequent imaging protocols for MR-based cell tracking in sheep and (iii) the MR-guided induction of

The neurosurgical approach for stereotaxy in sheep requires hornless subjects for easy accessibility of cranial structures. Merino sheep may be of advantage as many hornless strains can be found in this widely available race. Adult merino sheep weigh approximately 80 kilograms (ewe) to 130 kilograms (rams) and have a wither height of about 0.9 meter. This body size allows for relatively easy handling. Frequent and early contact to humans facilitates familiarization and improves the handling. Species appropriate housing, feeding

**3.1 General information about the species and handling requirements** 

heterogenic patient populations enrolled in clinical trials.

advantageous.

resolution and sensitivity.

cerebral hemorrhage in the species.

**3. Technical description of surgery** 

as well as thorough medical inspections and blood screening, medication and vaccination ensure a significant reduction of postoperative complications and thereby enhance study quality (Boltze et al., 2008).

Anesthesia is performed as described elsewhere (Boltze et al., 2008). Animals should be intubated after induction of anesthesia and placed in a prone ("sphinx") position during surgery and imaging. Vital parameters (electrocardiogram, oxygen saturation, blood pressure, rectal body temperature) should be continuously monitored during any surgical intervention.

### **3.2 Frameless stereotaxy in sheep – preparation and data acquisition**

The neuronavigation device, BrainSightTM, is a frameless system that allows for MRI data set based planning of surgical approaches as well as for surveillance and precision control of the surgical intervention with an optical position sensor (Frey et al., 2004). An individual 3D-reconstruction of the head, especially the brain, is required for the precise planning and performance of the sterotaxic injections.

### **3.2.1 Fiducial marker positioning and imaging**

The MR-compatible fiducial markers are attached to a maxillary splint (Fig. 1a). The use of the splint is different from neurosurgical approaches in human medicine, where fiducial markers can be fixed directly to cranial bones. This is not recommended in animals due to safety and welfare issues, especially when the animal is awake between MRI data set acquisition and surgery. The maxillary splint consists of a mouthpiece (Fig. 1a, 1) and two angled arms (Fig. 1a/b, 2) that hold the fiducial markers (Fig. 1a/b, 4). The mouthpiece is inserted carefully, avoiding damage to or constriction of the tracheal tube. It can be adapted to the individual shapes of the maxillary molars and the hard palate by using thermoplastic, which cures within a few minutes. The fiducial markers are usually placed in the area between the cheeks and the ears. The maxillary splint has to be adapted to each individual sheep to ensure maximum precision. The splint has to be inserted for imaging and surgery.

a) maxillary splint with mouthpiece (1), angeled arms (2), bar spacer to the skin (3) and fiducial marker (4); b) sheep before surgery, the maxillary splint is inserted and fixed with a bandage. The angeled arms (2) support the fiducial markers (4); c) 3D-MRI reconstruction of the skin illustrates the position of fiducial makers between cheek and ear (black arrow heads)

Frameless Stereotaxy in Sheep –

7. Choose the next slice.

completely selected.

Fig. 2. BrainSightTM software applications I

press "Compute Surface".

1. Switch the layout to "1|3 windows".

"Skin" of the select button (Fig. 3a, 1).

the fiducial marker for better view of their position.

Coordinate system is "Brainsight" (preselected).

"Landmarks" tab:

and bone. Use the "Reconstruction" tab as follows:

Neurosurgical and Imaging Techniques for Translational Stroke Research 27

8. Repeat points 5 to 7 for each MRI slice until all areas of the specific structure are

a) Graphical user interface with tab based modules (red rectangle), "Anatomical" tab (1), "Show Image & Detail" button (2); b) "ROIs": reconstruction of the brain from MRI data set,

3D reconstructions are created after defining the ROIs. A skin reconstruction is necessary to register the fiducial markers. It is recommended to render 3D reconstructions from brain

1. Skin reconstruction is performed automatically by choosing "Surface Skin". Use the threshold slider to suppress inclusion of air and/or wool in the skin rendering. Press

2. Bone and brain are calculated by selecting "Surface from ROIs". Label the generated surface as "bone" and "brain". Choose corresponding ROI from the "Select button" and

The MRI-positive fiducial markers need to be relocalized manually in the MRI data set. The tagging of these pivotal landmarks requires maximum precision. Use the

2. Skin surface reconstruction should be opened in the large left window by choosing

3. MRI data sets are now opened in three windows on the right side. Select "Inline & All Landmarks" in the upper, "Inline 90 & All Landmarks" in the middle and "Perpendicular & All landmarks" in the bottom window. You may wish to zoom out

4. Choose "sphere" from the select button (Fig. 3a, 2) as the shape of the fiducial markers.

6. Select a landmark position by choosing a fiducial marker in the skin reconstruction. It will be marked with a sphere. The crosshair in the MRI data now indicates the corresponding position in all views. Correct the relative position of the fiducial marker in each view using the sliders on the right hand side of the screen (Fig. 3a, 4). Position the cursor so that the crosshairs perfectly represents the middle of the fiducial marker in

5. Press "New" (Fig. 3a, 3). The compiled landmarks will be named automatically.

"Compute skin" and subsequently name the reconstruction (e.g. "skin").

9. Repeat steps 1 to 8 for any other target (e.g. bone, see below).

slider of grey value threshold (3), slider of opacity (4), icon tools (5)

It should be fixed with bandages to ensure an accurate, stable and reproducible position. Otherwise, even minor displacements of the fiducial markers may lead to large divergences between planned and realized target.

A 1.5 T scanner is sufficient for MRI data set acquisition even though the use of a 3 T scanner is recommended. The optimal time span between data acquisition and surgery should be long enough for animal recovery from imaging anesthesia. However, for cell transplantation after MCAO, this time span must not be too long to stay within a potential therapeutic time window. Usually, a recovery phase of one day is sufficient, but longer recovery phases should be scheduled if permitted by the experimental design.

After initial anesthesia and transport, the sheep is placed on the scanner table and is fixed using adhesive tape (cloth tape tesa®, Tesa SE, Hamburg, Germany) on shoulder and hip, which can be easily removed from the wool. The sheep is covered by a drape which offers limited protection from cooling and prevents soiling of the scanner. For subsequent target planning, a high-resolution T1-weighted 3D sequence is acquired with a minimum resolution of 1 x 1 x 1 mm. Acquisition time depends on the number of averages, but usually does not exceed 30 minutes. Additionally, an acquisition of an angiographic sequence is recommended in order to avoid damaging major intracerebral vessels during surgery (see paragraph 3.2.2).

### **3.2.2 Planning of surgery**

The BrainSightTM software (V2.1) consists of a graphical user interface with a tab based arrangement of modules (Fig. 2a, red rectangle). Pre-surgical image processing includes several steps that are explained in detail in the following paragraph.

After starting the software, the MRI data set has to be loaded. Therefore, click of the "Anatomical" tab (Fig. 2a, 1):


To avoid accidental intrasurgical damage of major cerebral arteries, an overlay with an angiographic data set is recommended. Therefore, use the "Overlays" tab:


In the next step, anatomical structures can be defined by segmentation. While the skin reconstruction is performed automatically, other relevant anatomical structures like the brain and cranial bones have to be identified separately and on each slice. Select the "ROIs" (=region of interest) tab to perform this operation:


7. Choose the next slice.

26 Advances in the Preclinical Study of Ischemic Stroke

It should be fixed with bandages to ensure an accurate, stable and reproducible position. Otherwise, even minor displacements of the fiducial markers may lead to large divergences

A 1.5 T scanner is sufficient for MRI data set acquisition even though the use of a 3 T scanner is recommended. The optimal time span between data acquisition and surgery should be long enough for animal recovery from imaging anesthesia. However, for cell transplantation after MCAO, this time span must not be too long to stay within a potential therapeutic time window. Usually, a recovery phase of one day is sufficient, but longer

After initial anesthesia and transport, the sheep is placed on the scanner table and is fixed using adhesive tape (cloth tape tesa®, Tesa SE, Hamburg, Germany) on shoulder and hip, which can be easily removed from the wool. The sheep is covered by a drape which offers limited protection from cooling and prevents soiling of the scanner. For subsequent target planning, a high-resolution T1-weighted 3D sequence is acquired with a minimum resolution of 1 x 1 x 1 mm. Acquisition time depends on the number of averages, but usually does not exceed 30 minutes. Additionally, an acquisition of an angiographic sequence is recommended in order to avoid damaging major intracerebral vessels during surgery (see

The BrainSightTM software (V2.1) consists of a graphical user interface with a tab based arrangement of modules (Fig. 2a, red rectangle). Pre-surgical image processing includes

After starting the software, the MRI data set has to be loaded. Therefore, click of the

3. Choose the radio button which corresponds to the correct animal position (e.g. "actual" orientation Sphinx Heads First; "scanner" orientation supine head first) To avoid accidental intrasurgical damage of major cerebral arteries, an overlay with an

In the next step, anatomical structures can be defined by segmentation. While the skin reconstruction is performed automatically, other relevant anatomical structures like the brain and cranial bones have to be identified separately and on each slice. Select the "ROIs"

3. Choose a threshold of grey value to localize the structure by moving the slider (Fig. 2b,

6. If necessary, correct the segmentation manually using the "Erase pencil". Cut the unwanted conjunction between target and non-target structures and clear all non-target

4. The opacity of the selection can be changed by the corresponding slider (Fig. 2b, 4). 5. Start segmentation of the threshold areas in the middle of the brain by pressing on the

"Seed Tool" icon (Fig. 2b, 5) and select the threshold area of the brain.

2. Identify positioning of animal by clicking on "Show Image & Detail" (Fig. 2a, 2).

recovery phases should be scheduled if permitted by the experimental design.

several steps that are explained in detail in the following paragraph.

1. Select MRI 3D data set by pressing "Choose" and load the data.

1. Implement the data set by clicking "Load Overlay".

structures using the "Erase Fill" tool (Fig. 2b, 5).

(=region of interest) tab to perform this operation:

1. Press "New ROI from Region Paint".

2. Name the ROI (e.g. "brain").

3).

angiographic data set is recommended. Therefore, use the "Overlays" tab:

2. Define the desired opacity by choosing the corresponding slider.

between planned and realized target.

paragraph 3.2.2).

**3.2.2 Planning of surgery** 

"Anatomical" tab (Fig. 2a, 1):


Fig. 2. BrainSightTM software applications I

a) Graphical user interface with tab based modules (red rectangle), "Anatomical" tab (1), "Show Image & Detail" button (2); b) "ROIs": reconstruction of the brain from MRI data set, slider of grey value threshold (3), slider of opacity (4), icon tools (5)

3D reconstructions are created after defining the ROIs. A skin reconstruction is necessary to register the fiducial markers. It is recommended to render 3D reconstructions from brain and bone. Use the "Reconstruction" tab as follows:


The MRI-positive fiducial markers need to be relocalized manually in the MRI data set. The tagging of these pivotal landmarks requires maximum precision. Use the "Landmarks" tab:


Frameless Stereotaxy in Sheep –

**3.3.1 General surgery** 

Fig. 4. BrainsightTM hardware

The marker is named automatically.

7. Repeat steps 3 to 6 for more trajectories and markers.

**3.3 Frameless stereotaxy in sheep – surgical procedure** 

Finally, the project should be saved to a specified folder (Menue "File").

Neurosurgical and Imaging Techniques for Translational Stroke Research 29

6. Choose "Marker" from the select button ("New" is preselect) to define the target point.

The BrainsightTM hardware comprises an optical position sensor and a number of surgical devices (Fig. 4). During surgery the anesthetized sheep is placed in the prone position on the

a) The peripheral hardware of BrainsightTM system comprises a computer and an optical position sensor (Polaris®); b) Additionally, a drill guide (not shown) and a ruler guide are

available on request; c) The surgical instruments and hardware

the upper "Inline & All Landmark" frame and the "Inline 90 & All Landmarks" using the using the "AP" (anterior/posterior) and the "Lat" (medial/lateral) slider.

7. Press "New" for a new landmark and repeat step 6 until all fiducial markers are identified as landmarks by the software.

Fig. 3. BrainSightTM software applications II

a) identification of landmarks by MR images using the "landmarks" tab, select button of window view (1), select button of the shape of the fiducial marker (2), "New" button (3), sliders for positioning (4); b) definition of targets and trajectories in MRI and 3D reconstructions of skin, bone and brain, sliders for positioning (5), select button of marker or trajectory (6)

The last step of the planning procedure is the identification of the surgical targets according to the study design. In addition, optimal trajectories are planned during this step. The following recommendations for accessing the target should be considered to ensure a minimal invasive surgical procedure and to avoid surgical problems and complications:


The route to the target (trajectory) and the target itself (marker) should be defined as follows using the "Targets" tab:


Finally, the project should be saved to a specified folder (Menue "File").

### **3.3 Frameless stereotaxy in sheep – surgical procedure**

### **3.3.1 General surgery**

28 Advances in the Preclinical Study of Ischemic Stroke

the using the "AP" (anterior/posterior) and the "Lat" (medial/lateral) slider. 7. Press "New" for a new landmark and repeat step 6 until all fiducial markers are

a) identification of landmarks by MR images using the "landmarks" tab, select button of window view (1), select button of the shape of the fiducial marker (2), "New" button (3),

reconstructions of skin, bone and brain, sliders for positioning (5), select button of marker or

The last step of the planning procedure is the identification of the surgical targets according to the study design. In addition, optimal trajectories are planned during this step. The following recommendations for accessing the target should be considered to ensure a minimal invasive surgical procedure and to avoid surgical problems and complications:

iii. Avoid a trajectory that crosses major vessels (as identified by the angiographic overlay).

The route to the target (trajectory) and the target itself (marker) should be defined as follows

1. Switch to a 2 x 3 layout and arrange the frames as follows: MRI data are shown in the upper row and the reconstructed objects in the bottom row. Select "Coronal & All Targets" in the 1st, "Inline 90 & All Targets" in the 2nd and "Inline & All Targets" in the 3rd upper frame. Select "Skin & All Targets", "Bone & All Targets" and "Brain & All

3. Place the crosshair at your target (since a homogeneous MRI signal from the target structure is preferred for the cell tracking experiments, the Corona radiata was used in

4. Use the sliders (Fig. 3b, 5) to place the trajectory (arrow in the crosshair) and make sure it is chosen with respect to the aforementioned recommendations. Ascertain the position of the trajectory in each frame and correct the access using the "AP", "Lat"

5. Choose "Trajectory" in the select button (Fig. 3b, 6) to confirm the planned trajectory.

sliders for positioning (4); b) definition of targets and trajectories in MRI and 3D

i. Avoid the perforation of the horn plate, even hornless animals have one.

identified as landmarks by the software.

Fig. 3. BrainSightTM software applications II

ii. Avoid the perforation of the frontal sinus.

trajectory (6)

iv. Avoid cerebral sulci.

using the "Targets" tab:

this example).

Targets" in the bottom row.

(lateral) and "Twist" sliders.

The trajectory is named automatically.

2. Coordinate system is "Brainsight" (preselected).

the upper "Inline & All Landmark" frame and the "Inline 90 & All Landmarks" using

The BrainsightTM hardware comprises an optical position sensor and a number of surgical devices (Fig. 4). During surgery the anesthetized sheep is placed in the prone position on the

Fig. 4. BrainsightTM hardware

a) The peripheral hardware of BrainsightTM system comprises a computer and an optical position sensor (Polaris®); b) Additionally, a drill guide (not shown) and a ruler guide are available on request; c) The surgical instruments and hardware

Frameless Stereotaxy in Sheep –

1. Arrange a 2 x 3 layout.

location of a fiducial markers. 5. Press "Sample & Go to next landmark".

Neurosurgical and Imaging Techniques for Translational Stroke Research 31

6. Repeat Point 3 to 5 until all landmarks are co-registered with the fiducial markers. 7. Press "Next Step" to enter "Validation" procedure of the registration (see below).

2. Place the pointer randomly in the divot of each fiducial marker. After ideal coregistration, the pointer tip should now precisely indicate the corresponding landmark. Check the distance of the pointer tip to the corresponding landmark. A vector drift of 1.6 mm can be tolerated, but a difference of <1.0 mm is strongly recommended while a deviation of <0.6 mm is optimal. Repeat the "Registration" in the case of >1.6 mm

3. To check for lateral deviation in registration of the subject's head, place the pointer on the skin surface at a virtual line between the eyes. Three points per side are recommended. Control the distance in the "Coronal" view, in the "Inline" view and in the "Skin" reconstruction. Repeat the registration in case a unilateral shift is noticed. 4. For longitudial deviation, place the pointer at several points on the skin (six are recommended) in the midline of the cranium. Control the distance in the "Sagital" view, in the "Inline" view and in the "Skin" reconstruction. Repeat the registration in case of a dorsoventral shift. NOTE: Skin thickness may be altered due to the animal being fixed in the c-clamp, causing the occipital skin to be pulled tautly or to bulge. This

a) Screenshots taken during the setting of the Polaris® optical position sensor. The tracker and the moveable pointer have to be located in the 3D field. Polaris status (1), "Next Step" button (2); b) For adjustment of trajectory and target (3) within the "Session" tab, the red

Following this step, adjust the surgical access and the targets. Recall the planned targets in

1. Arrange a 2 x 3 layout as follows: upper row comprises "Coronal & samples", "Inline 90 & samples" and "Inline & samples". Bottom row contains "Bullseye", "Skin & samples"

The allocation of the landmarks and fiducial makers is validated as follows:

vector deviation or to ensure demanded precision.

5. Press "Next Step" to continue with planning the surgery.

"Bullseye" (4) has to overlap with the green crosshair as shown

may alter the preceived skin thickness.

Fig. 5. BrainSightTM software applications III

the software interface:

and "Bone & samples".

optical position sensor. Make sure that the pointer does not move when recording the

operating table. On smaller tables, fix the animal with adhesive tape in the shoulder and hip region. The head rests on a foam rubber pad to ensure a stable and correct position. The upper head of the animal needs to be shaved and disinfected. Next, the maxillary splint has to be placed precisely in the mouth (ensure its correct position) and it should be fastened tightly with a bandage.

A rigid half-circular clamp (c-clamp) is used for fixation of the head and to provide support for the articulated arm. A similar holder system with skull pins is also used for neurosurgical procedures in humans (Olivier & Bertrand, 1983). The c-clamp is fixed to the skull bone by at least four adjustable skull screws. To avoid skull damage the screws are positioned at distinctive spots (e.g. beneath the Linea temporalis and ventral of the Protuberantia occipitalis externa, Fig. 6a). After fixing the c-clamp to the skull, the whole assembly is attached to the operating table using the adjustable mounting system. The system allows to lock the c-clamp, and thereby the skull in the appropriate position. Next the articulated arm is mounted at the c-clamp. Ensure that the entire field of surgery can be reached by the arm. Thereafter, the tracker (containing three track balls which can be recognized by the optical position sensor) is positioned at the c-clamp to act as a fixed optical reference for the coordination system. The system is now ready for merging the virtual MRI data with the spatial position of the sheep's head. The previously processed data sets (MR images, predefined landmarks and targets) have to be reopened by using the "File" option ("Open Project") in the menu. Choose the "Session" tab:


Next, the Polaris® optical position sensor has to be configured and adjusted:


Next steps require the spatial allocation of the surgical object (head) and MR images. Therefore, the registration of (physical) fiducial markers with the already defined virtual landmarks is performed as follows:


optical position sensor. Make sure that the pointer does not move when recording the location of a fiducial markers.


The allocation of the landmarks and fiducial makers is validated as follows:

1. Arrange a 2 x 3 layout.

30 Advances in the Preclinical Study of Ischemic Stroke

operating table. On smaller tables, fix the animal with adhesive tape in the shoulder and hip region. The head rests on a foam rubber pad to ensure a stable and correct position. The upper head of the animal needs to be shaved and disinfected. Next, the maxillary splint has to be placed precisely in the mouth (ensure its correct position) and it should be fastened

A rigid half-circular clamp (c-clamp) is used for fixation of the head and to provide support for the articulated arm. A similar holder system with skull pins is also used for neurosurgical procedures in humans (Olivier & Bertrand, 1983). The c-clamp is fixed to the skull bone by at least four adjustable skull screws. To avoid skull damage the screws are positioned at distinctive spots (e.g. beneath the Linea temporalis and ventral of the Protuberantia occipitalis externa, Fig. 6a). After fixing the c-clamp to the skull, the whole assembly is attached to the operating table using the adjustable mounting system. The system allows to lock the c-clamp, and thereby the skull in the appropriate position. Next the articulated arm is mounted at the c-clamp. Ensure that the entire field of surgery can be reached by the arm. Thereafter, the tracker (containing three track balls which can be recognized by the optical position sensor) is positioned at the c-clamp to act as a fixed optical reference for the coordination system. The system is now ready for merging the virtual MRI data with the spatial position of the sheep's head. The previously processed data sets (MR images, predefined landmarks and targets) have to be reopened by using the

2. Select all "Targets in project" in the opened frame and move them to "Targets to sample

3. Press "Next Step" to enter the "Polaris" submodule in the "Session". A new frame will

1. Make sure the "Polaris status" is "ok" (Fig. 5a, 1) and that all tools (pointer, subject tracker) are located in the detection space (visible field). Otherwise press "Reset

2. The tracker (fixed to the c-clamp) should be placed in the middle of the visible field by moving the Polaris camera. Ensure the correct position of the subject tracker in 3D, top, lateral and frontal view. Make sure that the visible field is wide enough to track the pointer when navigating in the periphery of the head. To check this move the Pointer in

Next steps require the spatial allocation of the surgical object (head) and MR images. Therefore, the registration of (physical) fiducial markers with the already defined virtual

1. Switch the layout to 1|3 windows and use the same window allocation as during

3. Select one landmark from the list. The correct selection will be confirmed acoustically.

4. Place the tip of the pointer in the divot of the corresponding fiducial marker. This is the most important step to ensure precision of the surgical approach. The pointer has to be placed orthogonally to the divot in each dimension. Avoid any pressure on the fiducial marker, which may alter its position. Make sure the pointer can be detected by the

The specific landmark is now displayed and marked in each window.

"File" option ("Open Project") in the menu. Choose the "Session" tab:

Next, the Polaris® optical position sensor has to be configured and adjusted:

3. Press "Next Step" (Fig. 5a, 2) to enter the "Registration" module.

1. Choose "New Online Session" from the select button.

in this session" via the drag and drop operations.

be opened automatically.

the 3D field of view).

landmark tagging.

landmarks is performed as follows:

2. Perform manual registration update.

Polaris".

tightly with a bandage.


Fig. 5. BrainSightTM software applications III

a) Screenshots taken during the setting of the Polaris® optical position sensor. The tracker and the moveable pointer have to be located in the 3D field. Polaris status (1), "Next Step" button (2); b) For adjustment of trajectory and target (3) within the "Session" tab, the red "Bullseye" (4) has to overlap with the green crosshair as shown

Following this step, adjust the surgical access and the targets. Recall the planned targets in the software interface:

1. Arrange a 2 x 3 layout as follows: upper row comprises "Coronal & samples", "Inline 90 & samples" and "Inline & samples". Bottom row contains "Bullseye", "Skin & samples" and "Bone & samples".

Frameless Stereotaxy in Sheep –

expanded during this step.

Neurosurgical and Imaging Techniques for Translational Stroke Research 33

the pointer tip in the drill hole. In case of minor deviation, the drill hole can be easily

Next, the distance from the drill hole to the intracerebral target must be measured. Place the pointer tip in the drill hole at the level of the skull surface, not the level of the dura. The distance between the pointer tip and the target should now be displayed on the computer. Thereafter, a ruler guide with digital display is placed in the articulated arm. The instrument to be inserted into the brain, for example a filled Hamilton syringe (see 3.3.2), is mounted at the ruler guide. The tip of the instrument (e.g. a syringe cannula, Hamilton Company USA, Reno, USA) has to be positioned at the level of the skull surface, as done with the pointer tip for distance measuring. Now, set the digital display to zero for

Fig. 7. Application equipment with ruler guide and Hamilton syringe. The syringe is

receives postoperative medication with antibiotics and analgesics for 5 days.

This should be done in a slow and steady manner to avoid microbleedings and injuries. Do not advance faster than 2 mm per minute. In case you want to inject any kind of substance or cell solution (as in the example given in 3.3.2) this should also be done slowly, ideally using a micropump at a maximum pump rate of 5 µl per minute. The cannula is left in position for another ten minutes to allow the injected substance to diffuse locally. The explantation of the cannula is done according to the implantation. Repeat all steps above for multiple insertions. Verify the trajectory for each individual injection. At the end of the surgery, the drill hole is plugged with sterile bone wax, the articulated arm and the c-clamp are removed carefully by detaching the screws, and the skin wounds are sutured. Before the animal awakes from the anesthesia, the maxillary splint is carefully removed and the sheep

inserted to a depth of 29.2 mm in the given example.

reference. The cannula can be inserted to the desired depth (Fig. 7).


#### Fig. 6. Surgical set-up in sheep

a) Position of the c-clamp, fixed with 4 skull screws beneath the Linea temporalis and Protuberantia occipitalis externa, note the tracker on the right side of the c-clamp; b) The articulated arm is mounted on the c-clamp and additionally fixed by a sharp stabilizing pin (black arrow). The pointer (white arrow) is placed in the articulated arm at the desired trajectory. c) Drill guide (white arrow) for manual drilling is placed in the arm (Please note that the subject's head is not covered by surgical drape for better illustration)

The skin above the desired access is locally incised. The incision usually does not exceed one centimeter. The periosteum is removed using a Willigers raspartory. You may use small retractors for better accessibility. For skull trepanation, adjust the drill guide to the measured bone thickness, mount it at the end of the articulated arm and place it directly on the exposed skull surface. Drilling is performed at lower speed (10,000 rpm or less, for example using a Microspeed® uni, Aesculap AG, Tuttlingen, Germany). Since minor deviation between measured and real bone thickness can occur, the dura mater may not be reached after drilling. In that case, carefully continue with manual drilling (Fig. 6c). The correct position of the drill hole within the planned trajectory should be checked by placing

2. Check that the coordinate system is "Brainsight", driver is "Pointer", and Crosshair is

4. Place the pointer in the articulated arm for adjustment of the trajectory (Fig. 6b). The pointer represents the needle to be inserted during surgery. Make sure the pointer is

5. Place the pointer tip next to the expected surgical access (drill hole) position by moving the articulated arm. After approximate adjustment, ascertain that the "Bullseye" (red

6. Use the x-y stage to optimize the position. Make sure the red dot of the "Bullseye"

7. Lock the articulated arm and place the stabilizing pin in the guide. Ensure that all

8. Measure the thickness of the cranial bone at the level of access in the "Inline 90 & samples" view on the computer. In order to avoid damage to the dura mater or the brain by drilling to deep you may wish to underestimate bone thickness by 0.5 mm.

a) Position of the c-clamp, fixed with 4 skull screws beneath the Linea temporalis and Protuberantia occipitalis externa, note the tracker on the right side of the c-clamp; b) The articulated arm is mounted on the c-clamp and additionally fixed by a sharp stabilizing pin (black arrow). The pointer (white arrow) is placed in the articulated arm at the desired

(Please note that the subject's head is not covered by surgical drape for better illustration) The skin above the desired access is locally incised. The incision usually does not exceed one centimeter. The periosteum is removed using a Willigers raspartory. You may use small retractors for better accessibility. For skull trepanation, adjust the drill guide to the measured bone thickness, mount it at the end of the articulated arm and place it directly on the exposed skull surface. Drilling is performed at lower speed (10,000 rpm or less, for example using a Microspeed® uni, Aesculap AG, Tuttlingen, Germany). Since minor deviation between measured and real bone thickness can occur, the dura mater may not be reached after drilling. In that case, carefully continue with manual drilling (Fig. 6c). The correct position of the drill hole within the planned trajectory should be checked by placing

trajectory. c) Drill guide (white arrow) for manual drilling is placed in the arm

hardware pieces are in the correct position and tightly locked now.

"Needle".

3. Select the first trajectory (Fig. 5b, 3).

circle) overlaps with the green crosshair.

aligns with the crosshair (Fig. 5b, 4).

Fig. 6. Surgical set-up in sheep

both locked in the chuck and located in the visible field.

the pointer tip in the drill hole. In case of minor deviation, the drill hole can be easily expanded during this step.

Next, the distance from the drill hole to the intracerebral target must be measured. Place the pointer tip in the drill hole at the level of the skull surface, not the level of the dura. The distance between the pointer tip and the target should now be displayed on the computer. Thereafter, a ruler guide with digital display is placed in the articulated arm. The instrument to be inserted into the brain, for example a filled Hamilton syringe (see 3.3.2), is mounted at the ruler guide. The tip of the instrument (e.g. a syringe cannula, Hamilton Company USA, Reno, USA) has to be positioned at the level of the skull surface, as done with the pointer tip for distance measuring. Now, set the digital display to zero for reference. The cannula can be inserted to the desired depth (Fig. 7).

Fig. 7. Application equipment with ruler guide and Hamilton syringe. The syringe is inserted to a depth of 29.2 mm in the given example.

This should be done in a slow and steady manner to avoid microbleedings and injuries. Do not advance faster than 2 mm per minute. In case you want to inject any kind of substance or cell solution (as in the example given in 3.3.2) this should also be done slowly, ideally using a micropump at a maximum pump rate of 5 µl per minute. The cannula is left in position for another ten minutes to allow the injected substance to diffuse locally. The explantation of the cannula is done according to the implantation. Repeat all steps above for multiple insertions. Verify the trajectory for each individual injection. At the end of the surgery, the drill hole is plugged with sterile bone wax, the articulated arm and the c-clamp are removed carefully by detaching the screws, and the skin wounds are sutured. Before the animal awakes from the anesthesia, the maxillary splint is carefully removed and the sheep receives postoperative medication with antibiotics and analgesics for 5 days.

Frameless Stereotaxy in Sheep –

6. Wash cells twice in PBS.

7. Count MNC.

Cell labeling

4. Centrifuge at 1,000 x g for 25 min.

using a sterile pipette (Fig. 8a).

Neurosurgical and Imaging Techniques for Translational Stroke Research 35

5. Carefully transfer the interface layer of mononuclear cells to a sterile centrifuge tube by

The BMSC population is isolated from MNC based on its ability to adhere to the cell culture flask. For that reason, MNC are seeded at a density of 5 x 10E4/cm2 in a culture flask. Cells are cultivated with DMEM (high glucose, PAA Laboratories GmbH, Pasching, Austria), 10% FCS (Invitrogen GmbH, Darmstadt, Germany) and 1% penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria) at 37°C and 5% CO2 for 14 days. Two days after

Fig. 8. Density gradient centrifugation of bone marrow and Prussian Blue staining of BMSC a) Density gradient centrifugation of diluted bone marrow: segmentation before and after centrifugation (supernatant: serum and platelets, sediment: erythrocytes and granulocytes) is displayed; b) cultivated, unlabeled, ovine BMSC stained with Prussian Blue (PB) and

Very small superparamagnetic iron oxide particles (VSOP, Ferropharm, Teltow, Germany) are used for magnetic cell labeling. VSOP consist of a 5 nm iron oxide core coated by monomer citrate (total diameter of 9 nm) with a negative surface charge. Iron particle incorporation by the cells causes a strong decrease in the transverse relaxation time, which results in signal loss in T2\*-weighted MR imaging (Arbab et al., 2003; Bowen et al., 2002; Renshaw et al., 1986). No apparent long-term cytotoxic effects were observed after using the particles both in vitro and in vivo (Fleige et al., 2002; Stroh et al., 2004 and 2009). For magnetic labeling sterile VSOP are added to the incubation medium. Addition of lipofection agents is not required for BMSC labeling. Cells are incubated with VSOP at 37°C and 5% CO2 for 90 minutes. After incubation, the cells are washed three times with PBS to remove any remaining VSOP not endocytosed by cells. The cells are harvested by incubation with

eosin; c) VSOP-labeled BMSC stained with PB and eosin; scale bar: 50 µm

seeding, all non-adherent cells are removed by two washing steps with PBS.

### **3.3.2 Exemplary applications**

Exemplary application for stereotactically guided (cell or blood) depostions following stroke and other cerebral disorders are an emerging field of research in regenerative medicine. This route of administration is expected to be used in upcoming clinical trials. The sheep model allows the simulation of autologous cell therapies, in particular using intracerebral stereotactic cell delivery. This may be required for example when cell depots need to be placed next to a lesion site, which may vary between individual cases.

In the described stereotactic model, autologous bone marrow stromal cells (BMSC) were chosen as an example since these cells were reported to be benefical after stroke treatment and neurodegenerative diseases (Joyce et al., 2010). Also other cell types like neural or embryonic stem cells were used for local transplantation close to the infracted area border (Guzman et al., 2007) or into the contralateral hemisphere (Hoehn et al., 2002) in rodent models. After purification and cultivation (see 4.1.1), a defined cell number is suspended and stored in a Hamilton syringe with a 15.6 cm long cannula. The technique of stereotactic transplantation is described in 3.3.1. For post-translational tracking, the cells can be labeled with iron oxide nanoparticles such as VSOP (very small superparamagnetic ironoxide particles, Ferropharm, Germany) (see 4.1.1). The localization and migration of transplanted cells is monitored by MRI (see 4.1.2).

Alternatively, the stereotactic device can be used to model cerebral hemorrhage by application of blood into the sheep brain. For that purpose, autologous blood is collected from an arterial (preferred) or venous line in a heparinized syringe and is transferred to a Hamilton syringe before application. The steps are technically identical with the injection of cells (see 3.3.1). The injected blood volume depends on the planed region and the target dimensions, but should not exceed two milliliters in sheep (approx. weight of sheep brain: 120 g).

### **4. Application examples**

### **4.1 Concept 1: Cell labeling for MRI-based tracking in vivo**

### **4.1.1 Harvesting and processing of Bone Marrow Stromal Cells (BMSC)**

Autologous cell harvest and cell processing

BMSC are commonly isolated from bone marrow aspirates. Bone marrow samples may be harvested from the iliac crest in humans and in sheep. Therefore, the puncture area on both iliac crests are shaved and disinfected while the anesthetized sheep is placed in a prone position. Samples of approximately 10 mL of bone marrow are taken from both sides by multiple punctures using a heparinized syringe. Higher aspiration volumes from single punctures may result in contamination with peripheral blood. In the next step, the mononuclear cells (MNC) are isolated by density gradient centrifugation, which should be done within one hour after bone marrow harvest. A protocol for the separation of ovine MNC is stated below.


Exemplary application for stereotactically guided (cell or blood) depostions following stroke and other cerebral disorders are an emerging field of research in regenerative medicine. This route of administration is expected to be used in upcoming clinical trials. The sheep model allows the simulation of autologous cell therapies, in particular using intracerebral stereotactic cell delivery. This may be required for example when cell depots need to be

In the described stereotactic model, autologous bone marrow stromal cells (BMSC) were chosen as an example since these cells were reported to be benefical after stroke treatment and neurodegenerative diseases (Joyce et al., 2010). Also other cell types like neural or embryonic stem cells were used for local transplantation close to the infracted area border (Guzman et al., 2007) or into the contralateral hemisphere (Hoehn et al., 2002) in rodent models. After purification and cultivation (see 4.1.1), a defined cell number is suspended and stored in a Hamilton syringe with a 15.6 cm long cannula. The technique of stereotactic transplantation is described in 3.3.1. For post-translational tracking, the cells can be labeled with iron oxide nanoparticles such as VSOP (very small superparamagnetic ironoxide particles, Ferropharm, Germany) (see 4.1.1). The localization and migration of transplanted

Alternatively, the stereotactic device can be used to model cerebral hemorrhage by application of blood into the sheep brain. For that purpose, autologous blood is collected from an arterial (preferred) or venous line in a heparinized syringe and is transferred to a Hamilton syringe before application. The steps are technically identical with the injection of cells (see 3.3.1). The injected blood volume depends on the planed region and the target dimensions, but should

BMSC are commonly isolated from bone marrow aspirates. Bone marrow samples may be harvested from the iliac crest in humans and in sheep. Therefore, the puncture area on both iliac crests are shaved and disinfected while the anesthetized sheep is placed in a prone position. Samples of approximately 10 mL of bone marrow are taken from both sides by multiple punctures using a heparinized syringe. Higher aspiration volumes from single punctures may result in contamination with peripheral blood. In the next step, the mononuclear cells (MNC) are isolated by density gradient centrifugation, which should be done within one hour after bone marrow harvest. A protocol for the separation of ovine

1. Dilute bone marrow with phosphate buffer saline (PBS, Biochrom KG seromed®, Berlin,

2. Merge Biocoll (1.077 g/mL, Biochrom KG seromed®, Berlin, Germany) and Pancoll (1.091 g/mL, PAN-Biotech GmbH, Aidenbach, Germany) in a ratio of 1:1 (v/v) to obtain a density of 1.084 g/mL (separation medium). Add 7 mL of mixed separation

3. Carefully place a layer of 10 mL of diluted bone marrow onto the gradient mixture.

Avoid commixture between cell layer and the separation medium.

placed next to a lesion site, which may vary between individual cases.

not exceed two milliliters in sheep (approx. weight of sheep brain: 120 g).

**4.1.1 Harvesting and processing of Bone Marrow Stromal Cells (BMSC)** 

**4.1 Concept 1: Cell labeling for MRI-based tracking in vivo** 

**3.3.2 Exemplary applications** 

cells is monitored by MRI (see 4.1.2).

**4. Application examples** 

MNC is stated below.

Autologous cell harvest and cell processing

Germany) at a 1:1 (v/v) ratio.

medium into a 50 mL falcon tube.

The BMSC population is isolated from MNC based on its ability to adhere to the cell culture flask. For that reason, MNC are seeded at a density of 5 x 10E4/cm2 in a culture flask. Cells are cultivated with DMEM (high glucose, PAA Laboratories GmbH, Pasching, Austria), 10% FCS (Invitrogen GmbH, Darmstadt, Germany) and 1% penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria) at 37°C and 5% CO2 for 14 days. Two days after seeding, all non-adherent cells are removed by two washing steps with PBS.

Fig. 8. Density gradient centrifugation of bone marrow and Prussian Blue staining of BMSC a) Density gradient centrifugation of diluted bone marrow: segmentation before and after centrifugation (supernatant: serum and platelets, sediment: erythrocytes and granulocytes) is displayed; b) cultivated, unlabeled, ovine BMSC stained with Prussian Blue (PB) and eosin; c) VSOP-labeled BMSC stained with PB and eosin; scale bar: 50 µm

### Cell labeling

Very small superparamagnetic iron oxide particles (VSOP, Ferropharm, Teltow, Germany) are used for magnetic cell labeling. VSOP consist of a 5 nm iron oxide core coated by monomer citrate (total diameter of 9 nm) with a negative surface charge. Iron particle incorporation by the cells causes a strong decrease in the transverse relaxation time, which results in signal loss in T2\*-weighted MR imaging (Arbab et al., 2003; Bowen et al., 2002; Renshaw et al., 1986). No apparent long-term cytotoxic effects were observed after using the particles both in vitro and in vivo (Fleige et al., 2002; Stroh et al., 2004 and 2009). For magnetic labeling sterile VSOP are added to the incubation medium. Addition of lipofection agents is not required for BMSC labeling. Cells are incubated with VSOP at 37°C and 5% CO2 for 90 minutes. After incubation, the cells are washed three times with PBS to remove any remaining VSOP not endocytosed by cells. The cells are harvested by incubation with

Frameless Stereotaxy in Sheep –

Neurosurgical and Imaging Techniques for Translational Stroke Research 37

particles or labeled cells, or even single cells, can be detected by high field MRI (Dodd et al.,

a) Viability during 24 hours after labeling, control treatment is set to 100%; b) Relaxometry of labeled and unlabeled BMSC. White bars: immediately; black bars: 24 hours after labeling

**sequence resolution TR TE duration**  T2\* 0.83 x 0.66 x 0.50 mm 620 20 2 h 07 min SWI 0.56 x 0.39 x 0.25 mm 60 20 1h 29 min Table 2. Parameters of MR imaging for detection of VSOP-labeled cells in ovine brain; TR –

For in vivo detection of VSOP-labeled BMSC in the ovine brain a 3T MRI scanner (Magnetom Trio, Siemens AG, Munich, Germany) was used. After initial anesthesia the sheep was placed on the scanner table as described in 3.1.1 and shown in Fig. 10a. A flexible head coil (Siemens AG, Munich, Germany) was placed centrally above the brain (Fig. 10b). After a brief T2-weighted Turbo Spin Echo (TSE) sequence for anatomical orientation, SWIand T2\*-sequences were acquired with the field of view set to the region of transplantation. The sequence parameters are listed in Table 2. Two hours after transplantation, a distinct, ellipsoidal to circular hypointensity was detectable at all injection sites in T2\* and SWI sequences (Fig. 11). This was not the case for control (saline) application without cells.

1999; Shapiro et al., 2005) taking advantage of the blooming effect.

Fig. 9. Viability and relaxometry of VSOP-labeled BMSC

(n=6 in each experiment)",\*\*p<0.01.

repetition time, TE – echo time

trypsin (Invitrogen GmbH, Darmstadt, Germany) for 5 minutes and centrifugated at 350 x g. To visualize the incorporated iron oxide particles, Prussian Blue (PB) staining can be used (Fig. 8c). A protocol for PB staining is given in Table 1.

The cells can be labeled additionally using fluorescent dyes as GFP or PKH26, which may be helpful for histological examination after in vivo applications. This allows discrimination between initially labeled cells and cells that secondarily took up iron by endocytosis.


Table 1. Protocol for Prussian Blue staining of VSOP-labeled BMSC

Impact of VSOP labeling on cell viability and T2 relaxation time

The influence of 3.0 mM VSOP on cellular viability and magnetic labeling has been investigated for ovine BMSC in previous experiments. The viability was evaluated by the trypan blue dye exclusion test before labeling, immediately thereafter, as well as after 4 and 24 hours. Viability was compared to that of unlabeled cells. The transverse relaxation time (T2) was measured at 0.47 T/ 20 MHz (Minispec, Bruker, Ettlingen, Germany) immediately and 24 hours after VSOP-labeling to examine labeling efficacy.

Our own results show that cellular viability decreased to 86 ± 16 % immediately after labeling. At 4 hours after labeling, the cell viability rate remained unaltered (89 ± 14 %), but slightly declined to 75 ± 3 % after 24 hours (Fig. 9a). Relaxometry measurements resulted in T2 relaxation times of 1,888 ± 171 ms for unlabeled cells as compared to 434 ± 147 ms for VSOP-labeled cells (p<0.01), indicating a reduction of T2-time to 23 ± 8% after VSOP labeling (Fig. 9b, white). This result was reproduced 24 hours after labeling (Fig. 9b, black).

### **4.1.2 BMSC detection in vivo**

Since VSOP shorten T2 relaxation time, the particles can be detected by MRI using gradient echo sequences, in particular T2\* weighted imaging. Alternatively, susceptibility weighted imaging (SWI) can be used. The main difference to T2\* is the inclusion of phase information into the image acquisition (Haacke et al., 2009). This is of particular advantage in case that dephasing particles such as iron-oxide particles are present in the respective voxel. The dephasing results in a hypointense signal at a given echo time. However, this signal loss is not specific, as there are a multitude of other sources of signal voids, such as blood and air.

The blooming effect is a well known phenomenon caused by iron oxide particles. Due to the augmented dephasing of proton spins, a major susceptibility artefact is depicted that exceeds the real dimension of the particle by a factor of up to 50. Hence, a small amount of

trypsin (Invitrogen GmbH, Darmstadt, Germany) for 5 minutes and centrifugated at 350 x g. To visualize the incorporated iron oxide particles, Prussian Blue (PB) staining can be used

The cells can be labeled additionally using fluorescent dyes as GFP or PKH26, which may be helpful for histological examination after in vivo applications. This allows discrimination between initially labeled cells and cells that secondarily took up iron by

**step Prussian Blue staining duration 1** Fixate in 4% paraformaldehyde 20 min

**3** Wash in PBS 5 min **4** Stain in eosin 4 min **5** Wash in PBS 5 min

The influence of 3.0 mM VSOP on cellular viability and magnetic labeling has been investigated for ovine BMSC in previous experiments. The viability was evaluated by the trypan blue dye exclusion test before labeling, immediately thereafter, as well as after 4 and 24 hours. Viability was compared to that of unlabeled cells. The transverse relaxation time (T2) was measured at 0.47 T/ 20 MHz (Minispec, Bruker, Ettlingen, Germany) immediately

Our own results show that cellular viability decreased to 86 ± 16 % immediately after labeling. At 4 hours after labeling, the cell viability rate remained unaltered (89 ± 14 %), but slightly declined to 75 ± 3 % after 24 hours (Fig. 9a). Relaxometry measurements resulted in T2 relaxation times of 1,888 ± 171 ms for unlabeled cells as compared to 434 ± 147 ms for VSOP-labeled cells (p<0.01), indicating a reduction of T2-time to 23 ± 8% after VSOP labeling (Fig. 9b, white). This result was reproduced 24 hours after labeling (Fig. 9b,

Since VSOP shorten T2 relaxation time, the particles can be detected by MRI using gradient echo sequences, in particular T2\* weighted imaging. Alternatively, susceptibility weighted imaging (SWI) can be used. The main difference to T2\* is the inclusion of phase information into the image acquisition (Haacke et al., 2009). This is of particular advantage in case that dephasing particles such as iron-oxide particles are present in the respective voxel. The dephasing results in a hypointense signal at a given echo time. However, this signal loss is not specific, as there are a multitude of other sources of signal

The blooming effect is a well known phenomenon caused by iron oxide particles. Due to the augmented dephasing of proton spins, a major susceptibility artefact is depicted that exceeds the real dimension of the particle by a factor of up to 50. Hence, a small amount of

**6** Conserve with glycerol (culture flasks) or mounting medium (slide)

1% hydrochloride acid in a 1:1 ratio. 20 min

(Fig. 8c). A protocol for PB staining is given in Table 1.

**<sup>2</sup>**Stain with 2% potassium ferrocyanide, Trihydrate and

Table 1. Protocol for Prussian Blue staining of VSOP-labeled BMSC Impact of VSOP labeling on cell viability and T2 relaxation time

and 24 hours after VSOP-labeling to examine labeling efficacy.

endocytosis.

black).

**4.1.2 BMSC detection in vivo** 

voids, such as blood and air.

particles or labeled cells, or even single cells, can be detected by high field MRI (Dodd et al., 1999; Shapiro et al., 2005) taking advantage of the blooming effect.

Fig. 9. Viability and relaxometry of VSOP-labeled BMSC

a) Viability during 24 hours after labeling, control treatment is set to 100%; b) Relaxometry of labeled and unlabeled BMSC. White bars: immediately; black bars: 24 hours after labeling (n=6 in each experiment)",\*\*p<0.01.


Table 2. Parameters of MR imaging for detection of VSOP-labeled cells in ovine brain; TR – repetition time, TE – echo time

For in vivo detection of VSOP-labeled BMSC in the ovine brain a 3T MRI scanner (Magnetom Trio, Siemens AG, Munich, Germany) was used. After initial anesthesia the sheep was placed on the scanner table as described in 3.1.1 and shown in Fig. 10a. A flexible head coil (Siemens AG, Munich, Germany) was placed centrally above the brain (Fig. 10b). After a brief T2-weighted Turbo Spin Echo (TSE) sequence for anatomical orientation, SWIand T2\*-sequences were acquired with the field of view set to the region of transplantation. The sequence parameters are listed in Table 2. Two hours after transplantation, a distinct, ellipsoidal to circular hypointensity was detectable at all injection sites in T2\* and SWI sequences (Fig. 11). This was not the case for control (saline) application without cells.

Frameless Stereotaxy in Sheep –

graft

Neurosurgical and Imaging Techniques for Translational Stroke Research 39

Fig. 11. MR and macroscopic view of 100,000 VSOP-labeled BMSC in the ovine brain a) susceptibility weighted imaging (SWI), b) phase image of SWI, c) T2\* image, d)

**4.1.3 Neuropathology and ex vivo BMSC detection** 

macroscopic view on brain slice. Black arrow indicates the macroscopically visible BMSC

Animals are sacrificed during deep anesthesia by a single, intravenously injection of 20 mL pentobarbital (Eutha 77, Essex Pharma LtD, Munich, Germany). After death is confirmed (abscence of cardiac, respiratory and reflexive activity over a period of at least two minutes) the sheep is rapidly decapitated at the atlanto-occipital junction. The carotid arteries are exposed for perfusion. Blunt perfusion cannulas are placed in each artery and are fixed with stout thread. Initially, the head is perfused with 3 L PBS followed by 20 L 4% paraformaldehyde (PFA, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). A roller pump system (Roth Cyclo II, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) can be used for that purpose. After removal of skin, muscles and adnexes by a sharp knife, the cranial cavity is opened using an oscillating saw (KM-40, Heraeus GmbH, Hanau, Germany). After careful removal of the dura, the open cranium is fixated in 4% PFA for at least 24 hours.

Afterwards, the brain is removed for immersion fixation for another 48 hours in PFA.

Macroscopic examination of the brain comprises weighing, measuring of circumferences, and photographic documentation from all directions. Next, the brain is cut into 4 mm thick, coronal slices which are each photographed from rostral and occipital direction.

Histological examination allows discrimination of cellular signals to other sources of hypointense signal change (4.1.3).

Fig. 10. MRI of sheep

a) Position of sheep in MR scanner. The animal is fixed with adhesive tap; b) Image taken by the scanner's monitoring camera. The head is placed on a position pad and covered with folio drape and a flexible head coil is used (white arrow). Potentially excreted saliva and rumen fluid are collected by a plastic bowl placed directly below the animal's mouth

The non-invasive detection of the transplanted cells allows longitudinal studies for up to six months in the individual animal for precise detection of migration and localization of transplanted cells (Jendelova et al., 2004; Stroh et al., 2004 and 2005). It can also be combined with stroke related MR-examinations and allows tracing of migration processes towards the lesion.

Histological examination allows discrimination of cellular signals to other sources of

hypointense signal change (4.1.3).

Fig. 10. MRI of sheep

lesion.

directly below the animal's mouth

a) Position of sheep in MR scanner. The animal is fixed with adhesive tap;

b) Image taken by the scanner's monitoring camera. The head is placed on a position pad and covered with folio drape and a flexible head coil is used (white arrow). Potentially excreted saliva and rumen fluid are collected by a plastic bowl placed

The non-invasive detection of the transplanted cells allows longitudinal studies for up to six months in the individual animal for precise detection of migration and localization of transplanted cells (Jendelova et al., 2004; Stroh et al., 2004 and 2005). It can also be combined with stroke related MR-examinations and allows tracing of migration processes towards the

Fig. 11. MR and macroscopic view of 100,000 VSOP-labeled BMSC in the ovine brain a) susceptibility weighted imaging (SWI), b) phase image of SWI, c) T2\* image, d) macroscopic view on brain slice. Black arrow indicates the macroscopically visible BMSC graft

### **4.1.3 Neuropathology and ex vivo BMSC detection**

Animals are sacrificed during deep anesthesia by a single, intravenously injection of 20 mL pentobarbital (Eutha 77, Essex Pharma LtD, Munich, Germany). After death is confirmed (abscence of cardiac, respiratory and reflexive activity over a period of at least two minutes) the sheep is rapidly decapitated at the atlanto-occipital junction. The carotid arteries are exposed for perfusion. Blunt perfusion cannulas are placed in each artery and are fixed with stout thread. Initially, the head is perfused with 3 L PBS followed by 20 L 4% paraformaldehyde (PFA, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). A roller pump system (Roth Cyclo II, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) can be used for that purpose. After removal of skin, muscles and adnexes by a sharp knife, the cranial cavity is opened using an oscillating saw (KM-40, Heraeus GmbH, Hanau, Germany). After careful removal of the dura, the open cranium is fixated in 4% PFA for at least 24 hours. Afterwards, the brain is removed for immersion fixation for another 48 hours in PFA.

Macroscopic examination of the brain comprises weighing, measuring of circumferences, and photographic documentation from all directions. Next, the brain is cut into 4 mm thick, coronal slices which are each photographed from rostral and occipital direction.

Frameless Stereotaxy in Sheep –

**4.2 Concept 2: Hemorrhage model 4.2.1 Hemorrhage detection** 

particular patient (black arrow)

application

Neurosurgical and Imaging Techniques for Translational Stroke Research 41

Fig. 13. Histological analysis of 100,000 transplanted, VSOP-labeled BMSC after striatal

microhemorrhages (e.g. due to cell injection) are detectable; bar: 100 µm

a) Overview of transplantation site stained with PB, scale bar: 500 µm; b) Magnification of PB staining, scale bar: 100 µm; c) hematoxylin/eosin staining of transplantation site, no

T1-, T2- and T2\*-weighted MR sequences can be used for hemorrhage detection. The injection of autologous blood into the sheep brain usually results in a spheric to ellipsoidshaped blood clot, with the depot size depending on the injected blood volume. The MR signal characteristics of the hemorrhage differ depending on the temporal stage of the hemorrhage and the pulse sequence used (Kidwell & Wintermark, 2008). In the hyperacute stage, hemoglobin is still oxygenated and therefore diamagnetic, thus appearing iso- to hypointense in T1-weighted sequences (Fig. 14a) and hyperintense in T2-weighted MRI (Fig. 15a). A hypointense rim is observable in T2\* sequence (Fig. 14b and c). In contrast, methemoglobin is present in the sub-acute stage. Methemoglobin is paramagnetic and generates a hyperintensive signal in T1-weighted and a hypointensive signal in T2-weighted MRI. Later, the signal changes become hypointensive in T1- and T2-weigthed MRI due to the progressive biodegradation of hemoglobin into superparamagnetic hemosiderin

Fig. 14. Imaging of stereotactically placed blood clot (1.8 mL, 3 hours old) in ovine brain for simulation of intracerebral hemorrhage using T1 weighted (a) and T2\* weighted sequences (b). An acute occipital intracerebral bleed in a human patient (T2\*, 2 hours after onset of visual disturbances, white arrow) is shown in c) for direct comparison. For more details, please refer to main text. Note an old left temporal ischemia (secondary diagnosis) in this

Usually, the transplantation procedure does not result in macroscopically visible brain tissue alterations. In a minor number of cases, a very small alteration is observable at the injection site (Fig. 12).

Fig. 12. Ovine brain after stereotatic transplantation

The insertion of the cannula may lead to a small alteration (black arrow) on the brain surface.

The transplantation area is cut out of the associated brain slice, embedded in paraffin and cut into 4 µm thick sections for histological analysis. Hematoxylin/eosin staining according to Table 3 is recommended for histological overview and exclusion of bleeding. Prussian Blue staining (according to Table 1, step 2 - 6) is used for detection of VSOPlabeled cells.


Table 3. Protocol for hematoxylin/eosin staining

Transplantation of 100,000 VSOP-labeled cells results in a brown spot at the transplantation site which was clearly visible in the coronal slice (Fig. 11d). Transplantation of PBS reveals no changes in the macroscopic view.

Histological investigation by hematoxylin/eosin staining usually reveals a small cavity containing the labeled cells with scattered mononuclear cells within the transplantation site 8 hours after transplantation. Occasionally, microbleedings may occur close to the injection site. In sheep transplanted with VSOP-labeled BMSC, PB-positive cells are clearly detected at the specified target region (Fig. 13). No migration of cells is observed 8 hours after transplantation.

Fig. 13. Histological analysis of 100,000 transplanted, VSOP-labeled BMSC after striatal application

a) Overview of transplantation site stained with PB, scale bar: 500 µm; b) Magnification of PB staining, scale bar: 100 µm; c) hematoxylin/eosin staining of transplantation site, no microhemorrhages (e.g. due to cell injection) are detectable; bar: 100 µm

### **4.2 Concept 2: Hemorrhage model 4.2.1 Hemorrhage detection**

40 Advances in the Preclinical Study of Ischemic Stroke

Usually, the transplantation procedure does not result in macroscopically visible brain tissue alterations. In a minor number of cases, a very small alteration is observable at the

The insertion of the cannula may lead to a small alteration (black arrow) on the brain

The transplantation area is cut out of the associated brain slice, embedded in paraffin and cut into 4 µm thick sections for histological analysis. Hematoxylin/eosin staining according to Table 3 is recommended for histological overview and exclusion of bleeding. Prussian Blue staining (according to Table 1, step 2 - 6) is used for detection of VSOP-

**step hematoxylin/eosin staining duration**  Stain in hematoxylin 2 min Wash in piped water 2 min Blue in warm piped water 10 min Stain in eosin 4 min Wash in pure water 5 min

Transplantation of 100,000 VSOP-labeled cells results in a brown spot at the transplantation site which was clearly visible in the coronal slice (Fig. 11d). Transplantation of PBS reveals

Histological investigation by hematoxylin/eosin staining usually reveals a small cavity containing the labeled cells with scattered mononuclear cells within the transplantation site 8 hours after transplantation. Occasionally, microbleedings may occur close to the injection site. In sheep transplanted with VSOP-labeled BMSC, PB-positive cells are clearly detected at the specified target region (Fig. 13). No migration of cells is observed 8 hours after

injection site (Fig. 12).

surface.

labeled cells.

transplantation.

Fig. 12. Ovine brain after stereotatic transplantation

**6** Conservation (dehydrating, mounting) Table 3. Protocol for hematoxylin/eosin staining

no changes in the macroscopic view.

T1-, T2- and T2\*-weighted MR sequences can be used for hemorrhage detection. The injection of autologous blood into the sheep brain usually results in a spheric to ellipsoidshaped blood clot, with the depot size depending on the injected blood volume. The MR signal characteristics of the hemorrhage differ depending on the temporal stage of the hemorrhage and the pulse sequence used (Kidwell & Wintermark, 2008). In the hyperacute stage, hemoglobin is still oxygenated and therefore diamagnetic, thus appearing iso- to hypointense in T1-weighted sequences (Fig. 14a) and hyperintense in T2-weighted MRI (Fig. 15a). A hypointense rim is observable in T2\* sequence (Fig. 14b and c). In contrast, methemoglobin is present in the sub-acute stage. Methemoglobin is paramagnetic and generates a hyperintensive signal in T1-weighted and a hypointensive signal in T2-weighted MRI. Later, the signal changes become hypointensive in T1- and T2-weigthed MRI due to the progressive biodegradation of hemoglobin into superparamagnetic hemosiderin

Fig. 14. Imaging of stereotactically placed blood clot (1.8 mL, 3 hours old) in ovine brain for simulation of intracerebral hemorrhage using T1 weighted (a) and T2\* weighted sequences (b). An acute occipital intracerebral bleed in a human patient (T2\*, 2 hours after onset of visual disturbances, white arrow) is shown in c) for direct comparison. For more details, please refer to main text. Note an old left temporal ischemia (secondary diagnosis) in this particular patient (black arrow)

Frameless Stereotaxy in Sheep –

**6. Acknowledgment** 

University of Leipzig.

*Cerebrovasc Dis.,* 18,4,281-287

56, 8, 1015-1020

1951-1964

*Med.,* 48, 1, 52-61

techniques. *Transplantation.,* 76, 7, 123-130

**7. References** 

Neurosurgical and Imaging Techniques for Translational Stroke Research 43

system BrainSightTM was successfully adapted to the ovine skull anatomy. It allows an individual and accurate planning as well as precise execution of stereotactic interventions. Next to a detailed description of the technique itself, two relevant applications of the experimental techniques are reported in this chapter. First, the magnetic labeling and stereotactic transplantation of a stem cell population into the ovine brain is described. The methodology allows for precise injection and tracking of autologous stem cell populations using a widely available 3T MRI scanner. Relatively simple but reliable pathohistological techniques for post-mortem brain assessment and detection of transplanted cells are also given. Thus, the reported methodology may be used for evaluation of cell-based treatment strategies of central nervous system disorders in the gyrencephalic brain. Second, the stereotactic technique can be used for precise modeling of intracerebral hemorrhages by autologous blood injection in sheep. The observed results are in good correlation to those seen in human patients, especially regarding relevant diagnostic findings in MRI. Summarizing, stereotactic interventions in sheep represent a well applicable and reliable

approach for translational research with a wide spectrum of possible applications.

The authors thank Dr. Stephen Frey from Rogue Research Inc. for technical support and the staff of the Institute of Anatomy, Histology and Embrology, Faculty of Veterinary Medicine,

Amiridze, N., Gullapalli, R., Hoffman, G. & Darwish, R. (2009). Experimental model of

Arbab, A.S., Bashaw, L.A., Miller, B.R., Jordan, E.K., Bulte, J.W. & Frank, J.A. (2003).

Barber, P.A., Zhang, J., Demchuk, A.M., Hill, M.D. & Buchan, A.M. (2001). Why are stroke

Blinzler, C., Breuer, L., Huttner, H.B., Schellinger, P.D., Schwab, S. & Köhrmann, M. (2011).

after thrombolysis for acute ischemic stroke. *Cerebrovasc Dis.*, 31, 2, 185-190 Boltze, J., Förschler, A., Nitzsche, B., Waldmin, D., Hoffmann, A., Boltze, C.M., Dreyer, A.Y.,

Bowen, C.V., Zhang, X., Saab, G., Gareau, P.J. & Rutt, B.K. (2002). Application of the static

brainstem stroke in rabbits via endovascular occlusion of the basilar artery. *J Stroke* 

Intracytoplasmic tagging of cells with ferumoxides and transfection agent for cellular magnetic resonance imaging after cell transplantation: methods and

patients excluded from TPA therapy? An analysis of patient eligibility. *Neurology*,

Characteristics and outcome of patients with early complete neurological recovery

Goldammer, A., Reischauer, A., Härtig, W., Geiger, K.D., Barthel, H., Emmrich, F. & Gille, U. (2008). Permanent middle cerebral artery occlusion in sheep: a novel large animal model of focal cerebral ischemia. *J Cereb Blood Flow Metab.*, 28, 12,

dephasing regime theory to superparamagnetic iron-oxide loaded cells. *Magn Reson* 

towards the chronic phase of a hemorrhage. Also, a blooming effect can be observed by the high iron oxide content of hemosiderin (4.1.2).

Fig. 14 gives a direct comparison between a stereotactically injected blood depot in the sheep model and a hyperacute bleeding in a human patient (with symptom onset of approx. 2 h before MRI) in T2\* MRI. A hyperintense signal is clearly observable in the center of the injected blood in the sheep brain (b) and the hyperacute intracerebral hemorrhage in the human patient (c), whereas a clear hypointense rim can be detected in the outer areas of the hemorrhage in both subjects. This difference in the signal intensity is caused by oxygenated hemoglobin in the central areas and deoxygenated hemoglobin in the peripheral areas of the hemorrhage. This signal behavior is typical for a hyperacute hemorrhage within a timeframe of approximately 3 to 9 h after symptom onset in human beings (Howells et al., 2010). In summary, the blood-induced MR signal 3 hours after autologous blood injection in the sheep model shows the same characteristics to the situation found in the human patient.

Fig. 15. MRI of induced, acute hemorrhage (1.5 mL) in the ovine brain a) T2-weighted TSE sequence with a clearly observable, hyperintensive area (blood clot), b) corresponding macroscopic view of brain slice

Tissue preparation for gross pathology after hemorrhage modeling can be performed according to 4.1.3. The clearly observable cavity in Fig. 15b indicates the injection site. The tissue around the injection site was compressed during blood injection. Similar findings are reported from autopsies of human patients who died from massive intracerebral hemorrhage in early stages.

### **5. Summary**

Stereotactic neurosurgery is a routine technique in human medicine. Stereotaxy-based local treatments of stroke (and other diseases) were effectively tested in rodents using strain- and weight-adapted coordinates and approaches. Hence, it is expected that intracerebral administration paradigms will be evaluated in upcoming clinical trials. However, a proof of concept of such treatment paradigms may be demanded in translational research, preferentially using large animal models. However, similar techniques were so far only available for primate models which are restricted to highly specialized centers.

The described, frameless stereotaxy in sheep represents a novel and translational approach for neurosurgical applications in a widely available species. The used neuronavigation system BrainSightTM was successfully adapted to the ovine skull anatomy. It allows an individual and accurate planning as well as precise execution of stereotactic interventions. Next to a detailed description of the technique itself, two relevant applications of the experimental techniques are reported in this chapter. First, the magnetic labeling and stereotactic transplantation of a stem cell population into the ovine brain is described. The methodology allows for precise injection and tracking of autologous stem cell populations using a widely available 3T MRI scanner. Relatively simple but reliable pathohistological techniques for post-mortem brain assessment and detection of transplanted cells are also given. Thus, the reported methodology may be used for evaluation of cell-based treatment strategies of central nervous system disorders in the gyrencephalic brain. Second, the stereotactic technique can be used for precise modeling of intracerebral hemorrhages by autologous blood injection in sheep. The observed results are in good correlation to those seen in human patients, especially regarding relevant diagnostic findings in MRI. Summarizing, stereotactic interventions in sheep represent a well applicable and reliable approach for translational research with a wide spectrum of possible applications.

### **6. Acknowledgment**

The authors thank Dr. Stephen Frey from Rogue Research Inc. for technical support and the staff of the Institute of Anatomy, Histology and Embrology, Faculty of Veterinary Medicine, University of Leipzig.

### **7. References**

42 Advances in the Preclinical Study of Ischemic Stroke

towards the chronic phase of a hemorrhage. Also, a blooming effect can be observed by the

Fig. 14 gives a direct comparison between a stereotactically injected blood depot in the sheep model and a hyperacute bleeding in a human patient (with symptom onset of approx. 2 h before MRI) in T2\* MRI. A hyperintense signal is clearly observable in the center of the injected blood in the sheep brain (b) and the hyperacute intracerebral hemorrhage in the human patient (c), whereas a clear hypointense rim can be detected in the outer areas of the hemorrhage in both subjects. This difference in the signal intensity is caused by oxygenated hemoglobin in the central areas and deoxygenated hemoglobin in the peripheral areas of the hemorrhage. This signal behavior is typical for a hyperacute hemorrhage within a timeframe of approximately 3 to 9 h after symptom onset in human beings (Howells et al., 2010). In summary, the blood-induced MR signal 3 hours after autologous blood injection in the sheep model shows the same characteristics to the situation found in the human patient.

Fig. 15. MRI of induced, acute hemorrhage (1.5 mL) in the ovine brain

b) corresponding macroscopic view of brain slice

hemorrhage in early stages.

**5. Summary** 

a) T2-weighted TSE sequence with a clearly observable, hyperintensive area (blood clot),

Tissue preparation for gross pathology after hemorrhage modeling can be performed according to 4.1.3. The clearly observable cavity in Fig. 15b indicates the injection site. The tissue around the injection site was compressed during blood injection. Similar findings are reported from autopsies of human patients who died from massive intracerebral

Stereotactic neurosurgery is a routine technique in human medicine. Stereotaxy-based local treatments of stroke (and other diseases) were effectively tested in rodents using strain- and weight-adapted coordinates and approaches. Hence, it is expected that intracerebral administration paradigms will be evaluated in upcoming clinical trials. However, a proof of concept of such treatment paradigms may be demanded in translational research, preferentially using large animal models. However, similar techniques were so far only

The described, frameless stereotaxy in sheep represents a novel and translational approach for neurosurgical applications in a widely available species. The used neuronavigation

available for primate models which are restricted to highly specialized centers.

high iron oxide content of hemosiderin (4.1.2).


Frameless Stereotaxy in Sheep –

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Neurosurgical and Imaging Techniques for Translational Stroke Research 45

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**3** 

*France* 

**A Master Key to Assess Stroke Consequences** 

Long-term consequences of stroke are often dramatically disabling for daily living but also costly for public health. Stroke research is therefore a priority for most industrialized countries. Therefore and paradoxically, the current situation is disappointing for the patients but stimulating for the researchers who have to figure out new therapies and assess their efficiency. The major initial problem to solve for clinicians when a stroke occurs is to rapidly distinguish between ischemia and hemorrhage to decide for the reperfusion of the brain or for the bleeding stop. Therefore, the first step of stroke treatment takes place within a very short-term and tiny time window (even if it was recently extended to 4 hours for thrombolysis). Next steps of the treatments are directed to avoid another stroke and to promote rehabilitation. For this latter, unfortunately, the treatments are scarce and although many different options have been tested (kinesiotherapy, speech therapy etc.), the situation is still unsatisfactory. News fields of research tried to find out new therapies aimed at improving neurogenesis, implanting stem cells or grafts, or stimulating trophic and growth factors, but in vain, transfer from animal to human is still disappointing. Another way to deal with treatment of stroke is to assess what happens in those patients who recover spontaneously. Indeed, around 33% of stroke patients spontaneously recover after stroke but the mechanisms involved in the recovery, not elucidated till now, could be interesting to promote. To assess these mechanisms, it is of a major importance to be able to measure longterm functional deficits in animal models of stroke, which is by the way a strong

recommendation from the STAIR Roundtables (STAIR, 1999; STAIRIII, 2001).

According to the brain structures infracted, stroke induces many different symptoms affecting physiological, sensori-motor, and/or cognitive functions. In experimental studies, it is possible to induce different models of stroke and to assess with many different ways their consequences. However, given the extraordinary spontaneous recovery displayed by animals, especially rodents, it is not always possible to highlight long-term deficits. Indeed, small brain lesions induce deficits that either cannot be detected by global behavioral tests or only for a short term period after stroke (limb placing test, neurological score). That is the reason why news tests have been designed to assess functional recovery in animals. Among the various behavioral tests developed so far, the adhesive removal test and all its variations (sticky-tape test, bilateral asymmetry test) have been proved to be one of the most efficient to highlight tiny long term sensori-motor deficits. Originally brought back from bedside to

**1. Introduction** 

**1.1 History of the adhesive removal test** 

**Across Species: The Adhesive Removal Test** 

Valentine Bouet and Thomas Freret *Université de Caen Basse-Normandie* 


### **A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test**

Valentine Bouet and Thomas Freret *Université de Caen Basse-Normandie France* 

### **1. Introduction**

46 Advances in the Preclinical Study of Ischemic Stroke

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Willems, P.W., van der Sprenkel, J.W., Tulleken, C.A., Viergever, M.A. & Taphoorn, M.J.

Zhu, H., Li, Q., Feng, M., Chen, Y.X., Li, H., Sun, J.J., Zhao, C.H., Wang, R.Z., Bezard, E. &

(CASES) (2011). Thrombolysis at 3-4.5 hours after acute ischemic stroke onset- evidence from the Canadian Alteplase for Stroke Effectiveness Study (CASES)

Grune, T. (2004). Iron oxide particles for molecular magnetic resonance imaging cause transient oxidative stress in rat macrophages. *Free Radic Biol Med*., 36, 8, 976-

H., Schober, R., Pohl, E.E. & Zimmer, C. (2005). In vivo detection limits of magnetically labeled embryonic stem cells in the rat brain using high-field (17.6 T)

Grune. T. & Zimmer, C. (2009). Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of

(STEPS): bridging basic and clinical science for cellular and neurogenic factor

(2006). Neuronavigation and surgery of intracerebral tumours. J Neurol., 253, 9,

Qin, C. (2011). A new cerebral hemorrhage model in cynomolgus macaques created by injection of autologous anticoagulated blood into the brain. *J Clin Neurosci.*, 18, 7,

and neurorecovery. *Neurotherapeutics*, 8, 3, 434-451

sized iron oxide particles. *Magn Reson Med.*, 53, 2, 329-338

hemorrhage. *Neurosurg Rev.*, 32, 1, 15-22

registry. *Cerebrovasc Dis.*, 31, 3, 223-228

984

1123-1136

955-960

Parkinson's disease. *Neurosurgery*, 43, 5, 989-1013

magnetic resonance imaging. *Neuroimage*, 24, 3, 635-645

Parkinson disease. *Mol Imaging*, 8, 3, 166-178

therapy in treating stroke. *Stroke,* 40, 2, 510-515

### **1.1 History of the adhesive removal test**

Long-term consequences of stroke are often dramatically disabling for daily living but also costly for public health. Stroke research is therefore a priority for most industrialized countries. Therefore and paradoxically, the current situation is disappointing for the patients but stimulating for the researchers who have to figure out new therapies and assess their efficiency. The major initial problem to solve for clinicians when a stroke occurs is to rapidly distinguish between ischemia and hemorrhage to decide for the reperfusion of the brain or for the bleeding stop. Therefore, the first step of stroke treatment takes place within a very short-term and tiny time window (even if it was recently extended to 4 hours for thrombolysis). Next steps of the treatments are directed to avoid another stroke and to promote rehabilitation. For this latter, unfortunately, the treatments are scarce and although many different options have been tested (kinesiotherapy, speech therapy etc.), the situation is still unsatisfactory. News fields of research tried to find out new therapies aimed at improving neurogenesis, implanting stem cells or grafts, or stimulating trophic and growth factors, but in vain, transfer from animal to human is still disappointing. Another way to deal with treatment of stroke is to assess what happens in those patients who recover spontaneously. Indeed, around 33% of stroke patients spontaneously recover after stroke but the mechanisms involved in the recovery, not elucidated till now, could be interesting to promote. To assess these mechanisms, it is of a major importance to be able to measure longterm functional deficits in animal models of stroke, which is by the way a strong recommendation from the STAIR Roundtables (STAIR, 1999; STAIRIII, 2001).

According to the brain structures infracted, stroke induces many different symptoms affecting physiological, sensori-motor, and/or cognitive functions. In experimental studies, it is possible to induce different models of stroke and to assess with many different ways their consequences. However, given the extraordinary spontaneous recovery displayed by animals, especially rodents, it is not always possible to highlight long-term deficits. Indeed, small brain lesions induce deficits that either cannot be detected by global behavioral tests or only for a short term period after stroke (limb placing test, neurological score). That is the reason why news tests have been designed to assess functional recovery in animals. Among the various behavioral tests developed so far, the adhesive removal test and all its variations (sticky-tape test, bilateral asymmetry test) have been proved to be one of the most efficient to highlight tiny long term sensori-motor deficits. Originally brought back from bedside to

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 49

have to be adjusted, notably the size of adhesives tapes, but also the animal's body part that will be concerned by tactile stimulation depending on whether the species is purely a

Although training sessions are not mandatory, they are highly recommended. Training decreases anxiety related to the test, and therefore decreases the probability that the animal will urinate and defecate during the subsequent test phase, allowing researchers to obtain an optimal level of performance. It is beside of prime importance to maintain consistency in the testing environment because small changes may impact on animals' emotional state and consequently on the functional outcome. Additionally, training allows indentifying any preoperative asymmetries that would induce a bias in the interpretation of the data. Finally, training also decreases inter-individual variability, making performances homogenous. Once the animals display good performances, only deficits due to the insult are compared, without learning effect. Furthermore, it is important that sham-operated and experimental animals are trained to the task, because surgery itself could induce a slight change in performances. Besides, doing so allow to use each animal performance as its own control

Fig. 1. Time to remove (s) an adhesive tape in four non-injured species during training. Data are expressed as mean (±SD). In all species, ANOVA indicates a significant decrease with time (p<0.0001 in NMRI mice, p=0.0005 in marmosets, p=0.0015 in young Wistar rats, p<0.0001 in adult Sprague Dawley rats). In most cases there is a decrease in performances

D1 D2 D3 D4 D5

\*

**Adult SPRAGUE-DAWLEY rat**

**Marmoset**

\*

D1 D2 D3 D4

(training phase performances compared to those obtained after surgery).

D1 D2 D3 D4 D5

**Young WISTAR rat**

PND20 PND21 PND22 PND23 PND24

between the first and the second day of training.

*P=0.08*

**SWISS mice**

quadruped or not (see below).

**2.2 Training and age** 

\*

\*

bench by Schallert *et al.* (Schallert et al., 1982), this test is derived from a neurologic exam routinely used by clinicians, namely the Double Simultaneous Stimulation (DSS). In fact, the DSS has been initially developed by clinicians to highlight the contralateral neglect syndrome which is observed in patients after damage to the parietal cortex (Heilman et al., 2000). During the DSS examination, the patient has to attend to and identify paired of sensory stimuli that are applied to both sides of the body at the same time. Stimuli can be visual, auditive or tactile. Extinction to double simultaneous tactile stimulation (tactile DSS) was first reported at the beginning of the 80's (Schallert et al., 1982). Tactile extinction is the failure to perceive and/or report tactile stimulation on the body side contralateral to a brain lesion when the homologous, ipsilesioned region is stimulated simultaneously; whereas no defect in tactile perception occurs with unilateral stimulation. Interestingly, tactile extinction showed to be promising as a predictor of post-stroke functional outcome (Rose et al., 1994). In the way of improving the functional assessment in experimental stroke models, adhesive removal test has been first adapted to rats about a quarter of century ago (Schallert et al., 1982). The stimulus employed was then tactile, consisting of adhesive tapes applied to different parts of the animal body (forelimbs, hindlimbs and snout). It measures sensory functions, sensory neglect and motor functions independently for the left and the right side. Initially designed to highlight deficit induced by unilateral nigrostriatal damage, its range of application has been extended to several brain disorders (Parkinson disease, brain trauma, spinal cord lesion), including stroke. Giving advantage to assess sensory and motor deficits, free from postural bias and circling behaviors, the adhesive removal test has been highly used in rats to a large extent. Interestingly, long-term after stroke, while many tests are not sensitive enough to measure any deficit because of an apparent full recovery, the adhesive removal test is one of the rare tests powerful enough to give an objective score of the functional deficit. Besides, the deficits observed after stroke very well mimics the extinction to tactile double simultaneous stimuli reported in stroke patients (Schallert et al., 1982). Owing to its success in rats, the adhesive removal test has been afterward developed in other animal species. Thus, in the beginning of the 90's, the test was adapted to a nonhuman primate species, the marmoset (*Callithrix jacchus*) (Annett et al., 1992; Marshall &Ridley, 1996). Used then in gerbils (Ishibashi et al., 2003) and even dogs (Quaranta et al., 2004), it has been lastly developed in mice (Bouet et al., 2009; Bouet et al., 2007; Starkey et al., 2005). Thus, beyond its effectiveness to highlight long term, and consequently tiny, sensory and motor deficit, the adhesive removal test is adaptable to several animal models, which is a fundamental advantage to translate animal research to clinical application.

Convinced that this test could offer even more than it has already did, we will along this chapter give an overlook of the way it has to be performed and the results you may obtained in several animal models of stroke: mice, rats, and marmosets (Bouet et al., 2009; Bouet et al., 2010; Bouet et al., 2007; Freret et al., 2009; Freret et al., 2008; Freret et al., 2006; Freret et al., 2006), *i.e.* the most used species in neuroscience research.

### **2. Achievement of adhesive removal test across laboratory species**

#### **2.1 Species**

As stated before, the adhesive removal test has been already developed in a wide range of animal species, from nonhuman primate to rodents (rats, mice, gerbils). This test can theoretically be performed in any species that is anatomically able to remove a piece of adhesive pasted on its body. However, according to the species concerned, technical details have to be adjusted, notably the size of adhesives tapes, but also the animal's body part that will be concerned by tactile stimulation depending on whether the species is purely a quadruped or not (see below).

#### **2.2 Training and age**

48 Advances in the Preclinical Study of Ischemic Stroke

bench by Schallert *et al.* (Schallert et al., 1982), this test is derived from a neurologic exam routinely used by clinicians, namely the Double Simultaneous Stimulation (DSS). In fact, the DSS has been initially developed by clinicians to highlight the contralateral neglect syndrome which is observed in patients after damage to the parietal cortex (Heilman et al., 2000). During the DSS examination, the patient has to attend to and identify paired of sensory stimuli that are applied to both sides of the body at the same time. Stimuli can be visual, auditive or tactile. Extinction to double simultaneous tactile stimulation (tactile DSS) was first reported at the beginning of the 80's (Schallert et al., 1982). Tactile extinction is the failure to perceive and/or report tactile stimulation on the body side contralateral to a brain lesion when the homologous, ipsilesioned region is stimulated simultaneously; whereas no defect in tactile perception occurs with unilateral stimulation. Interestingly, tactile extinction showed to be promising as a predictor of post-stroke functional outcome (Rose et al., 1994). In the way of improving the functional assessment in experimental stroke models, adhesive removal test has been first adapted to rats about a quarter of century ago (Schallert et al., 1982). The stimulus employed was then tactile, consisting of adhesive tapes applied to different parts of the animal body (forelimbs, hindlimbs and snout). It measures sensory functions, sensory neglect and motor functions independently for the left and the right side. Initially designed to highlight deficit induced by unilateral nigrostriatal damage, its range of application has been extended to several brain disorders (Parkinson disease, brain trauma, spinal cord lesion), including stroke. Giving advantage to assess sensory and motor deficits, free from postural bias and circling behaviors, the adhesive removal test has been highly used in rats to a large extent. Interestingly, long-term after stroke, while many tests are not sensitive enough to measure any deficit because of an apparent full recovery, the adhesive removal test is one of the rare tests powerful enough to give an objective score of the functional deficit. Besides, the deficits observed after stroke very well mimics the extinction to tactile double simultaneous stimuli reported in stroke patients (Schallert et al., 1982). Owing to its success in rats, the adhesive removal test has been afterward developed in other animal species. Thus, in the beginning of the 90's, the test was adapted to a nonhuman primate species, the marmoset (*Callithrix jacchus*) (Annett et al., 1992; Marshall &Ridley, 1996). Used then in gerbils (Ishibashi et al., 2003) and even dogs (Quaranta et al., 2004), it has been lastly developed in mice (Bouet et al., 2009; Bouet et al., 2007; Starkey et al., 2005). Thus, beyond its effectiveness to highlight long term, and consequently tiny, sensory and motor deficit, the adhesive removal test is adaptable to several animal models, which is a

fundamental advantage to translate animal research to clinical application.

**2. Achievement of adhesive removal test across laboratory species** 

2006), *i.e.* the most used species in neuroscience research.

**2.1 Species** 

Convinced that this test could offer even more than it has already did, we will along this chapter give an overlook of the way it has to be performed and the results you may obtained in several animal models of stroke: mice, rats, and marmosets (Bouet et al., 2009; Bouet et al., 2010; Bouet et al., 2007; Freret et al., 2009; Freret et al., 2008; Freret et al., 2006; Freret et al.,

As stated before, the adhesive removal test has been already developed in a wide range of animal species, from nonhuman primate to rodents (rats, mice, gerbils). This test can theoretically be performed in any species that is anatomically able to remove a piece of adhesive pasted on its body. However, according to the species concerned, technical details Although training sessions are not mandatory, they are highly recommended. Training decreases anxiety related to the test, and therefore decreases the probability that the animal will urinate and defecate during the subsequent test phase, allowing researchers to obtain an optimal level of performance. It is beside of prime importance to maintain consistency in the testing environment because small changes may impact on animals' emotional state and consequently on the functional outcome. Additionally, training allows indentifying any preoperative asymmetries that would induce a bias in the interpretation of the data. Finally, training also decreases inter-individual variability, making performances homogenous. Once the animals display good performances, only deficits due to the insult are compared, without learning effect. Furthermore, it is important that sham-operated and experimental animals are trained to the task, because surgery itself could induce a slight change in performances. Besides, doing so allow to use each animal performance as its own control (training phase performances compared to those obtained after surgery).

Fig. 1. Time to remove (s) an adhesive tape in four non-injured species during training. Data are expressed as mean (±SD). In all species, ANOVA indicates a significant decrease with time (p<0.0001 in NMRI mice, p=0.0005 in marmosets, p=0.0015 in young Wistar rats, p<0.0001 in adult Sprague Dawley rats). In most cases there is a decrease in performances between the first and the second day of training.

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 51

Fig. 2. Pictures depicting a young rat, an adult rat, a mouse and a marmoset being removing the adhesive tapes. All three species use the mouth to remove the adhesive. When very well trained, rats sometimes keep a three-feet posture to remove the tape. The training is very useful to render the animals accustomed to the procedure, and therefore to obtain homogenous data. When the training is not possible (for instance, if a brain injury is performed in young animals), the test is still possible, but the authors have to discuss the

According to the species, the size of the tape has to be adapted and very tightly controlled. Indeed, Schallert and Whishaw (Schallert &Whishaw, 1984) showed that the size of the adhesive could strongly influence the performances. In rats, Komotar *et al.* (Komotar et al., 2007) advised to use very large adhesive tapes and pasted them around the wrist, in order to make a kind of sleeve that cannot be removed for the duration of 30 s (the main parameter is the time the animals attempts to remove the adhesive). Most of the times, in rats and mice adhesive tape is small and pasted on the paw with the aim to cover the three pads, thenar and hypothenar (size: 1x1cm in rats (Freret et al., 2006); 0.3x0.4cm in mice (Bouet et al., 2007)). In primates, the tape has to be rectangular and should roll up the foot (2x4.5 cm in marmosets, (Freret et al., 2008)). Unavoidably, there are many differences between labs in the achievement of this test, and therefore the most important things to keep in mind is to

fact that the recovery and the learning of the task are mandatory overlapping.

**2.4 Adhesive tape size** 

Even though adult animals are used in most studies, we recently showed that it is possible to perform the adhesive removal test in young rats (from post-natal day 20) (Bouet et al., 2010) – enlarging thus the range of application of the test to perinatal ischemic stroke. In this case, pups were submitted to a brain injury at the age of 7-days old. Thus, training to the task before injury was not feasible making confounding training and deficits related to the injury. We observed the first day that young rats (20 days old) were unable to perform the task within the given time. Afterwards, sham animals showed better and better performances with time while lesioned animals stayed on the plateau level. This indicates that in particular cases, it is possible to obtain satisfactory data even without training. Our data showed that one week of training (1 trial per day) is sufficient for adult rats, mice and marmosets to reach a plateau level (Figure 1), whereas it is not the case for rat pups (PND20). This increased duration of training with young animals is partly due to the fact that rearing is required to remove adhesive and at this age, hindlegs weight bearing is still unstable. Of note, in marmosets, the stress of the procedure induces high values in the time to remove the adhesive the first days that is the reason why the maximal time given to the animal to perform the task is of 10 min (2 min for rodents). However, the plateau is reached by the 3rd day of training.

#### **2.3 Body parts**

According to the species, adhesive removal is performed on different ways, related to the body parts on which adhesive tapes are placed, the parameters collected the place in which the test is performed… Concerning the body parts concerned, they often vary according to the location of the brain lesion and because of the differential sensitivity of different body parts of the species considered. For instance in rats, placing the adhesive on forepaws is the most efficient; indeed they do not really take care of adhesive placed on their back paws, probably because of weight support that induces a large stimulation of the plantar surface and strongly diminishes the discrimination. Additionally, sticking the adhesive tape on the forepaws of the animals will drive it to naturally remove the adhesive for its grooming. The same holds true for mice, in which adhesive test has been first described with positioning on the snout (Fleming et al., 2004), and then on forepaws (Bouet et al., 2009; Bouet et al., 2010; Bouet et al., 2007; Freret et al., 2009; Freret et al., 2006; Freret et al., 2006; Starkey et al., 2005), alike rats. In gerbils, and also in most of the studies performed in rats, the adhesive is placed on the wrist (Ishibashi et al., 2003). In primates, because of the role of hands in objects manipulation, adhesives are preferably placed around the feet (Annett et al., 1992; Freret et al., 2008; Marshall &Ridley, 1996).

Consequently, while removing an adhesive pasted on the forepaws seems quite easy to do for rodents after training, it seems to be more tricky for marmosets. Figure 2 shows that while rodent stand on their hindpaws to remove the adhesive with the mouth, the marmoset has to stand on his bottom and raise the leg to bring the foot to the mouth. Such postures are rather close to the grooming postures for rodents, but they are more scarse in marmosets. This is one of the reasons that explain the increase in time to remove the adhesive for marmosets compared to rodents. The other reason is due to the fact that for marmosets the adhesive tapes enroll all the foot, making hard, therefore time-consuming to unroll. Such a long adhesive tape unrolling the limb of the animal can also be done in rodents, making it even more difficult to remove (Komotar et al., 2007).

Fig. 2. Pictures depicting a young rat, an adult rat, a mouse and a marmoset being removing the adhesive tapes. All three species use the mouth to remove the adhesive. When very well trained, rats sometimes keep a three-feet posture to remove the tape. The training is very useful to render the animals accustomed to the procedure, and therefore to obtain homogenous data. When the training is not possible (for instance, if a brain injury is performed in young animals), the test is still possible, but the authors have to discuss the fact that the recovery and the learning of the task are mandatory overlapping.

### **2.4 Adhesive tape size**

50 Advances in the Preclinical Study of Ischemic Stroke

Even though adult animals are used in most studies, we recently showed that it is possible to perform the adhesive removal test in young rats (from post-natal day 20) (Bouet et al., 2010) – enlarging thus the range of application of the test to perinatal ischemic stroke. In this case, pups were submitted to a brain injury at the age of 7-days old. Thus, training to the task before injury was not feasible making confounding training and deficits related to the injury. We observed the first day that young rats (20 days old) were unable to perform the task within the given time. Afterwards, sham animals showed better and better performances with time while lesioned animals stayed on the plateau level. This indicates that in particular cases, it is possible to obtain satisfactory data even without training. Our data showed that one week of training (1 trial per day) is sufficient for adult rats, mice and marmosets to reach a plateau level (Figure 1), whereas it is not the case for rat pups (PND20). This increased duration of training with young animals is partly due to the fact that rearing is required to remove adhesive and at this age, hindlegs weight bearing is still unstable. Of note, in marmosets, the stress of the procedure induces high values in the time to remove the adhesive the first days that is the reason why the maximal time given to the animal to perform the task is of 10 min (2 min for rodents). However, the plateau is reached

According to the species, adhesive removal is performed on different ways, related to the body parts on which adhesive tapes are placed, the parameters collected the place in which the test is performed… Concerning the body parts concerned, they often vary according to the location of the brain lesion and because of the differential sensitivity of different body parts of the species considered. For instance in rats, placing the adhesive on forepaws is the most efficient; indeed they do not really take care of adhesive placed on their back paws, probably because of weight support that induces a large stimulation of the plantar surface and strongly diminishes the discrimination. Additionally, sticking the adhesive tape on the forepaws of the animals will drive it to naturally remove the adhesive for its grooming. The same holds true for mice, in which adhesive test has been first described with positioning on the snout (Fleming et al., 2004), and then on forepaws (Bouet et al., 2009; Bouet et al., 2010; Bouet et al., 2007; Freret et al., 2009; Freret et al., 2006; Freret et al., 2006; Starkey et al., 2005), alike rats. In gerbils, and also in most of the studies performed in rats, the adhesive is placed on the wrist (Ishibashi et al., 2003). In primates, because of the role of hands in objects manipulation, adhesives are preferably placed around the feet (Annett et al., 1992; Freret et

Consequently, while removing an adhesive pasted on the forepaws seems quite easy to do for rodents after training, it seems to be more tricky for marmosets. Figure 2 shows that while rodent stand on their hindpaws to remove the adhesive with the mouth, the marmoset has to stand on his bottom and raise the leg to bring the foot to the mouth. Such postures are rather close to the grooming postures for rodents, but they are more scarse in marmosets. This is one of the reasons that explain the increase in time to remove the adhesive for marmosets compared to rodents. The other reason is due to the fact that for marmosets the adhesive tapes enroll all the foot, making hard, therefore time-consuming to unroll. Such a long adhesive tape unrolling the limb of the animal can also be done in

rodents, making it even more difficult to remove (Komotar et al., 2007).

by the 3rd day of training.

al., 2008; Marshall &Ridley, 1996).

**2.3 Body parts** 

According to the species, the size of the tape has to be adapted and very tightly controlled. Indeed, Schallert and Whishaw (Schallert &Whishaw, 1984) showed that the size of the adhesive could strongly influence the performances. In rats, Komotar *et al.* (Komotar et al., 2007) advised to use very large adhesive tapes and pasted them around the wrist, in order to make a kind of sleeve that cannot be removed for the duration of 30 s (the main parameter is the time the animals attempts to remove the adhesive). Most of the times, in rats and mice adhesive tape is small and pasted on the paw with the aim to cover the three pads, thenar and hypothenar (size: 1x1cm in rats (Freret et al., 2006); 0.3x0.4cm in mice (Bouet et al., 2007)). In primates, the tape has to be rectangular and should roll up the foot (2x4.5 cm in marmosets, (Freret et al., 2008)). Unavoidably, there are many differences between labs in the achievement of this test, and therefore the most important things to keep in mind is to

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 53

Fig. 3. Positioning of the adhesive tapes in mice, rats and marmosets. The piece of adhesive has to be positioned on fore paws for mice and rats, and on hind paws in marmosets (alternation between left and right for the first positioning and between trials should be

Overall, time to contact and time to remove crudely separate out sensory *versus* motor deficits (Schaar et al., 2010). On a side point, it should be noted that the adhesive removal test can also be used to measure animals sensory asymmetries. The magnitude of sensory

**Rats**

**Marmosets**

achieved).

**Mice**

usually take a small piece between teeth and remove it in one movement (Figure 2), while marmosets often try several times to remove it by biting. The time to remove reflects as well as sensory and motor abilities, since it requires a correct dexterity.

always have a control group, always apply the adhesive with the same pressure on the right and left hand (an experimenter blind to the treatment is an obvious necessity), and always buy the adhesive tape from the same supplier (same brand). Our experience is that the sewed-adhesives used for bandage are the best (from Sogiphar, Urgo or BSN Medicals in France for example).

### **2.5 Positioning of the adhesive**

Adhesive positioning requires animal contention to ensure a very good precision (Figure 3). Contention has to be performed as gently as possible, because any increase in stress can totally biases the results by increasing the time to perform the task, and this is true whatever the species. For the rat and the marmoset, the animal can be held by the torso by an experimenter, while the other experimenter places the tapes (Bouet et al., 2010; Freret et al., 2008; Freret et al., 2006). For the mouse and the gerbil, the animal can be held by the back skin in order to let the forepaw free (about the same way used to make intraperitoneal injection) (Bouet et al., 2009; Ishibashi et al., 2003). The experimenters have to get used with the contention before starting the experiments, by a previous training if necessary. The contention has to be gentle but firm. Rodents have to be held by the back skin as close to the neck as possible to maintain the head and thus prevent any biting. Once the animal is quietly held, the experimenter has to place an adhesive on each paw (alternating right and left between days and between animals), in a way that the pressure is identical on both paws and that the same skin portion is covered on both paws. To avoid the experimenter who is in charge to place the adhesive to be bite, it is important to maintain the foreleg as extended as possible by pulling the fingers before placing the adhesive tape. For rodents, the adhesive is rectangular or squared, and for marmosets it is rectangular and has to be placed around the foot in order to make a small overlap of the tissue.

The animal is then placed back in his own cage (without congeners) or in an experimental box (if this last is used, a habituation period has to be performed before positioning the adhesive by giving to the animal 1 or 2 minute free exploration of the box). Indeed, the test can be either performed in the home cage or in a testing box, depending on the animals housing conditions. If animals are single-housed, the first solution should be preferred, since it avoids any supplementary stress for animal. As a contrary, carrying out the adhesive removal in a testing box rather than in the home cage, should be preferred when animal are group-housed to avoid interaction between individuals that can alter performances. Nevertheless, in the former case, a habituation period to the testing box should be respected.

### **2.6 Collecting the data**

Once the animal is placed back in the cage with the two adhesive pasted on the paws, four different values have to be collected: time to contact and time to remove the adhesive for each paw.


usually take a small piece between teeth and remove it in one movement (Figure 2), while marmosets often try several times to remove it by biting. The time to remove reflects as well as sensory and motor abilities, since it requires a correct dexterity.

**Mice**

52 Advances in the Preclinical Study of Ischemic Stroke

always have a control group, always apply the adhesive with the same pressure on the right and left hand (an experimenter blind to the treatment is an obvious necessity), and always buy the adhesive tape from the same supplier (same brand). Our experience is that the sewed-adhesives used for bandage are the best (from Sogiphar, Urgo or BSN Medicals in

Adhesive positioning requires animal contention to ensure a very good precision (Figure 3). Contention has to be performed as gently as possible, because any increase in stress can totally biases the results by increasing the time to perform the task, and this is true whatever the species. For the rat and the marmoset, the animal can be held by the torso by an experimenter, while the other experimenter places the tapes (Bouet et al., 2010; Freret et al., 2008; Freret et al., 2006). For the mouse and the gerbil, the animal can be held by the back skin in order to let the forepaw free (about the same way used to make intraperitoneal injection) (Bouet et al., 2009; Ishibashi et al., 2003). The experimenters have to get used with the contention before starting the experiments, by a previous training if necessary. The contention has to be gentle but firm. Rodents have to be held by the back skin as close to the neck as possible to maintain the head and thus prevent any biting. Once the animal is quietly held, the experimenter has to place an adhesive on each paw (alternating right and left between days and between animals), in a way that the pressure is identical on both paws and that the same skin portion is covered on both paws. To avoid the experimenter who is in charge to place the adhesive to be bite, it is important to maintain the foreleg as extended as possible by pulling the fingers before placing the adhesive tape. For rodents, the adhesive is rectangular or squared, and for marmosets it is rectangular and has to be placed around

The animal is then placed back in his own cage (without congeners) or in an experimental box (if this last is used, a habituation period has to be performed before positioning the adhesive by giving to the animal 1 or 2 minute free exploration of the box). Indeed, the test can be either performed in the home cage or in a testing box, depending on the animals housing conditions. If animals are single-housed, the first solution should be preferred, since it avoids any supplementary stress for animal. As a contrary, carrying out the adhesive removal in a testing box rather than in the home cage, should be preferred when animal are group-housed to avoid interaction between individuals that can alter performances. Nevertheless, in the former case, a habituation period to the testing box should be respected.

Once the animal is placed back in the cage with the two adhesive pasted on the paws, four different values have to be collected: time to contact and time to remove the adhesive for



France for example).

**2.6 Collecting the data** 

each paw.

**2.5 Positioning of the adhesive** 

the foot in order to make a small overlap of the tissue.

scratching of the leg on the side of the adhesive.

Fig. 3. Positioning of the adhesive tapes in mice, rats and marmosets. The piece of adhesive has to be positioned on fore paws for mice and rats, and on hind paws in marmosets (alternation between left and right for the first positioning and between trials should be achieved).

Overall, time to contact and time to remove crudely separate out sensory *versus* motor deficits (Schaar et al., 2010). On a side point, it should be noted that the adhesive removal test can also be used to measure animals sensory asymmetries. The magnitude of sensory

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 55

al., 2006; van Lookeren Campagne et al., 1999). The reason for this lack in literature is that long-term behavioral deficits (*i.e.*, several weeks after surgery) are difficult to detect in mice. Iadecola and colleagues even explained that they had to proximally occlude the artery ''because dMCA occlusion produced no neurological deficits'' in the mouse (Iadecola et al., 1997). This is presumably because of the low sensitivity of the behavioral testing available in the literature. Nevertheless, mice are of real interest in experimental studies because of their low cost and possible transgenic alterations. To further argue for the usefulness of the adhesive removal test, some data illustrating deficits observed in mice after d-MCAo are presented below (see figure 4). Surgical procedures used to induce stroke are described in

As a rule, a highly significant impairment on the contralateral side is commonly observed, whereas the deficit is usually more or less important on the ipsilateral side. Besides, the contralateral deficit is often long-lasting with a delay that will vary according to the species considered and the duration of the occlusion of the middle cerebral artery, *i.e.* the severity of the injury (Figure 4). As regards to spontaneous recovery phenomenon, the ipsilateral deficit often disappears during the first stage after the insult. Quite a contrary, improvement of performances on the contralateral side is not always observed, once again according to the design of the study. This discrepancy of rate of recovery between ipsi- and controlateral side makes the adhesive removal test an efficient tool for assessing the kinetic of functional outcome after cerebral ischemia (acute and long-term phase). Thus, this task may be suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote regeneration. In rats, while no somatosensory impairment is observed after a 30-min duration of occlusion of the middle cerebral artery (MCAo), 60-min of occlusion induces a bilateral and long lasting deficit, as reflected by an increased time to contact (Figure 4; (Freret et al., 2006)). This deficit tends however to partially recover over time, as attested by the decreasing slope of the time to contact curves. To ensure any potent ischemia-induced somatosensory asymmetry, an index giving the difference between ipsi- and controlateral performances can be calculated (see Freret et al., 2006; Bouet et al., 2007). Thus, comparing contro- and ipsilateral side, it should be note that the rats display a preference towards the ipsilateral,

As regards to motor abilities estimated by the time to remove the adhesive, a unilateral and transient deficit can be observed after a 30-min MCAo (up to 1 week after surgery). By contrast, 60-min MCAo will drastically impact on motor functions; a bilateral motor coordination deficit – albeit only transient on the ipsilateral side - can be observed. This long-lasting deficit on the contralateral side reflects a failure to respond to a novel tactile

In mice, considering a 60-min duration of occlusion of the MCA (p-MCAo), a bilateral somatensory deficit can be highlighted – as attested by the increased time to contact the adhesives (Bouet et al., 2009; Bouet et al., 2007). Besides, as regards to the time to remove the adhesives, a bilateral impairment can be observed after ischemia. Our group has observed that this deficit in motor coordination appears to be long lasting, longer time to remove the adhesive on the contralateral side of the lesion, since it is still observable up to 6 weeks after

Bouet et al., 2007, Bouet et al., 2010, Freret et al., 2008, Freret et al., 2009.

**3.1 Expected results after cerebral ischemia across animals' species** 

thus non paretic side.

surgery (unpublished data).

stimulus.

asymmetry is measured by adjusting the ratio of the size of the adhesive tapes on each limb. This test can thus reveal asymmetrical biases in stimulus-directed activity after focal ischemia (Schallert et al., 2000).

### **2.7 Analyzing of the data collected**

Statistical analyses have to be conducted on several parameters to check first some possible discrepancies but also to measure the intensity of the deficits and of the recovery. The first important point is to check, before any brain injury, if there is no initial asymmetry between left and right side. Indeed, it has been showed that for certain sensorimotor tasks, rodents could display an important asymmetry (Bulman-Fleming et al., 1997). The statistical analysis will therefore compare contralateral and ipsilateral removal and contact times. The second point concerns the appraisal of the deficit. To this, two different possibilities exist: data obtained after the injury could be compared to those obtained before the insult (this is also important in sham-operated animals to assess if surgery in itself induces or not any deficit). In this case, it is interesting to express data in percentages compared to pre-surgery. Otherwise, comparisons could also concern data obtained on the controlateral *versus* ipsilateral side. This allows comparing two measurements performed on the same animals within the same session. Moreover, this allows calculating an asymmetry index (contralateral – ipsilateral time).

As regards to number of animals per group, previous studies from the literature have proved that even a small number of animals per group allow to show statistical difference in the adhesive removal test (Freret et al., 2006; Zhang et al., 2011).

### **3. Deficits in adhesive removal test after stroke in rodents and marmosets**

The adhesive-removal test is classically used to detect forepaw somatosensory *– time to contact –* and sensorimotor *– time to remove –* deficits that are not attributable to postural bias (Schallert et al., 1983). Its sensitivity to ischemia-induced deficits has already been reported in the literature (Modo et al., 2000) for every species in which it has been developed, and in most experimental models of cerebral ischemia. For best comparisons between species, most of the results presented here are related to the intra-luminal model of cerebral ischemia. This model has been chosen to illustrate our purpose, because it concerns the middle cerebral artery (MCA) – the most frequently affected artery in stroke patients, because duration of occlusion can be modified, but mostly because a reperfusion event is feasible and thus this closely mimics what happens in patients. In this model, the site of the occlusion of the MCA (MCAo) could either be proximal (close to the origin of the artery, p-MCAo) or distal (after the lenticulostriate branches, d-MCAo). In case of p-MCAo, behavioral deficits in the adhesive removal test are readily observable – even in mice, a species in which subtle behavioral changes are particularly difficult to detect. However, p-MCAo is not representative of all clinical situations because it leads to brain infarctions that are relatively larger than those observed in human stroke. As it induces smaller infarct, d-MCAo is more relevant to those clinical situations. Behavioral alterations after d-MCAo have been largely explored in rats or in marmosets, but, unfortunately, most of the studies using this model in mice considered time points early after surgery and mostly addressed lesion size. Only motor coordination difficulties, attentional deficits and a low increase in eye movement during the dark phase of sleep have been reported in mice (Baumann et al., 2006; Guegan et

asymmetry is measured by adjusting the ratio of the size of the adhesive tapes on each limb. This test can thus reveal asymmetrical biases in stimulus-directed activity after focal

Statistical analyses have to be conducted on several parameters to check first some possible discrepancies but also to measure the intensity of the deficits and of the recovery. The first important point is to check, before any brain injury, if there is no initial asymmetry between left and right side. Indeed, it has been showed that for certain sensorimotor tasks, rodents could display an important asymmetry (Bulman-Fleming et al., 1997). The statistical analysis will therefore compare contralateral and ipsilateral removal and contact times. The second point concerns the appraisal of the deficit. To this, two different possibilities exist: data obtained after the injury could be compared to those obtained before the insult (this is also important in sham-operated animals to assess if surgery in itself induces or not any deficit). In this case, it is interesting to express data in percentages compared to pre-surgery. Otherwise, comparisons could also concern data obtained on the controlateral *versus* ipsilateral side. This allows comparing two measurements performed on the same animals within the same session. Moreover, this allows calculating an asymmetry index

As regards to number of animals per group, previous studies from the literature have proved that even a small number of animals per group allow to show statistical difference in

**3. Deficits in adhesive removal test after stroke in rodents and marmosets** 

The adhesive-removal test is classically used to detect forepaw somatosensory *– time to contact –* and sensorimotor *– time to remove –* deficits that are not attributable to postural bias (Schallert et al., 1983). Its sensitivity to ischemia-induced deficits has already been reported in the literature (Modo et al., 2000) for every species in which it has been developed, and in most experimental models of cerebral ischemia. For best comparisons between species, most of the results presented here are related to the intra-luminal model of cerebral ischemia. This model has been chosen to illustrate our purpose, because it concerns the middle cerebral artery (MCA) – the most frequently affected artery in stroke patients, because duration of occlusion can be modified, but mostly because a reperfusion event is feasible and thus this closely mimics what happens in patients. In this model, the site of the occlusion of the MCA (MCAo) could either be proximal (close to the origin of the artery, p-MCAo) or distal (after the lenticulostriate branches, d-MCAo). In case of p-MCAo, behavioral deficits in the adhesive removal test are readily observable – even in mice, a species in which subtle behavioral changes are particularly difficult to detect. However, p-MCAo is not representative of all clinical situations because it leads to brain infarctions that are relatively larger than those observed in human stroke. As it induces smaller infarct, d-MCAo is more relevant to those clinical situations. Behavioral alterations after d-MCAo have been largely explored in rats or in marmosets, but, unfortunately, most of the studies using this model in mice considered time points early after surgery and mostly addressed lesion size. Only motor coordination difficulties, attentional deficits and a low increase in eye movement during the dark phase of sleep have been reported in mice (Baumann et al., 2006; Guegan et

the adhesive removal test (Freret et al., 2006; Zhang et al., 2011).

ischemia (Schallert et al., 2000).

(contralateral – ipsilateral time).

**2.7 Analyzing of the data collected** 

al., 2006; van Lookeren Campagne et al., 1999). The reason for this lack in literature is that long-term behavioral deficits (*i.e.*, several weeks after surgery) are difficult to detect in mice. Iadecola and colleagues even explained that they had to proximally occlude the artery ''because dMCA occlusion produced no neurological deficits'' in the mouse (Iadecola et al., 1997). This is presumably because of the low sensitivity of the behavioral testing available in the literature. Nevertheless, mice are of real interest in experimental studies because of their low cost and possible transgenic alterations. To further argue for the usefulness of the adhesive removal test, some data illustrating deficits observed in mice after d-MCAo are presented below (see figure 4). Surgical procedures used to induce stroke are described in Bouet et al., 2007, Bouet et al., 2010, Freret et al., 2008, Freret et al., 2009.

#### **3.1 Expected results after cerebral ischemia across animals' species**

As a rule, a highly significant impairment on the contralateral side is commonly observed, whereas the deficit is usually more or less important on the ipsilateral side. Besides, the contralateral deficit is often long-lasting with a delay that will vary according to the species considered and the duration of the occlusion of the middle cerebral artery, *i.e.* the severity of the injury (Figure 4). As regards to spontaneous recovery phenomenon, the ipsilateral deficit often disappears during the first stage after the insult. Quite a contrary, improvement of performances on the contralateral side is not always observed, once again according to the design of the study. This discrepancy of rate of recovery between ipsi- and controlateral side makes the adhesive removal test an efficient tool for assessing the kinetic of functional outcome after cerebral ischemia (acute and long-term phase). Thus, this task may be suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote regeneration.

In rats, while no somatosensory impairment is observed after a 30-min duration of occlusion of the middle cerebral artery (MCAo), 60-min of occlusion induces a bilateral and long lasting deficit, as reflected by an increased time to contact (Figure 4; (Freret et al., 2006)). This deficit tends however to partially recover over time, as attested by the decreasing slope of the time to contact curves. To ensure any potent ischemia-induced somatosensory asymmetry, an index giving the difference between ipsi- and controlateral performances can be calculated (see Freret et al., 2006; Bouet et al., 2007). Thus, comparing contro- and ipsilateral side, it should be note that the rats display a preference towards the ipsilateral, thus non paretic side.

As regards to motor abilities estimated by the time to remove the adhesive, a unilateral and transient deficit can be observed after a 30-min MCAo (up to 1 week after surgery). By contrast, 60-min MCAo will drastically impact on motor functions; a bilateral motor coordination deficit – albeit only transient on the ipsilateral side - can be observed. This long-lasting deficit on the contralateral side reflects a failure to respond to a novel tactile stimulus.

In mice, considering a 60-min duration of occlusion of the MCA (p-MCAo), a bilateral somatensory deficit can be highlighted – as attested by the increased time to contact the adhesives (Bouet et al., 2009; Bouet et al., 2007). Besides, as regards to the time to remove the adhesives, a bilateral impairment can be observed after ischemia. Our group has observed that this deficit in motor coordination appears to be long lasting, longer time to remove the adhesive on the contralateral side of the lesion, since it is still observable up to 6 weeks after surgery (unpublished data).

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 57

on the ipsilateral side of the lesion and might be due, at least partly, to dizziness of the animal due to surgery and/or the anesthesia. With respect to the time to remove the adhesive, a bilateral motor coordination deficit is observed whatever the duration of the occlusion. Of note, it has been demonstrated in this same model of cerebral ischemia a spontaneous functional recovery on the ipsilateral side, while controlateral time to remove

the adhesive remains hardly affected up to 4 weeks after surgery (Bihel et al.).

adhesive removal motor deficits (time to remove) (Freret et al., 2006).

**removal test** 

**3.2 Correlations between brain histological damage and deficit in the adhesive** 

Correlations between the cortical and striatal histological lesions and the ischemia-induced behavioral impairments in the adhesive removal test have been well investigated in the literature, mostly in rodents (Grabowski et al., 1991; Hudzik et al., 2000; Hunter et al., 1998; Rogers et al., 1997; Virley et al., 2000). We and other authors have demonstrated a close correlation between contralateral contact and removal latencies on this task and abnormal changes in the ipsilateral caudate putamen, lower parietal cortex and forelimb cortex following transient MCAo (30; 60 or 90 min; see Figure 5). Each of these regions of interest contributed to functional impairments on this task across an extended time course (up to several months post-ischemia) (Virley et al., 2000). As regards to the final cortical damage, it seems particularly correlated to both transient and long-lasting sensorimotor deficits measured by adhesive removal test. Conversely, the final striatal lesion appears to be consistently related to the

Fig. 5. Schemes showing extent of the lesion after distal and proximal models of Middle Cerebral Artery occlusion (MCAo) in rodents and marmoset. Distal MCAo refers to the occlusion of the artery in its distal portion, *i.e.* after lenticulostriate branches (usually

Time to contact contralateral adhesive after stroke (% pre-surgery)

Time to remove contralateral adhesive after stroke (% pre-surgery)

Fig. 4. Adhesive removal performances in rat, mouse and marmoset after stroke (% pre-surgical values). Mean percentage of time to contact the adhesive tapes (upper graphs), positioned on the controlateral side (left) and on the ipsilateral side (right). Time to remove the adhesive tapes (lower graphs), positioned on the controlateral side (left) and on the ipsilateral side (right). All stroke models presented concern the occlusion the middle cerebral artery (MCAo). For rats, stroke was induced by the intraluminal occlusion of the MCA (30 or 60 min). For mice, MCAo was occluded either by distal permanent electrocoagulation or by intraluminal occlusion (60 min - proximal). In marmosets, MCA was occluded by intraluminal approach either permanently of transiently (3 h). Each point represents the mean of three trials performed on three consecutive days.

Take a look now at the impairments associated to relatively small brain lesion (d-MCAo), adhesive removal test is very useful to detect functional contralateral deficits, even 3 weeks after surgery (Freret et al., 2009). Whereas alteration in the somatosensory perception of the contralateral adhesive (time to contact) partially recovers within 3 weeks after ischemia, the sensorimotor contralateral impairment (time to remove) is still strongly present on the 3rd week after surgery.

In marmoset, an early bilateral somatosensory deficit (time to contact) (Freret et al., 2009) is induced by either transient (3h) or permanent proximal MCAo. This deficit is often transient

Time to contact contralateral adhesive after stroke (% pre-surgery)

Time to remove contralateral adhesive after stroke (% pre-surgery)

**IPSILATERAL**

Week 1 Week 2 Week 3 Week 4 Week 9

Week 1 Week 2 Week 3 Week 4 Week 9

Rat - MCAo 30 min Rat - MCAo 60 min Mouse - distal MCAo Mouse - proximal MCAo Marmoset - transient MCAo Marmoset - permanent MCAo

Rat - MCAo 30 min Rat - MCAo 60 min Mouse - distal MCAo Mouse - proximal MCAo Marmoset - transient MCAo Marmoset - permanent MCAo

Rat - MCAo 30 min Rat - MCAo 60 min Mouse - distal MCAo Mouse - proximal MCAo Marmoset - transient MCAo Marmoset - permanent MCAo

Fig. 4. Adhesive removal performances in rat, mouse and marmoset after stroke (% pre-surgical values). Mean percentage of time to contact the adhesive tapes

was occluded by intraluminal approach either permanently of transiently (3 h). Each point represents the mean of three trials performed on three consecutive days.

(upper graphs), positioned on the controlateral side (left) and on the ipsilateral side (right). Time to remove the adhesive tapes (lower graphs), positioned on the controlateral side (left) and on the ipsilateral side (right). All stroke models presented concern the occlusion the middle cerebral artery (MCAo). For rats, stroke was induced by the intraluminal occlusion of the MCA (30 or 60 min). For mice, MCAo was occluded either by distal permanent electrocoagulation or by intraluminal occlusion (60 min - proximal). In marmosets, MCA

Take a look now at the impairments associated to relatively small brain lesion (d-MCAo), adhesive removal test is very useful to detect functional contralateral deficits, even 3 weeks after surgery (Freret et al., 2009). Whereas alteration in the somatosensory perception of the contralateral adhesive (time to contact) partially recovers within 3 weeks after ischemia, the sensorimotor contralateral impairment (time to remove) is still strongly present on the 3rd

In marmoset, an early bilateral somatosensory deficit (time to contact) (Freret et al., 2009) is induced by either transient (3h) or permanent proximal MCAo. This deficit is often transient

Rat - MCAo 30 min Rat - MCAo 60 min Mouse - distal MCAo Mouse - proximal MCAo Marmoset - transient MCAo Marmoset - permanent MCAo

**CONTROLATERAL IPSILATERAL**

week after surgery.

Week 1 Week 2 Week 3 Week 4 Week 9

Week 1 Week 2 Week 3 Week 4 Week 9

**CONTROLATERAL**

on the ipsilateral side of the lesion and might be due, at least partly, to dizziness of the animal due to surgery and/or the anesthesia. With respect to the time to remove the adhesive, a bilateral motor coordination deficit is observed whatever the duration of the occlusion. Of note, it has been demonstrated in this same model of cerebral ischemia a spontaneous functional recovery on the ipsilateral side, while controlateral time to remove the adhesive remains hardly affected up to 4 weeks after surgery (Bihel et al.).

### **3.2 Correlations between brain histological damage and deficit in the adhesive removal test**

Correlations between the cortical and striatal histological lesions and the ischemia-induced behavioral impairments in the adhesive removal test have been well investigated in the literature, mostly in rodents (Grabowski et al., 1991; Hudzik et al., 2000; Hunter et al., 1998; Rogers et al., 1997; Virley et al., 2000). We and other authors have demonstrated a close correlation between contralateral contact and removal latencies on this task and abnormal changes in the ipsilateral caudate putamen, lower parietal cortex and forelimb cortex following transient MCAo (30; 60 or 90 min; see Figure 5). Each of these regions of interest contributed to functional impairments on this task across an extended time course (up to several months post-ischemia) (Virley et al., 2000). As regards to the final cortical damage, it seems particularly correlated to both transient and long-lasting sensorimotor deficits measured by adhesive removal test. Conversely, the final striatal lesion appears to be consistently related to the adhesive removal motor deficits (time to remove) (Freret et al., 2006).

Fig. 5. Schemes showing extent of the lesion after distal and proximal models of Middle Cerebral Artery occlusion (MCAo) in rodents and marmoset. Distal MCAo refers to the occlusion of the artery in its distal portion, *i.e.* after lenticulostriate branches (usually

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 59

al., 2008). This test has also proven to be useful for cellular therapies (Minnerup et al., 2011; Shen et al., 2007), or other pharmacological approaches (testosterone injection – (Morris et al., 2010)), Thymosin beta4 I – (Pan et al., 2005)). In mice, Rehni et al., 2007 (Rehni et al., 2007) showed that intracerebroventricular administration of stem cells derived from amniotic fluid is beneficial for adhesive removal after 60-min MCAo in mice. In marmosets, till now only few studies have assessed the effects of pharmacological agents on adhesive

Overall, those results argue in favor of the use of the adhesive removal test to accurately assess the effects of pharmacological agents on functional outcome. Indeed, this task is suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote

Data reported here show that adhesive removal test *i.* can be easily performed in most species used in experimental research *ii.* is powerful to point out functional deficits on a long-term duration after brain injury. This is a particularly interesting point because it allows assessing efficiency of new therapeutic strategies. Those deficits are in human patients those for which no real efficient therapy exists, excepted rehabilitative strategies like kinesiotherapy. Measuring their importance in animals give models to go further in

Contact and removal times give complementary information about the nervous system deficiency. Although the measurement of contact time is not a perfect reflect of sensory system functioning, because for obvious reasons it is related to a motor action (shaking paw, bringing paw to the mouth), it reflects nevertheless a sensory system stimulation. The animal feels or not that something is stuck on its paw and reacts by a movement. From experimental data obtained with adhesive removal, it is not possible to distinguish if the deficit in contact time is related to a primary somatosensory dysfunction (Ward et al., 1997), a sensory hemi-neglect (related to attentional deficit of a body part and involving striatum (Heilman et al., 2000; Reep et al., 2004)), or a tactile extinction phenomenon (related to

Removal of the tape is a rather tricky task for all the animals presented here because it is not *per se* a natural situation. Rodents and marmosets use their mouth to do it, and amazingly after stroke rats often hold their deficient paw with the intact one to bring it to the mouth as if they were not able to maintain at the right position the deficient/controlateral hand. The data presented here show that for equivalent brain lesions (parietal cortex and striatum affected by intraluminal model of stroke) all species tested display long-lasting deficits in removal time, at least at the dates tested, which is however rather long compared to delays usually assessed in stroke investigations. Time to contact the contralateral adhesive is also increased for few weeks after stroke, and comes back to normal levels for the less severe brain injury, while it stays abnormal for more severe injuries. It is important to note that the severity of the deficit is quite well related with the extent of the lesion, but also to the structures affected. When the parietal cortex alone is affected (30-min MCAo in rat, distal model in mouse), the deficit is less severe than when other structures are also involved in the lesioned area. Indeed, when the striatum is also affected (60-min MCAo in rat, proximal

removal test after stroke, but much on Parkinson's disease (Annett et al., 1994).

**4. General overview and limitations of the adhesive removal test** 

regeneration.

researches on brain injury recovery.

bilateral stimulation (Schallert &Whishaw, 1984)).

obtained by electrocoagulation or administration of pharmacological agent, *e.g.* endothelin-1 (Virley et al., 2004)), while proximal MCAo refers to an occlusion at the origin of the artery (intraluminal approach). Extent of the lesion has been drawn from our data and literature. Of note, these schemes are not fully representative of all experimental models of middle cerebral artery ischemia, but give an overview of most frequently obtained brain lesion.

On a side point, the potential relationship between impairments in the adhesive removal test and brain regions that undergo secondary degeneration has been less investigated so far. Indeed, ischemia is known to cause secondary degeneration in non-ischemic remote brain areas such as the ipsilateral thalamus (mostly the ventroposteromedial and ventroposterolateral nuclei (Iizuka et al., 1990)) as well as in the substantia nigra (Tamura et al., 1990). These regions that do not belong to the territory of the MCA, are connected to the primary lesion site. As regards to secondary thalamus shrinkage, retrograde as well as anterograde degeneration due, respectively, to cortical lesion (Iizuka et al., 1990) (Wallerian) and to basal ganglia lesion and/or extensive vasogenic edema (Dihne et al., 2002) are thought to be responsible. Whether the extent of these degenerative changes is or not directly correlated to the size of primary lesion is still under controversy (Freret et al., 2006; Iizuka et al., 1990). No matter how, in a rat stroke model, the final thalamic atrophy appears to be correlated with ischemia-induced deficits observed in the adhesive-removal test (especially its sensory component) (Freret et al., 2006). This correlation suggests that the thalamus makes, albeit moderately, a significant contribution to the ischemia-induced longlasting somatosensory (ipsilateral bias) and sensorimotor deficits (dexterity alteration). These findings are in agreement with the known involvement of thalamic ventroposterior nuclei in the somatosensory pathways and sensory processing (Tracey &Waite, 1995). In addition, correlations appeared at the late chronic stage (*i.e.*, several weeks after ischemic insult, which is consistent with the delay of thalamic shrinkage development; (Fujie et al., 1990; Jones &Schallert, 1992). Regarding substantia nigra, damage to this brain structure is also correlated with contralateral deficits in the adhesive removal test (Virley et al., 2000). Finally, talking about correlations between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, one must keep in mind that although the presence of such a correlation would make the interpretation of performances in this behavioral test easier, the absence of a linear relationship between both parameters does not mean the absence of involvement of the structure in the behavioral task. Indeed, one can imagine that beyond a single and direct relationship between one or several brain structures and the behavioral performances, those latter might depend upon the integrity of a cerebral network that can be influenced by others brain structures. If so, a lesion limited only to this cerebral network or to the related brain structures will have consequences on behavioral performances; even though the one or the other remains unaffected.

### **3.3 Sensitivity of the adhesive removal test to pharmacological treatment**

In line with this consideration of a relationship between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, our group have demonstrated that a delayed and chronic administration of deferoxamine (an iron chelator) reduces the secondary thalamic atrophy and improves functional recovery in the adhesive removal test after focal ischemia in the rat (Freret et al., 2006). Similarly, a neuroprotective effect of D-JNKi (a peptide inhibitor of JNK (c-Jun-N-terminal kinase)) have been highlighted and corroborated with adhesive removal improvement in the rat (Esneault et

obtained by electrocoagulation or administration of pharmacological agent, *e.g.* endothelin-1 (Virley et al., 2004)), while proximal MCAo refers to an occlusion at the origin of the artery (intraluminal approach). Extent of the lesion has been drawn from our data and literature. Of note, these schemes are not fully representative of all experimental models of middle cerebral artery ischemia, but give an overview of most frequently obtained brain lesion.

On a side point, the potential relationship between impairments in the adhesive removal test and brain regions that undergo secondary degeneration has been less investigated so far. Indeed, ischemia is known to cause secondary degeneration in non-ischemic remote brain areas such as the ipsilateral thalamus (mostly the ventroposteromedial and ventroposterolateral nuclei (Iizuka et al., 1990)) as well as in the substantia nigra (Tamura et al., 1990). These regions that do not belong to the territory of the MCA, are connected to the primary lesion site. As regards to secondary thalamus shrinkage, retrograde as well as anterograde degeneration due, respectively, to cortical lesion (Iizuka et al., 1990) (Wallerian) and to basal ganglia lesion and/or extensive vasogenic edema (Dihne et al., 2002) are thought to be responsible. Whether the extent of these degenerative changes is or not directly correlated to the size of primary lesion is still under controversy (Freret et al., 2006; Iizuka et al., 1990). No matter how, in a rat stroke model, the final thalamic atrophy appears to be correlated with ischemia-induced deficits observed in the adhesive-removal test (especially its sensory component) (Freret et al., 2006). This correlation suggests that the thalamus makes, albeit moderately, a significant contribution to the ischemia-induced longlasting somatosensory (ipsilateral bias) and sensorimotor deficits (dexterity alteration). These findings are in agreement with the known involvement of thalamic ventroposterior nuclei in the somatosensory pathways and sensory processing (Tracey &Waite, 1995). In addition, correlations appeared at the late chronic stage (*i.e.*, several weeks after ischemic insult, which is consistent with the delay of thalamic shrinkage development; (Fujie et al., 1990; Jones &Schallert, 1992). Regarding substantia nigra, damage to this brain structure is also correlated with contralateral deficits in the adhesive removal test (Virley et al., 2000). Finally, talking about correlations between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, one must keep in mind that although the presence of such a correlation would make the interpretation of performances in this behavioral test easier, the absence of a linear relationship between both parameters does not mean the absence of involvement of the structure in the behavioral task. Indeed, one can imagine that beyond a single and direct relationship between one or several brain structures and the behavioral performances, those latter might depend upon the integrity of a cerebral network that can be influenced by others brain structures. If so, a lesion limited only to this cerebral network or to the related brain structures will have consequences on

behavioral performances; even though the one or the other remains unaffected.

**3.3 Sensitivity of the adhesive removal test to pharmacological treatment** 

In line with this consideration of a relationship between impairments in the adhesive removal test and brain damages in experimental models of focal ischemia, our group have demonstrated that a delayed and chronic administration of deferoxamine (an iron chelator) reduces the secondary thalamic atrophy and improves functional recovery in the adhesive removal test after focal ischemia in the rat (Freret et al., 2006). Similarly, a neuroprotective effect of D-JNKi (a peptide inhibitor of JNK (c-Jun-N-terminal kinase)) have been highlighted and corroborated with adhesive removal improvement in the rat (Esneault et al., 2008). This test has also proven to be useful for cellular therapies (Minnerup et al., 2011; Shen et al., 2007), or other pharmacological approaches (testosterone injection – (Morris et al., 2010)), Thymosin beta4 I – (Pan et al., 2005)). In mice, Rehni et al., 2007 (Rehni et al., 2007) showed that intracerebroventricular administration of stem cells derived from amniotic fluid is beneficial for adhesive removal after 60-min MCAo in mice. In marmosets, till now only few studies have assessed the effects of pharmacological agents on adhesive removal test after stroke, but much on Parkinson's disease (Annett et al., 1994).

Overall, those results argue in favor of the use of the adhesive removal test to accurately assess the effects of pharmacological agents on functional outcome. Indeed, this task is suitable for assessing both neuroprotective therapies which target early intervention as well as those aimed at the prevention of delayed damage and therapies which promote regeneration.

### **4. General overview and limitations of the adhesive removal test**

Data reported here show that adhesive removal test *i.* can be easily performed in most species used in experimental research *ii.* is powerful to point out functional deficits on a long-term duration after brain injury. This is a particularly interesting point because it allows assessing efficiency of new therapeutic strategies. Those deficits are in human patients those for which no real efficient therapy exists, excepted rehabilitative strategies like kinesiotherapy. Measuring their importance in animals give models to go further in researches on brain injury recovery.

Contact and removal times give complementary information about the nervous system deficiency. Although the measurement of contact time is not a perfect reflect of sensory system functioning, because for obvious reasons it is related to a motor action (shaking paw, bringing paw to the mouth), it reflects nevertheless a sensory system stimulation. The animal feels or not that something is stuck on its paw and reacts by a movement. From experimental data obtained with adhesive removal, it is not possible to distinguish if the deficit in contact time is related to a primary somatosensory dysfunction (Ward et al., 1997), a sensory hemi-neglect (related to attentional deficit of a body part and involving striatum (Heilman et al., 2000; Reep et al., 2004)), or a tactile extinction phenomenon (related to bilateral stimulation (Schallert &Whishaw, 1984)).

Removal of the tape is a rather tricky task for all the animals presented here because it is not *per se* a natural situation. Rodents and marmosets use their mouth to do it, and amazingly after stroke rats often hold their deficient paw with the intact one to bring it to the mouth as if they were not able to maintain at the right position the deficient/controlateral hand.

The data presented here show that for equivalent brain lesions (parietal cortex and striatum affected by intraluminal model of stroke) all species tested display long-lasting deficits in removal time, at least at the dates tested, which is however rather long compared to delays usually assessed in stroke investigations. Time to contact the contralateral adhesive is also increased for few weeks after stroke, and comes back to normal levels for the less severe brain injury, while it stays abnormal for more severe injuries. It is important to note that the severity of the deficit is quite well related with the extent of the lesion, but also to the structures affected. When the parietal cortex alone is affected (30-min MCAo in rat, distal model in mouse), the deficit is less severe than when other structures are also involved in the lesioned area. Indeed, when the striatum is also affected (60-min MCAo in rat, proximal

A Master Key to Assess Stroke Consequences Across Species: The Adhesive Removal Test 61

study reporting the usefulness of the tongue protrusion test in a rat model of proximal cerebral ischemia (Gulyaeva et al., 2003). Furthermore, it suggests that permanent distal stroke could be a relevant model of oral and facial impairments, which are also tremendous

To conclude on the usefulness of the adhesive removal test, one must admit that this test:

3. is useful to assess recovery, since it is capable of measuring long-lasting deficits,

2. is an accurate test to assess somatosensory and motor dysfunctions, even if they are

4. allows for longitudinal studies through adaptation of the size of the adhesive tape according to the age of the individual tested. Indeed, as previously described, we adapted the adhesive removal test to in 20 days old rat pups by reducing the size of the

5. Finally, the adhesive removal test allows for interspecies comparison (marmosets, rats, dogs and mice), as strongly advised by the expert committees for preclinical studies.

Annett LE, Martel FL, Rogers DC, Ridley RM, Baker HF, Dunnett SB. Behavioral assessment

Annett LE, Rogers DC, Hernandez TD, Dunnett SB. Behavioural analysis of unilateral monoamine depletion in the marmoset. Brain 1992;115 ( Pt 3):825-56. Baumann CR, Kilic E, Petit B, Werth E, Hermann DM, Tafti M, Bassetti CL. Sleep EEG

Bihel E, Pro-Sistiaga P, Letourneur A, Toutain J, Saulnier R, Insausti R, Bernaudin M,

Bouet V, Boulouard M, Toutain J, Divoux D, Bernaudin M, Schumann-Bard P, Freret T. The

Bouet V, Freret T, Ankri S, Bezault M, Renolleau S, Boulouard M, Jacotot E, Chauvier D,

Bouet V, Freret T, Toutain J, Divoux D, Boulouard M, Schumann-Bard P. Sensorimotor and

Bulman-Fleming MB, Bryden MP, Rogers TT. Mouse paw preference: effects of variations in

Denes G, Semenza C, Stoppa E, Lis A. Unilateral spatial neglect and recovery from

ischemic stroke with reperfusion in the rat. Behav Brain Res 2010.

lesions of the nigrostriatal pathway. Exp Neurol 1994;125(2):228-46.

cortical lesions. Sleep 2006;29(10):1339-44.

Nat Protoc 2009;4(10):1560-4.

Neurol 2007;203(2):555-67.

investigations. J Cereb Blood Flow Metab;30(2):273-85.

testing protocol. Behav Brain Res 1997;86(1):79-87.

hemiplegia: a follow-up study. Brain 1982;105 (Pt 3):543-52.

of the effects of embryonic nigral grafts in marmosets with unilateral 6-OHDA

changes after middle cerebral artery infarcts in mice: different effects of striatal and

Roussel S, Touzani O. Permanent or transient chronic ischemic stroke in the nonhuman primate: behavioral, neuroimaging, histological, and immunohistochemical

adhesive removal test: a sensitive method to assess sensorimotor deficits in mice.

Schumann-Bard P. Predicting sensorimotor and memory deficits after neonatal

cognitive deficits after transient middle cerebral artery occlusion in the mouse. Exp

1. is relevant because it was developed in relation to clinical evaluations,

problems in human stroke.

**5. Conclusion** 

very tiny,

adhesive tape.

**6. References** 

model in mouse, both models in marmoset), the recovery is even longer and most of the time not complete within the delays tested.

Mechanisms implied in the recovery of the ability to remove the adhesive tape are not well determined but some of them have to be considered: structural modifications are undoubted (sprouting, synaptogenesis,..), functional modifications (synaptic plasticity, use of brain areas close to the lesion site to fulfill the roles of destroyed tissue…), sensory substitution (increasing role of deep cutaneous modality to replace for the superficial one…), or even behavioral strategies (as for instance, the rat holding the deficient paw with the intact paw to bring it to its mouth).

### **4.1 Sensory hemi-neglect or tactile extinction syndrome**

In rats, a 60-min MCAo induces a long-lasting motor coordination deficit on the contralateral side, reflecting a failure to respond to a novel tactile stimulus. Whether this phenomenon reflects either a primary somatosensory dysfunction (Ward et al., 1997), a truly sensory hemi-neglect (*i.e.*, an attention deficit, as it is classically invoked in the clinic; (Heilman et al., 2000)), or a tactile extinction syndrome (*i.e.*, an interhemispheric perceptual interaction between both stimuli; (Schallert &Whishaw, 1984) is hard to say. Indeed, such duration of occlusion induces a lesion that includes the primary sensory cortical area (S1FL) and, in a secondary degenerative manner, part of the thalamus. These two regions are involved in the somatosensory information processing. On the other hand, it has been shown that unilateral lesions of the posterior parietal cortex and of the dorsocentral striatum (i.e., two structures that are at least partly affected by ischemia) result in multimodal neglect in the rat (Reep et al., 2004). Clinical studies on healthy subjects have reported that the vigilance component of attention to sensory stimuli involves, at least in part, the parietal cortex (Pardo et al., 1991). Thus, a lesion of the parietal cortex could also contribute to a sensory neglect syndrome in the rat after ischemia. An alteration of the cortical– basalganglia–thalamic network could lead to a sensory neglect syndrome in the rat. Nevertheless, a tactile extinction syndrome occurs frequently in stroke patients with right cerebral hemisphere damage (Rose et al., 1994), and the presence of neglect is not only debilitating for patient's ability for independent daily life but is also a significant predictor of poor outcome for recovery from hemiplegia in stroke patients (Denes et al., 1982). The longlasting correlation described between the contralateral time to contact and the final cortical damage (Freret et al., 2006) reinforces the idea that this structure makes significant contribution to the sensory impairment observed after ischemia (Virley et al., 2000).

#### **4.2 Facial and/or limb impairments**

After d-MCAo in mice, even though the extension of the lesion is relatively limited, the adhesive removal test is efficient to highlight sensorimotor contralateral impairment (time to removal) 3 weeks after the surgery. This deficit may combine both sensory feedback alterations and motor coordination that are not attributable to postural bias (Schallert et al., 1983). Given the spatial distribution of the cerebral lesion (Figure 5), this impairment might also reflect in this case a face-related somatososensory perception alteration (*i.e.*, a difficulty for the animal to sense the adhesive with its ipsilateral whiskers when it approaches the tape to his face and/or with its tongue when he licks the adhesive in order to facilitate the removal) rather than a forelimb motor or sensory alteration (because the related brain region, *i.e.*, S1 FL, is mainly spared by ischemia). This hypothesis is in accordance with a study reporting the usefulness of the tongue protrusion test in a rat model of proximal cerebral ischemia (Gulyaeva et al., 2003). Furthermore, it suggests that permanent distal stroke could be a relevant model of oral and facial impairments, which are also tremendous problems in human stroke.

### **5. Conclusion**

60 Advances in the Preclinical Study of Ischemic Stroke

model in mouse, both models in marmoset), the recovery is even longer and most of the

Mechanisms implied in the recovery of the ability to remove the adhesive tape are not well determined but some of them have to be considered: structural modifications are undoubted (sprouting, synaptogenesis,..), functional modifications (synaptic plasticity, use of brain areas close to the lesion site to fulfill the roles of destroyed tissue…), sensory substitution (increasing role of deep cutaneous modality to replace for the superficial one…), or even behavioral strategies (as for instance, the rat holding the deficient paw with the intact paw

In rats, a 60-min MCAo induces a long-lasting motor coordination deficit on the contralateral side, reflecting a failure to respond to a novel tactile stimulus. Whether this phenomenon reflects either a primary somatosensory dysfunction (Ward et al., 1997), a truly sensory hemi-neglect (*i.e.*, an attention deficit, as it is classically invoked in the clinic; (Heilman et al., 2000)), or a tactile extinction syndrome (*i.e.*, an interhemispheric perceptual interaction between both stimuli; (Schallert &Whishaw, 1984) is hard to say. Indeed, such duration of occlusion induces a lesion that includes the primary sensory cortical area (S1FL) and, in a secondary degenerative manner, part of the thalamus. These two regions are involved in the somatosensory information processing. On the other hand, it has been shown that unilateral lesions of the posterior parietal cortex and of the dorsocentral striatum (i.e., two structures that are at least partly affected by ischemia) result in multimodal neglect in the rat (Reep et al., 2004). Clinical studies on healthy subjects have reported that the vigilance component of attention to sensory stimuli involves, at least in part, the parietal cortex (Pardo et al., 1991). Thus, a lesion of the parietal cortex could also contribute to a sensory neglect syndrome in the rat after ischemia. An alteration of the cortical– basalganglia–thalamic network could lead to a sensory neglect syndrome in the rat. Nevertheless, a tactile extinction syndrome occurs frequently in stroke patients with right cerebral hemisphere damage (Rose et al., 1994), and the presence of neglect is not only debilitating for patient's ability for independent daily life but is also a significant predictor of poor outcome for recovery from hemiplegia in stroke patients (Denes et al., 1982). The longlasting correlation described between the contralateral time to contact and the final cortical damage (Freret et al., 2006) reinforces the idea that this structure makes significant

contribution to the sensory impairment observed after ischemia (Virley et al., 2000).

After d-MCAo in mice, even though the extension of the lesion is relatively limited, the adhesive removal test is efficient to highlight sensorimotor contralateral impairment (time to removal) 3 weeks after the surgery. This deficit may combine both sensory feedback alterations and motor coordination that are not attributable to postural bias (Schallert et al., 1983). Given the spatial distribution of the cerebral lesion (Figure 5), this impairment might also reflect in this case a face-related somatososensory perception alteration (*i.e.*, a difficulty for the animal to sense the adhesive with its ipsilateral whiskers when it approaches the tape to his face and/or with its tongue when he licks the adhesive in order to facilitate the removal) rather than a forelimb motor or sensory alteration (because the related brain region, *i.e.*, S1 FL, is mainly spared by ischemia). This hypothesis is in accordance with a

time not complete within the delays tested.

**4.1 Sensory hemi-neglect or tactile extinction syndrome** 

to bring it to its mouth).

**4.2 Facial and/or limb impairments** 

To conclude on the usefulness of the adhesive removal test, one must admit that this test:


### **6. References**


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**4** 

**Variations in Origin of Arteries Supplying** 

Animal testing, also known as animal experimentation, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million used annually (Cohn, 2010). The number of mice and rats used in the United States alone in 2001 was 80 million. Most animals are euthanized after being used in an experiment

It includes pure research such as genetics, developmental biology, behavioral studies, as well as applied research such as biomedical research, xenotransplantation, drug testing and toxicology tests, including cosmetics testing. Animals are also used for education, breeding and defense research. The practice is regulated to various degrees in different

The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE. Aristotle (384–322 BCE) and Erasistratus (304–258 BCE) were among

The ability of humans to change the genetics of animals took a large step forwards in 1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice (Jaenisch & Mintz, 1974). This genetic research progressed rapidly and in 1996 Dolly the sheep was born, the first mammal to be cloned

Toxicology testing became important in the 20th century. In the 19th century laws regulating drugs were more relaxed. For example, in the U.S. the government could only ban a drug after a company had been prosecuted for selling products that harmed customers. In the 1960s, in reaction to the Thalidomide tragedy, further laws were passed requiring safety testing on pregnant animals before a drug can be sold (Burkholz, 1997). Albino rabbits are used in eye irritancy tests because rabbits have less tear flow than other animals and the lack of eye pigment in albinos make the effects easier to visualize. Rabbits

the first to perform experiments on living animals (Cohen & Loew, 1984).

are also frequently used for the production of polyclonal antibodies.

**1. Introduction** 

(Carbone, 2004).

countries.

**1.1 Laboratory animals** 

from an adult cell (Wilmut et al., 1997).

**the Brain in Rabbit and Their Impact** 

**on Total Cerebral Ischemia** 

Eva Petrovova and Lenka Luptakova

*The Slovak Republic* 

David Mazensky, Jan Danko, Emil Pilipcinec,

*University of Veterinary Medicine and Pharmacy, Kosice* 

ablation, parkinsonism and spinal cord injury. Neuropharmacology 2000;39(5):777- 87.


## **Variations in Origin of Arteries Supplying the Brain in Rabbit and Their Impact on Total Cerebral Ischemia**

David Mazensky, Jan Danko, Emil Pilipcinec, Eva Petrovova and Lenka Luptakova *University of Veterinary Medicine and Pharmacy, Kosice The Slovak Republic* 

### **1. Introduction**

64 Advances in the Preclinical Study of Ischemic Stroke

Schallert T, Upchurch M, Lobaugh N, Farrar SB, Spirduso WW, Gilliam P, Vaughn D,

Schallert T, Upchurch M, Wilcox RE, Vaughn DM. Posture-independent sensorimotor

Schallert T, Whishaw IQ. Bilateral cutaneous stimulation of the somatosensory system in

Shen LH, Li Y, Chen J, Cui Y, Zhang C, Kapke A, Lu M, Savant-Bhonsale S, Chopp M. One-

STAIR. Recommendations for standards regarding preclinical neuroprotective and

STAIRIII. Recommendations for clinical trial evaluation of acute stroke therapies. Stroke

Starkey ML, Barritt AW, Yip PK, Davies M, Hamers FP, McMahon SB, Bradbury EJ.

Tamura A, Kirino T, Sano K, Takagi K, Oka H. Atrophy of the ipsilateral substantia nigra following middle cerebral artery occlusion in the rat. Brain Res 1990;510(1):154-7. Tracey DJ, Waite ME. Somatosensory System. In: Press A, editor. The rat nervous system

van Lookeren Campagne M, Thibodeaux H, van Bruggen N, Cairns B, Gerlai R, Palmer JT,

Virley D, Beech JS, Smart SC, Williams SC, Hodges H, Hunter AJ. A temporal MRI

hemidecorticate rats. Behav Neurosci 1984;98(3):518-40.

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2nd Edition. Sydney: Academic Press; 1995. p 689-700.

corticospinal tract in adult mice. Exp Neurol 2005;195(2):524-39.

cerebral ischemia. Proc Natl Acad Sci U S A 1999;96(22):12870-5.

a rodent model of focal ischemia. Stroke 2011;42(5):1437-44.

87.

Behav 1982;16(3):455-62.

2001;32(7):1598-606.

Biochem Behav 1983;18(5):753-9.

rats with stroke. Stroke 2007;38(7):2150-6.

ablation, parkinsonism and spinal cord injury. Neuropharmacology 2000;39(5):777-

Wilcox RE. Tactile extinction: distinguishing between sensorimotor and motor asymmetries in rats with unilateral nigrostriatal damage. Pharmacol Biochem

analysis of inter-hemispheric receptor asymmetries in neostriatum. Pharmacol

year follow-up after bone marrow stromal cell treatment in middle-aged female

Assessing behavioural function following a pyramidotomy lesion of the

Williams SP, Lowe DG. Evidence for a protective role of metallothionein-1 in focal

assessment of neuropathology after transient middle cerebral artery occlusion in the rat: correlations with behavior. J Cereb Blood Flow Metab 2000;20(3):563-82. Ward NM, Sharkey J, Brown VJ. Assessment of sensorimotor neglect after occlusion of the middle cerebral artery in the rat. Behav Neurosci 1997;111(5):1133-45. Zhang L, Li Y, Zhang C, Chopp M, Gosiewska A, Hong KW. Delayed administration of

human umbilical tissue-derived cells improved neurological functional recovery in

### **1.1 Laboratory animals**

Animal testing, also known as animal experimentation, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million used annually (Cohn, 2010). The number of mice and rats used in the United States alone in 2001 was 80 million. Most animals are euthanized after being used in an experiment (Carbone, 2004).

It includes pure research such as genetics, developmental biology, behavioral studies, as well as applied research such as biomedical research, xenotransplantation, drug testing and toxicology tests, including cosmetics testing. Animals are also used for education, breeding and defense research. The practice is regulated to various degrees in different countries.

The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE. Aristotle (384–322 BCE) and Erasistratus (304–258 BCE) were among the first to perform experiments on living animals (Cohen & Loew, 1984).

The ability of humans to change the genetics of animals took a large step forwards in 1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice (Jaenisch & Mintz, 1974). This genetic research progressed rapidly and in 1996 Dolly the sheep was born, the first mammal to be cloned from an adult cell (Wilmut et al., 1997).

Toxicology testing became important in the 20th century. In the 19th century laws regulating drugs were more relaxed. For example, in the U.S. the government could only ban a drug after a company had been prosecuted for selling products that harmed customers. In the 1960s, in reaction to the Thalidomide tragedy, further laws were passed requiring safety testing on pregnant animals before a drug can be sold (Burkholz, 1997).

Albino rabbits are used in eye irritancy tests because rabbits have less tear flow than other animals and the lack of eye pigment in albinos make the effects easier to visualize. Rabbits are also frequently used for the production of polyclonal antibodies.

Variations in Origin of Arteries Supplying

**1.3.3 Truncus brachiocephalicus** 

Fig. 1. Basis cordis

**1.3.4 Arteria carotis communis** 

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 67

aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases and the origin of the a. carotis communis sinistra in 4% of cases from the arcus aortae. White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra and the a.

The truncus brachiocephalicus runs behind the cartilage of the first rib and the v. cava cranialis dextra on the left side of the trachea. Near the midline it courses to the right and then partly dorsally. Immediatelly after its origin gives off the a. carotis communis sinistra. The truncus brachiocephalicus is very short and divides into the a. carotis communis dextra and the a. subclavia dextra (Nejedlý, 1965). By other author the a. subclavia dextra was

described as the first branch of the truncus brachiocephalicus (Popesko et al., 1990).

7. a. subclavia sinistra; 8. truncus brachiocephalicus; 9. a. subclavia dextra; 10. truncus bicaroticus; 13. n. vagus dexter; 14. n. recurrens dexter; 15. n. recurrens sinister; 20. v. cava cranialis sinistra; 21. v. cava cranialis dextra; 22. v. cava caudalis; 23. v. azygos dextra; 24. truncus pulmonalis; 25. a. pulmonalis sinistra; 26. a. pulmonalis dextra; 27. vv. pulmonales; 28. atrium sinistrum; 29. atrium dextrum; 30. ventriculus sinister; 31. ventriculus dexter; 32. ligamentum arteriosum; 33. lymphonodi thoracici aortici; 34. a. bronchalis. v. Vena. Vv.

In the neck region the a. carotis communis is covered by m. sternohyoideus (musculus), more cranially also by the m. sternothyroideus. It lies on the lateral surface of the trachea. The a. carotis communis lies dorsally to the esophagus, then laterally to the larynx. It reaches the maxillar angle laterally from the cranial end of the m. sternohyoideus and under the parotid gland. In this area it divides into the a. carotis interna and a. carotis externa (Nejedlý, 1965).

Venae. n. Nervus. a. Arteria. Dorsal view (Popesko et al., 1990)

subclavia dextra et sinistra as independent branches of the arcus aortae.

### **1.2 Cerebral ischemia**

It is the deficiency of blood and metabolic substrates in the brain due to insufficient arterial supply or venous drainage, causing disruption of cerebral functions and partially reversible or irreversible damage to neurons.

Ischemia leads to alterations in brain metabolism, reduction in metabolic rates and energy crisis (Vespa et al., 2005).

There are two types of ischemia: focal ischemia, which is confined to a specific region of the brain; and global ischemia, which encompasses wide areas of brain tissue.

The main symptoms involve impairments in vision, body movement, and speaking. The causes of brain ischemia vary from sickle cell anemia to congenital heart defects. Symptoms of brain ischemia can include unconsciousness, blindness, problems with coordination, and weakness in the body. Other effects that may result from brain ischemia are stroke, cardiorespiratory arrest and irreversible brain damage.

An interruption of blood flow to the brain for more 10 seconds causes unconsciousness and an interruption in flow for more than a few minutes generally results in irreversible brain damage. In 1974, Hossmann and Zimmerman demonstrated that ischemia induced in mammalian brains for up to an hour can be at least partially recovered. Accordingly, this discovery raised the possibility of intervening after brain ischemia before the damage becomes irreversible.

Global brain ischemia occurs when blood flow to the brain is halted or drastically reduced. This is commonly caused by cardiac arrest. If sufficient circulation is restored within a short period of time, symptoms may be transient. However, if a significant amount of time passes before restoration, brain damage may be permanent. While reperfusion may be essential to protecting as much brain tissue as possible, it may also lead to reperfusion injury. Reperfusion injury is classified as the damage that ensues after restoration of blood supply to ischemic tissue.

### **1.3 Anatomy of the arteries supplying the brain in the rabbit**

### **1.3.1 Aorta ascendens**

The aorta ascendens arises almost linearly and dorsally in the midline. It extends from the cranial margin of the second rib cartilage to the cranial margin of the first rib cartilage. At first runs inside the pericard, left to beginning of the pulmonary trunk, to the left and cranially to the dorsal part of the right auricle and to the left and cranially to the right ventricle. Then it runs inside the thymus dorsally to the left side of the v. cava cranialis dextra (vena) and continues to the arcus aortae (Nejedlý, 1965).

### **1.3.2 Arcus aortae**

The arcus aortae is running from the right to the left transversally and also a little caudally from the point of the second thoracic vertebra. Its dorsal wall is convex, the ventral wall is concave. It lies to the left from the v. cava cranialis dextra. The left part turns around the bronchus principalis sinister and ends on the left side of the third thoracic vertebra behind the v. cava cranialis sinistra. Very close to the midline of the body from its arise to the right the truncus brachiocephalicus and to the left the a. subclavia sinistra (arteria) (Popesko et al., 1990; Fig. 1). The truncus brachiocephalicus, the a. carotis communis sinistra and the a. subclavia dextra as branches of the arcus aortae were described by Nejedlý (1965). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases and the origin of the a. carotis communis sinistra in 4% of cases from the arcus aortae. White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra and the a. subclavia dextra et sinistra as independent branches of the arcus aortae.

### **1.3.3 Truncus brachiocephalicus**

66 Advances in the Preclinical Study of Ischemic Stroke

It is the deficiency of blood and metabolic substrates in the brain due to insufficient arterial supply or venous drainage, causing disruption of cerebral functions and partially reversible

Ischemia leads to alterations in brain metabolism, reduction in metabolic rates and energy

There are two types of ischemia: focal ischemia, which is confined to a specific region of the

The main symptoms involve impairments in vision, body movement, and speaking. The causes of brain ischemia vary from sickle cell anemia to congenital heart defects. Symptoms of brain ischemia can include unconsciousness, blindness, problems with coordination, and weakness in the body. Other effects that may result from brain ischemia are stroke,

An interruption of blood flow to the brain for more 10 seconds causes unconsciousness and an interruption in flow for more than a few minutes generally results in irreversible brain damage. In 1974, Hossmann and Zimmerman demonstrated that ischemia induced in mammalian brains for up to an hour can be at least partially recovered. Accordingly, this discovery raised the possibility of intervening after brain ischemia before the damage

Global brain ischemia occurs when blood flow to the brain is halted or drastically reduced. This is commonly caused by cardiac arrest. If sufficient circulation is restored within a short period of time, symptoms may be transient. However, if a significant amount of time passes before restoration, brain damage may be permanent. While reperfusion may be essential to protecting as much brain tissue as possible, it may also lead to reperfusion injury. Reperfusion injury is classified as the damage that ensues after restoration of blood supply

The aorta ascendens arises almost linearly and dorsally in the midline. It extends from the cranial margin of the second rib cartilage to the cranial margin of the first rib cartilage. At first runs inside the pericard, left to beginning of the pulmonary trunk, to the left and cranially to the dorsal part of the right auricle and to the left and cranially to the right ventricle. Then it runs inside the thymus dorsally to the left side of the v. cava cranialis

The arcus aortae is running from the right to the left transversally and also a little caudally from the point of the second thoracic vertebra. Its dorsal wall is convex, the ventral wall is concave. It lies to the left from the v. cava cranialis dextra. The left part turns around the bronchus principalis sinister and ends on the left side of the third thoracic vertebra behind the v. cava cranialis sinistra. Very close to the midline of the body from its arise to the right the truncus brachiocephalicus and to the left the a. subclavia sinistra (arteria) (Popesko et al., 1990; Fig. 1). The truncus brachiocephalicus, the a. carotis communis sinistra and the a. subclavia dextra as branches of the arcus aortae were described by Nejedlý (1965). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus

brain; and global ischemia, which encompasses wide areas of brain tissue.

cardiorespiratory arrest and irreversible brain damage.

**1.3 Anatomy of the arteries supplying the brain in the rabbit** 

dextra (vena) and continues to the arcus aortae (Nejedlý, 1965).

**1.2 Cerebral ischemia** 

crisis (Vespa et al., 2005).

becomes irreversible.

to ischemic tissue.

**1.3.1 Aorta ascendens** 

**1.3.2 Arcus aortae** 

or irreversible damage to neurons.

The truncus brachiocephalicus runs behind the cartilage of the first rib and the v. cava cranialis dextra on the left side of the trachea. Near the midline it courses to the right and then partly dorsally. Immediatelly after its origin gives off the a. carotis communis sinistra. The truncus brachiocephalicus is very short and divides into the a. carotis communis dextra and the a. subclavia dextra (Nejedlý, 1965). By other author the a. subclavia dextra was described as the first branch of the truncus brachiocephalicus (Popesko et al., 1990).

### Fig. 1. Basis cordis

7. a. subclavia sinistra; 8. truncus brachiocephalicus; 9. a. subclavia dextra; 10. truncus bicaroticus; 13. n. vagus dexter; 14. n. recurrens dexter; 15. n. recurrens sinister; 20. v. cava cranialis sinistra; 21. v. cava cranialis dextra; 22. v. cava caudalis; 23. v. azygos dextra; 24. truncus pulmonalis; 25. a. pulmonalis sinistra; 26. a. pulmonalis dextra; 27. vv. pulmonales; 28. atrium sinistrum; 29. atrium dextrum; 30. ventriculus sinister; 31. ventriculus dexter; 32. ligamentum arteriosum; 33. lymphonodi thoracici aortici; 34. a. bronchalis. v. Vena. Vv. Venae. n. Nervus. a. Arteria. Dorsal view (Popesko et al., 1990)

### **1.3.4 Arteria carotis communis**

In the neck region the a. carotis communis is covered by m. sternohyoideus (musculus), more cranially also by the m. sternothyroideus. It lies on the lateral surface of the trachea. The a. carotis communis lies dorsally to the esophagus, then laterally to the larynx. It reaches the maxillar angle laterally from the cranial end of the m. sternohyoideus and under the parotid gland. In this area it divides into the a. carotis interna and a. carotis externa (Nejedlý, 1965).

Variations in Origin of Arteries Supplying

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 69

cervical vertebrae cranially. It passes through the foramen tranversarium of the atlas, courses medially, then cranially and a little dorsally. It runs through the foramen vertebrale laterale of the atlas and gives off the a. spinalis dorsalis and ventralis. After this it penetrates the dura mater and continues to the cranial part of the medulla oblongata. On the caudal margin of the dorsal surface of the pars basilaris ossis occipitalis it is fused together with the contralateral a. vertebralis. This fusion forms the a. basilaris (Fig. 3). A. basilaris continues rostrally on the ventral surface of the medulla oblongata and pons. This artery gives off some branches which praticipate on the formation of the circle of Willis and by this way it

A. vertebralis dextra et sinistra located inside canalis transversarius of the cervical vertebrae.

a. A. cerebelli caudalis is the largest of the transverse branches arising from the a. basilaris on the ventral surface of the hindbrain. It originates about half way along the a. basilaris

b. A. cerebri caudalis is a paired vessel formed at the level of the rostral margin of the pons by the bifurcation of the a. basilaris. It passes at each side laterally and dorsally to the caudal portion of the cerebral hemisphere, giving secondary branches to the

c. A. cerebelli rostralis is a relatively large branch of the a. cerebri caudalis, arising near the origin of the latter and passing to the rostral portion of the cerebellum after giving

d. The end of the a. carotis interna lies on either side of the tuber cinereum. It turns forward, but is connected backwards with the a. cerebri caudalis by an a. communicans

e. A. cerebri media is given off from the a. carotis interna, branching over the middle

portion of the hemispere to supply most of its lateral and dorsal surfaces

The fusion of the bilateral aa. vertebrales to the a. basilaris. a. Arteria. Dorsal view

and passes laterally and up the side of the caudal part of the cerebellum

The arteries of the brain may be described on its ventral surface as follows:

participates on the arterial supplying of the brain (Nejedlý, 1965).

Fig. 3. Corrosion cast of the vertebral arteries

**1.3.8 The arteries of the brain** 

diencephalon

caudalis

branches to the midbrain

Fig. 2. Scheme of the origin of the large arteries supplying the brain in the rabbit 1. arcus aortae; 2. truncus brachiocephalicus; 3. truncus bicaroticus; 4. a. subclavia dextra; 5. a. subclavia sinistra; 6. a. carotis communis dextra; 7. a. carotis communis sinistra; 8. a. vertebralis sinistra; 9. a. vertebralis dextra. a. Arteria. Lateral view

### **1.3.5 Arteria carotis interna**

The a. carotis interna as poorly developed artery it arises by the division of the a. carotis communis into the a. carotis externa and the a. carotis interna. From the a. carotis externa it is divided by m. styloglossus and m. stylopharyngeus. It is running dorsally on the medial side of the bulla tympanica ossis temporalis. It enters the canalis caroticus ossis temporalis and inside this canal continues into the skull cavity. It is running rostrally on the medial surface of the n. trigeminus (nervus) and on the lateral surface of the corpus ossis basisphenoidalis is directed into the sulcus caroticus. It turns dorsally on the medial surface on the place of the entrance of the n. oculomotorius into the fissura orbitalis. It crosses the n. oculomotorius to the right. In this way it makes three bends. After this it enters the ventral end of the canalis caroticus. Here it gives off the a. communicans caudalis and a. ophtalmica dorsalis and subsequently is divided into the a. carebri rostralis and a. cerebri media (Nejedlý, 1965).

### **1.3.6 Arteria subclavia**

The a. subclavia runs caudally and dorsally to the v. subclavia, above the n. cervicalis VIII., behind the origin of the m. sternomastoideus and m. pectoralis superficialis. The branches of a. subclavia are: truncus costocervicalis, a. vertebralis, a. cervicalis superficialis, a. mammaria interna, a. intercostalis suprema, a. cervicalis profunda and a. transversa colli. The direct continuation is a. axillaris (Nejedlý, 1965).

#### **1.3.7 Arteria vertebralis**

The a. vertebralis enters the foramen transversarium of the sixth cervical vertebra. It gives off rr. musculares (rami) and rr. spinales. It continues inside the canalis transversarius of the

Fig. 2. Scheme of the origin of the large arteries supplying the brain in the rabbit

vertebralis sinistra; 9. a. vertebralis dextra. a. Arteria. Lateral view

**1.3.5 Arteria carotis interna** 

**1.3.6 Arteria subclavia** 

**1.3.7 Arteria vertebralis** 

The direct continuation is a. axillaris (Nejedlý, 1965).

1. arcus aortae; 2. truncus brachiocephalicus; 3. truncus bicaroticus; 4. a. subclavia dextra; 5. a. subclavia sinistra; 6. a. carotis communis dextra; 7. a. carotis communis sinistra; 8. a.

The a. carotis interna as poorly developed artery it arises by the division of the a. carotis communis into the a. carotis externa and the a. carotis interna. From the a. carotis externa it is divided by m. styloglossus and m. stylopharyngeus. It is running dorsally on the medial side of the bulla tympanica ossis temporalis. It enters the canalis caroticus ossis temporalis and inside this canal continues into the skull cavity. It is running rostrally on the medial surface of the n. trigeminus (nervus) and on the lateral surface of the corpus ossis basisphenoidalis is directed into the sulcus caroticus. It turns dorsally on the medial surface on the place of the entrance of the n. oculomotorius into the fissura orbitalis. It crosses the n. oculomotorius to the right. In this way it makes three bends. After this it enters the ventral end of the canalis caroticus. Here it gives off the a. communicans caudalis and a. ophtalmica dorsalis and subsequently is divided into the a. carebri rostralis and a. cerebri media (Nejedlý, 1965).

The a. subclavia runs caudally and dorsally to the v. subclavia, above the n. cervicalis VIII., behind the origin of the m. sternomastoideus and m. pectoralis superficialis. The branches of a. subclavia are: truncus costocervicalis, a. vertebralis, a. cervicalis superficialis, a. mammaria interna, a. intercostalis suprema, a. cervicalis profunda and a. transversa colli.

The a. vertebralis enters the foramen transversarium of the sixth cervical vertebra. It gives off rr. musculares (rami) and rr. spinales. It continues inside the canalis transversarius of the cervical vertebrae cranially. It passes through the foramen tranversarium of the atlas, courses medially, then cranially and a little dorsally. It runs through the foramen vertebrale laterale of the atlas and gives off the a. spinalis dorsalis and ventralis. After this it penetrates the dura mater and continues to the cranial part of the medulla oblongata. On the caudal margin of the dorsal surface of the pars basilaris ossis occipitalis it is fused together with the contralateral a. vertebralis. This fusion forms the a. basilaris (Fig. 3). A. basilaris continues rostrally on the ventral surface of the medulla oblongata and pons. This artery gives off some branches which praticipate on the formation of the circle of Willis and by this way it participates on the arterial supplying of the brain (Nejedlý, 1965).

Fig. 3. Corrosion cast of the vertebral arteries

A. vertebralis dextra et sinistra located inside canalis transversarius of the cervical vertebrae. The fusion of the bilateral aa. vertebrales to the a. basilaris. a. Arteria. Dorsal view

### **1.3.8 The arteries of the brain**

The arteries of the brain may be described on its ventral surface as follows:


Variations in Origin of Arteries Supplying

**1.4 Rabbit as experimental animal of the brain ischemia** 

arteries supplying the brain with blood in the rabbit.

**2. Material and methods 2.1 Experimental animals** 

ad libitum.

**2.2 Material** 

**2.3 Methods**

**2.3.1 Surgical preparation of the rabbit** 

rostrales.

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 71

The a. cerebri rostralis unites with that of the other side to form a short common trunk between the hemispheres, which redivides into the paired vessels supplying the medial surfaces. A complete anastomic loop is thus formed round the hypothalamus by the a. carotis interna, a. cerebri rostralis, a. communicans caudalis and a. cerebri caudalis. This is the circle of Willis or the circulus arteriosus cerebri (Popesko et al., 1990). Nejedlý (1965) described the a. communicans rostralis as a connection between the bilateral aa. cerebri

Over 20,000 rabbits were used for animal testing only in the UK in 2004. Examples include restricting blood flow to the brain to induce cerebral ischemia (Tolwani et al., 1999). This is most often carried out by ligation of major vessels, e.g. the truncus brachiocephalicus and the a. subclavia sinistra, in their place of origin (Hossmann 1998; Iwama et al., 2000; Pluta 1987). Harukuni and Bhardwaj (2006) reported ligation of the truncus brachiocephalicus and the a. subclavia sinistra as one way to induce total cerebral ischemia. Ischemia within the arteries branching from the vertebral arteries in the back of the brain may result in symptoms such as dizziness, vertigo, double vision, or weakness on both sides of the body. Other symptoms include, difficulty speaking, slurred speech and the loss of coordination (Beers et al., 2003). The aim of this study was to verify whether experimentally induced total cerebral ischemia in rabbits actually corresponds to total ischemia on the basis of the origin of certain vessels. We observed morphological variations in the origin and course of the

The study was carried out on 50 adult (age=140 days) New Zealand white rabbits (breed HY+), females (n=25) and males (n=25) of weight range 2.5-3 kg in an accredited experimental laboratory at the University of Veterinary Medicine in Kosice, Slovak Republic. The animals were kept in cages under standard conditions (temperature 15-20 °C, relative humidity 45 %, 12 hours light period) and fed granular mixed feed (O-10 NORM TYP, Spišské krmné zmesi, Spišské Vlachy, Slovak Republic). Drinking water was provided

The Batson΄s No. 17 Plastic Replica and Corrosion Kit (Polysciences Europe GmbH, Germany) was used as a casting medium. This consist of Base Solution A (2-Propenoic acid, 2-methyl-, 1,2-ethanediyl 1 ester, Dibutyl phthalate, Methyl methacrylate, Polymethyl methacrylate ), Catalyst (Acetone, Benzoyl peroxide, Dibutyl phthalate), Promoter C (Dibutyl 1 phthalate, N,N-Dimethyl-4-toluidine) and red pigment (1,2-Benzenedicarboxylic acid, bis[2-ethylhexyl ester], epoxidized soybean oil and 2-Naphthenecarboxylic acid).

The animals were injected intravenously with heparine (50, 000 UI/kg) 30 minutes before they were sacrified with intravenous injection of Embutramide (T-61, 0.3 mL/kg). The skin

Fig. 4. The arteries of the brain

1. bulbus olfactorius; 2 n. opticus; 3. n. oculomotorius; 4. n. trochlearis; 5. n. trigeminus; 6. n. abducens; 7. n. facialis; 8. n. vestibulocochlearis; 9. n. glossopharyngeus; 10. n. vagus; 11. n. accessorius; 12. n. hypoglossus; 18. corpus mamillare; 19. lobus piriformis; 20. chiasma opticum; 21. sulcus rhinalis lateralis; 24. paraflocculus; 26. a. carotis interna; 27., 28. a. cerebri media; 27. r. caudalis; 28. r. rostralis; 29. a. cerebri rostralis; 30. a. ethmoidalis interna; 31. a. communicans caudalis; 32. r. corporis mamillaris; 33. a. cerebri caudalis; 34. a. cerebelli rostralis; 35. rami ad pontem; 36. a. cerebelli caudalis; 37. a. basilaris; 38. a. vertebralis. a. Arteria. r. Ramus. Ventral View (Popesko et al., 1990)

f. A. cerebri rostralis is the continuation of the a. carotis interna after the origin of the a. cerebri media. It passes to the rostral portion of the ventral surface of the cerebral hemisphere and to the olfactory bulb.

The a. cerebri rostralis unites with that of the other side to form a short common trunk between the hemispheres, which redivides into the paired vessels supplying the medial surfaces. A complete anastomic loop is thus formed round the hypothalamus by the a. carotis interna, a. cerebri rostralis, a. communicans caudalis and a. cerebri caudalis. This is the circle of Willis or the circulus arteriosus cerebri (Popesko et al., 1990). Nejedlý (1965) described the a. communicans rostralis as a connection between the bilateral aa. cerebri rostrales.

### **1.4 Rabbit as experimental animal of the brain ischemia**

Over 20,000 rabbits were used for animal testing only in the UK in 2004. Examples include restricting blood flow to the brain to induce cerebral ischemia (Tolwani et al., 1999). This is most often carried out by ligation of major vessels, e.g. the truncus brachiocephalicus and the a. subclavia sinistra, in their place of origin (Hossmann 1998; Iwama et al., 2000; Pluta 1987). Harukuni and Bhardwaj (2006) reported ligation of the truncus brachiocephalicus and the a. subclavia sinistra as one way to induce total cerebral ischemia. Ischemia within the arteries branching from the vertebral arteries in the back of the brain may result in symptoms such as dizziness, vertigo, double vision, or weakness on both sides of the body. Other symptoms include, difficulty speaking, slurred speech and the loss of coordination (Beers et al., 2003). The aim of this study was to verify whether experimentally induced total cerebral ischemia in rabbits actually corresponds to total ischemia on the basis of the origin of certain vessels. We observed morphological variations in the origin and course of the arteries supplying the brain with blood in the rabbit.

### **2. Material and methods**

### **2.1 Experimental animals**

The study was carried out on 50 adult (age=140 days) New Zealand white rabbits (breed HY+), females (n=25) and males (n=25) of weight range 2.5-3 kg in an accredited experimental laboratory at the University of Veterinary Medicine in Kosice, Slovak Republic. The animals were kept in cages under standard conditions (temperature 15-20 °C, relative humidity 45 %, 12 hours light period) and fed granular mixed feed (O-10 NORM TYP, Spišské krmné zmesi, Spišské Vlachy, Slovak Republic). Drinking water was provided ad libitum.

### **2.2 Material**

70 Advances in the Preclinical Study of Ischemic Stroke

1. bulbus olfactorius; 2 n. opticus; 3. n. oculomotorius; 4. n. trochlearis; 5. n. trigeminus; 6. n. abducens; 7. n. facialis; 8. n. vestibulocochlearis; 9. n. glossopharyngeus; 10. n. vagus; 11. n. accessorius; 12. n. hypoglossus; 18. corpus mamillare; 19. lobus piriformis; 20. chiasma opticum; 21. sulcus rhinalis lateralis; 24. paraflocculus; 26. a. carotis interna; 27., 28. a. cerebri media; 27. r. caudalis; 28. r. rostralis; 29. a. cerebri rostralis; 30. a. ethmoidalis interna; 31. a. communicans caudalis; 32. r. corporis mamillaris; 33. a. cerebri caudalis; 34. a. cerebelli rostralis; 35. rami ad pontem; 36. a. cerebelli caudalis; 37. a. basilaris; 38. a. vertebralis. a.

f. A. cerebri rostralis is the continuation of the a. carotis interna after the origin of the a. cerebri media. It passes to the rostral portion of the ventral surface of the cerebral

Fig. 4. The arteries of the brain

Arteria. r. Ramus. Ventral View (Popesko et al., 1990)

hemisphere and to the olfactory bulb.

The Batson΄s No. 17 Plastic Replica and Corrosion Kit (Polysciences Europe GmbH, Germany) was used as a casting medium. This consist of Base Solution A (2-Propenoic acid, 2-methyl-, 1,2-ethanediyl 1 ester, Dibutyl phthalate, Methyl methacrylate, Polymethyl methacrylate ), Catalyst (Acetone, Benzoyl peroxide, Dibutyl phthalate), Promoter C (Dibutyl 1 phthalate, N,N-Dimethyl-4-toluidine) and red pigment (1,2-Benzenedicarboxylic acid, bis[2-ethylhexyl ester], epoxidized soybean oil and 2-Naphthenecarboxylic acid).

### **2.3 Methods**

### **2.3.1 Surgical preparation of the rabbit**

The animals were injected intravenously with heparine (50, 000 UI/kg) 30 minutes before they were sacrified with intravenous injection of Embutramide (T-61, 0.3 mL/kg). The skin

Variations in Origin of Arteries Supplying

Fig. 5. Aortic arch of the rabbit without variations

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 73

a. subclavia dextra by terminal division of truncus brachiocephalicus (Fig. 5). The a. carotis

Fig. 6. Aortic arch of the rabbit with variation in origin of a. carotis communis sinistra

communis sinistra originated from arcus aortae in 6 % (3 animals; Fig. 6).

was subsequently removed as far as possible to prevent it from sticking to the corrosive cast in the maceration process. The thoracic cavity was opened from the left side by removing of the ribs. After the opening of the pericardial cavity a ligature was introduced to the ascending aorta. The aorta was cannulated through the left ventricle. The perfusion started after the fixation of the cannula in the ascending aorta with the ligature. The right vestibule was opened to lower the pressure in the vessels to ensure good injection. The vascular network was manually perfused through the fixed cannula in the ascending aorta for approximately 15-20 minutes with 2.5-3 l of warm (37 °C), 0.9 % NaOH in 0.01 M phosphate, pH 7.3 (Hossler & Monson, 1995).

### **2.3.2 Preparation of the casting medium**

The red pigment was added to the Base solution A prior to mixing the catalyst and promoter. The pigment was added in the amount of 5 %. It was mixed and divided into two equal parts (each part=25 mL). To the first half Catalyst in amount of 12 mL was added and mixed. To the second half Promoter C in amount of 12 drops was added and mixed. Then these two parts were mixed together.

### **2.3.3 Application of the casting medium**

The arterial network was filled with the casting medium manually through the same cannula inserted in the ascending aorta. Adequate filling was determined by the visualization of an even distribution of the casting medium (red) throughout the superficial vessels of the body. After the vascular casting is complete, the animals must not be manipulated for at least 30 minutes and then must be submersed in water at a temperature ranging from 40 °C to 60 °C for a period of 24 hours for full polymerization of the casting medium (Lametschwandtner et al., 1990).

### **2.3.4 Corrosion**

The corrosion as the dissolution of tissues surrounding the cast was performed by potassium hydroxide (KOH) at the concentration of solution 2–4 % for a period of 2 days. For the corrosion to be faster, the solution must remain at a constant temperature of 40 °C (Lametschwandtner et al., 1990). The solution for the corrosion was changed every 12 hours. After the surrounding tissue was dissolved vascular castings were rinsed in running water for removing the rests of the soft tissues. Specimens were dried at the room temperature by air exposure (Flešárová et al., 2003).

### **3. Results and discussion**

### **3.1 Variations in origin**

### **3.1.1 A. carotis communis**

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus and the a. subclavia sinistra from the arcus aortae in 92 % of cases (46 animals). a. Arteria. Ventral view. Macroscopic image

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus, the a. subclavia sinistra and the a. carotis communis sinistra from the arcus aortae in 6 % (3 animals). a. Arteria. Ventral view. Macroscopic image

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as the first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the

was subsequently removed as far as possible to prevent it from sticking to the corrosive cast in the maceration process. The thoracic cavity was opened from the left side by removing of the ribs. After the opening of the pericardial cavity a ligature was introduced to the ascending aorta. The aorta was cannulated through the left ventricle. The perfusion started after the fixation of the cannula in the ascending aorta with the ligature. The right vestibule was opened to lower the pressure in the vessels to ensure good injection. The vascular network was manually perfused through the fixed cannula in the ascending aorta for approximately 15-20 minutes with 2.5-3 l of warm (37 °C), 0.9 % NaOH in 0.01 M phosphate,

The red pigment was added to the Base solution A prior to mixing the catalyst and promoter. The pigment was added in the amount of 5 %. It was mixed and divided into two equal parts (each part=25 mL). To the first half Catalyst in amount of 12 mL was added and mixed. To the second half Promoter C in amount of 12 drops was added and mixed. Then

The arterial network was filled with the casting medium manually through the same cannula inserted in the ascending aorta. Adequate filling was determined by the visualization of an even distribution of the casting medium (red) throughout the superficial vessels of the body. After the vascular casting is complete, the animals must not be manipulated for at least 30 minutes and then must be submersed in water at a temperature ranging from 40 °C to 60 °C for a period of 24 hours for full polymerization of the casting

The corrosion as the dissolution of tissues surrounding the cast was performed by potassium hydroxide (KOH) at the concentration of solution 2–4 % for a period of 2 days. For the corrosion to be faster, the solution must remain at a constant temperature of 40 °C (Lametschwandtner et al., 1990). The solution for the corrosion was changed every 12 hours. After the surrounding tissue was dissolved vascular castings were rinsed in running water for removing the rests of the soft tissues. Specimens were dried at the room temperature by

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus and the a. subclavia sinistra from the arcus aortae in 92 % of cases (46

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus, the a. subclavia sinistra and the a. carotis communis sinistra from the

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as the first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the

arcus aortae in 6 % (3 animals). a. Arteria. Ventral view. Macroscopic image

pH 7.3 (Hossler & Monson, 1995).

**2.3.2 Preparation of the casting medium** 

these two parts were mixed together.

**2.3.3 Application of the casting medium** 

medium (Lametschwandtner et al., 1990).

air exposure (Flešárová et al., 2003).

animals). a. Arteria. Ventral view. Macroscopic image

**3. Results and discussion** 

**3.1 Variations in origin 3.1.1 A. carotis communis** 

**2.3.4 Corrosion** 

a. subclavia dextra by terminal division of truncus brachiocephalicus (Fig. 5). The a. carotis communis sinistra originated from arcus aortae in 6 % (3 animals; Fig. 6).

Fig. 5. Aortic arch of the rabbit without variations

Fig. 6. Aortic arch of the rabbit with variation in origin of a. carotis communis sinistra

Variations in Origin of Arteries Supplying

Fig. 8. A. vertebralis with its typical origin

Fig. 9. Atypical origin of the a. vertebralis

Macroscopic image

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 75

Vascular corrosion cast displaying the origin of the a. vertebralis dextra et sinistra from the a. subclavia dextra et sinistra in 86 % of cases (43 animals). a. Arteria. Ventral view.

Vascular corrosion cast displaying the origin of the a. vertebralis sinistra from the arcus

aortae in 10 % of cases (5 animals). a. Arteria. Dorsal view. Macroscopic image

### **3.1.2 A. subclavia**

In 98 % of cases (49 animals) the a. subclavia sinistra originated from the arcus aortae (Fig. 5). In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra (Fig. 6).

Fig. 7. Aortic arch of the rabbit with variation in origin of a. subclavia dextra

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra from the arcus aortae in 2 % (1 animal). a. Arteria. Dorsolateral view. Macroscopic image

### **3.1.3 A. vertebralis**

In 86 % of cases (43 animals) the a. vertebralis sinistra originated directly from the a. subclavia sinistra (Fig. 8) in 10 % of cases (5 animals) it originated from the arcus aortae as an independent branch (Fig. 9) and in 4 % of cases (2 animals) it arose from the arcus aortae as a common trunk with the a. scapularis descendens. The a. vertebralis dextra originated from the a. subclavia dextra in 98 % (49 animals) of cases. In that case we observed two aa. vertebralis dextrae (arteriae) with two different origins.

The a. vertebralis dextra I originated from the a. subclavia dextra and the a. vertebralis dextra II arose from the common trunk with the a. cervicalis superficialis dextra that originated from the a. carotis communis dextra.

After a short distance, they merged between the fifth and sixth cervical vertebrae into a single a. vertebralis dextra, which then entered the canalis transversarius at the level of the fifth cervical vertebra (Fig. 10). In summary, the origin of both aa. vertebrales varied in 16 % of cases (8 animals). In 8 % (4 animals) we found a bypass between the a. vertebralis sinistra and the a. basilaris (Fig. 11). This a. basilaris was also formed by the fusion of the a. vertebralis dextra et sinistra.

In 98 % of cases (49 animals) the a. subclavia sinistra originated from the arcus aortae (Fig. 5). In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus, the a. subclavia

Fig. 7. Aortic arch of the rabbit with variation in origin of a. subclavia dextra

(1 animal). a. Arteria. Dorsolateral view. Macroscopic image

vertebralis dextrae (arteriae) with two different origins.

originated from the a. carotis communis dextra.

Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra from the arcus aortae in 2 %

In 86 % of cases (43 animals) the a. vertebralis sinistra originated directly from the a. subclavia sinistra (Fig. 8) in 10 % of cases (5 animals) it originated from the arcus aortae as an independent branch (Fig. 9) and in 4 % of cases (2 animals) it arose from the arcus aortae as a common trunk with the a. scapularis descendens. The a. vertebralis dextra originated from the a. subclavia dextra in 98 % (49 animals) of cases. In that case we observed two aa.

The a. vertebralis dextra I originated from the a. subclavia dextra and the a. vertebralis dextra II arose from the common trunk with the a. cervicalis superficialis dextra that

After a short distance, they merged between the fifth and sixth cervical vertebrae into a single a. vertebralis dextra, which then entered the canalis transversarius at the level of the fifth cervical vertebra (Fig. 10). In summary, the origin of both aa. vertebrales varied in 16 % of cases (8 animals). In 8 % (4 animals) we found a bypass between the a. vertebralis sinistra and the a. basilaris (Fig. 11). This a. basilaris was also formed by the fusion of the a.

**3.1.2 A. subclavia** 

**3.1.3 A. vertebralis** 

vertebralis dextra et sinistra.

dextra and the a. subclavia sinistra (Fig. 6).

Fig. 8. A. vertebralis with its typical origin

Vascular corrosion cast displaying the origin of the a. vertebralis dextra et sinistra from the a. subclavia dextra et sinistra in 86 % of cases (43 animals). a. Arteria. Ventral view. Macroscopic image

Fig. 9. Atypical origin of the a. vertebralis

Vascular corrosion cast displaying the origin of the a. vertebralis sinistra from the arcus aortae in 10 % of cases (5 animals). a. Arteria. Dorsal view. Macroscopic image

Variations in Origin of Arteries Supplying

Fig. 12. Typical origin of a. carotis interna

Fig. 13. Atypical origin of a. carotis interna

caroticus. a. Arteria. Ventrolateral view. Macroscopic image

**3.1.4 A. carotis interna** 

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 77

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna by the terminal division of the a. carotis communis together with the a. carotis externa in 94 % (47 animals). Note the entrance of the a. carotis interna to the canalis

### Fig. 10. Doubled a. vertebralis

Vascular corrosion cast displaying two aa. vertebrales dextrae. Note merging of the two arteriae into a single vessel. a. Arteria. aa. Arteriae. Lateral view. Macroscopic image

Fig. 11. Variation in formation of a. basilaris

Vascular corrosion cast displaying the bypass between the a. vertebralis sinistra and a. basilaris in 8 % (4 animals). a. Arteria. Dorsal view. Macroscopic image

### **3.1.4 A. carotis interna**

76 Advances in the Preclinical Study of Ischemic Stroke

Vascular corrosion cast displaying two aa. vertebrales dextrae. Note merging of the two

Vascular corrosion cast displaying the bypass between the a. vertebralis sinistra and a.

basilaris in 8 % (4 animals). a. Arteria. Dorsal view. Macroscopic image

arteriae into a single vessel. a. Arteria. aa. Arteriae. Lateral view. Macroscopic image

Fig. 10. Doubled a. vertebralis

Fig. 11. Variation in formation of a. basilaris

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna by the terminal division of the a. carotis communis together with the a. carotis externa in 94 % (47 animals). Note the entrance of the a. carotis interna to the canalis caroticus. a. Arteria. Ventrolateral view. Macroscopic image

Fig. 12. Typical origin of a. carotis interna

Fig. 13. Atypical origin of a. carotis interna

Variations in Origin of Arteries Supplying

Fig. 15. Variation in arrangement of a. cerebelli caudalis

dextra et sinistra as independent branches of the arcus aortae.

**3.2 Discussion** 

**3.2.2 A. subclavia** 

**3.2.1 A. carotis communis** 

Vascular corrosion cast. The a. cerebelli rostralis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %). Dorsal view. Macroscopic image

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the a. subclavia dextra by the terminal division of the truncus brachiocephalicus. Popesko et al. (1990) described a. carotis communis dextra et sinistra as branches arising from truncus bicaroticus by its terminal division. Truncus bicaroticus was described as the direct continuation of the truncus brachiocephalicus. The a. carotis communis sinistra as branch of the arcus aortae in 4 % (2 animals) was also described by Ding (2006). By Nejedlý (1965) the origin of the a. carotis communis sinistra from the arcus aortae was described as a typical arrangement of branches of the arcus aortae. In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus. Nellie (1930) described this arrangement in all studied animals. White (1893) by studying 700 rabbits found in one case the a. carotis communis

In 98 % (49 animals) the a. subclavia dextra arise together with the a. carotis communis dextra by the terminal division of the truncus brachiocephalicus. By some authors the a.

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 79

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna from the common trunk with the a. occipitalis in 6 % (3 animals). a. Arteria. Caudoventral view. Macroscopic image

In 94 % (47 animals) the a. carotis interna arised by terminal division of the a. carotis communis together with the a. carotis externa (Fig. 12). In 6 % (3 animals) the a. carotis interna originated from a common trunk with the a. occipitalis (Fig. 13). The trunk is a branch of the a. carotis communis.

### **3.1.5 The arteries of the brain**

In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level (Fig. 14). In 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra (Fig. 15). In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra.

The a. cerebri caudalis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %).

The a. cerebri media dextra et sinistra originated from the a. carotis interna at the same level in all studied animals (100 %).

Fig. 14. Typical arrangement of a. cerebelli caudalis

Vascular corrosion cast. In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level from the a. basilaris. Dorsal view. Macroscopic image

The a. cerebri rostralis dextra et sinistra as the direct continuation from the a. carotis interna originated at the same level in all studied animals (100 %).

These all arteries were divided into the r. rostralis and r. caudalis. In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r. rostralis, r. medius and r. caudalis.

Fig. 15. Variation in arrangement of a. cerebelli caudalis

Vascular corrosion cast. The a. cerebelli rostralis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %). Dorsal view. Macroscopic image

### **3.2 Discussion**

78 Advances in the Preclinical Study of Ischemic Stroke

Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna from the common trunk with the a. occipitalis in 6 % (3 animals). a. Arteria.

In 94 % (47 animals) the a. carotis interna arised by terminal division of the a. carotis communis together with the a. carotis externa (Fig. 12). In 6 % (3 animals) the a. carotis interna originated from a common trunk with the a. occipitalis (Fig. 13). The trunk is

In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level (Fig. 14). In 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra (Fig. 15). In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra. The a. cerebri caudalis dextra et sinistra originated from the a. basilaris at the same level in

The a. cerebri media dextra et sinistra originated from the a. carotis interna at the same level

Caudoventral view. Macroscopic image

a branch of the a. carotis communis.

**3.1.5 The arteries of the brain**

all studied animals (100 %).

in all studied animals (100 %).

Fig. 14. Typical arrangement of a. cerebelli caudalis

originated at the same level in all studied animals (100 %).

Vascular corrosion cast. In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level from the a. basilaris. Dorsal view. Macroscopic image

cerebri caudalis sinistra was divided into the r. rostralis, r. medius and r. caudalis.

The a. cerebri rostralis dextra et sinistra as the direct continuation from the a. carotis interna

These all arteries were divided into the r. rostralis and r. caudalis. In 10 % (5 animals) the a.

### **3.2.1 A. carotis communis**

In 92 % of cases (46 animals) the a. carotis communis sinistra originated as first branch from the truncus brachiocephalicus. The a. carotis communis dextra arise together with the a. subclavia dextra by the terminal division of the truncus brachiocephalicus. Popesko et al. (1990) described a. carotis communis dextra et sinistra as branches arising from truncus bicaroticus by its terminal division. Truncus bicaroticus was described as the direct continuation of the truncus brachiocephalicus. The a. carotis communis sinistra as branch of the arcus aortae in 4 % (2 animals) was also described by Ding (2006). By Nejedlý (1965) the origin of the a. carotis communis sinistra from the arcus aortae was described as a typical arrangement of branches of the arcus aortae. In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus. Nellie (1930) described this arrangement in all studied animals. White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra as independent branches of the arcus aortae.

### **3.2.2 A. subclavia**

In 98 % (49 animals) the a. subclavia dextra arise together with the a. carotis communis dextra by the terminal division of the truncus brachiocephalicus. By some authors the a.

Variations in Origin of Arteries Supplying

occurrence of found variations.

also in a smaller number of animals used in experiments.

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978-0-230-60014-0, Baltimore, USA

Oxford, USA

8865

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possible variations.

**5. References** 

the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 81

The variations in the origin of arteries supplying the brain which we found point to the possibility of induction only a partial brain ischemia in a given set of animals. The probability of causing the partial brain ischemia may be the same as the percentage of the

One way to avoid obtaining of distorted results is the ligation of the arteries before their entering to the target organ, in this case to the brain. These arteries can be the a. basilaris or the a. carotis interna. The best results probably would have been achieved by ligation of the arteries on the ventral surface of the brain that are directly involved in the blood supply of the nerve tissue. However, this method is time consuming and surgically very difficult in. Another possibility is the detailed preparation of arteries in the place of their origin to avoid

With this work we tried to emphasize the need for more detailed knowledge of the circulatory system of the rabbit, which is one of the ways to achieve more objective results

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subclavia dextra was described as the first branch arising from the truncus brachiocephalicus (Popesko et al., 1990). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases from the arcus aortae and Nejedlý (1965) described it as typical arrangement of the branches of the arcus aortae. White (1893) by studying 700 pieces rabbits found in one case a. subclavia dextra et sinistra as independent branches of the arcus aortae.

### **3.2.3 A. vertebralis**

Until now the scientific literature has cited almost exclusively the uniform origin of the a. vertebralis from the a. subclavia (Nejedlý, 1965; Popesko et al., 1990). It was not described independent origin from the arcus aortae or the doubled a. vertebralis like in our cases. In 8 % (4 animals) we found the bypass between the a. vertebralis sinistra and the a. basilaris at the place of fusion of the a. vertebralis dextra et sinistra to the a. basilaris.

### **3.2.4 A. carotis interna**

We found the origin of the a. carotis interna and the a. carotis externa by the terminal division of the a. carotis communis in 92 % (46 animals). The same origin was described by Nejedlý (1965) and Popesko et al. (1990). But we found except this typical arrangement the origin of the a. carotis interna from the a. occipitalis in 8 % (4 animals).

### **3.2.5 The arteries of the brain**

Until now the origin of the bilateral a. cerebri rostralis et caudalis and a. cerebelli rostralis et caudalis was described at the same level from the a. basilaris and the a. carotis interna (Nejedlý, 1965; Popesko et al., 1990). We found that in 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra. In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra.

These all arteries were divided into the r. rostralis and r. caudalis (Nejedlý, 1965; Popesko et al., 1990). In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r. rostralis, r. medius and r. caudalis.

The bilateral aa. cerebri rostrales are fused together. The same arrangement was described by Popesko et al. (1990). The a. communicans rostralis as a connection between bilateral aa. cerebri rostrales was described by Nejedlý (1965).

### **4. Conclusion**

The effect of various chemical substances (Cantu **&** Hegsted**, 1970**) on the brain nerve tissue damaged by ischemia as well as various pathological and pathophysiological changes induced by the total cerebral ischemia in rabbits and other laboratory animals are the subject of many studies (Ishiyama et al., 2010).

The place of origin of the truncus brachiocephalicus and the a. subclavia sinistra are most commonly used to induce the total cerebral ischemia by ligation (Hossmann 1998; Iwama et al., 2000; Pluta, 1987). Harukuni and Bhardwaj (2006) present also the ligation of the truncus brachiocephalicus and the a. subclavia sinistra as a possible way to induce the total cerebral ischemia. The question is, whether this method of induction of the total cerebral ischemia is correct.

The variations in the origin of arteries supplying the brain which we found point to the possibility of induction only a partial brain ischemia in a given set of animals. The probability of causing the partial brain ischemia may be the same as the percentage of the occurrence of found variations.

One way to avoid obtaining of distorted results is the ligation of the arteries before their entering to the target organ, in this case to the brain. These arteries can be the a. basilaris or the a. carotis interna. The best results probably would have been achieved by ligation of the arteries on the ventral surface of the brain that are directly involved in the blood supply of the nerve tissue. However, this method is time consuming and surgically very difficult in. Another possibility is the detailed preparation of arteries in the place of their origin to avoid possible variations.

With this work we tried to emphasize the need for more detailed knowledge of the circulatory system of the rabbit, which is one of the ways to achieve more objective results also in a smaller number of animals used in experiments.

### **5. References**

80 Advances in the Preclinical Study of Ischemic Stroke

subclavia dextra was described as the first branch arising from the truncus brachiocephalicus (Popesko et al., 1990). Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae. Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases from the arcus aortae and Nejedlý (1965) described it as typical arrangement of the branches of the arcus aortae. White (1893) by studying 700 pieces rabbits found in one case a. subclavia dextra et sinistra as

Until now the scientific literature has cited almost exclusively the uniform origin of the a. vertebralis from the a. subclavia (Nejedlý, 1965; Popesko et al., 1990). It was not described independent origin from the arcus aortae or the doubled a. vertebralis like in our cases. In 8 % (4 animals) we found the bypass between the a. vertebralis sinistra and the a. basilaris at

We found the origin of the a. carotis interna and the a. carotis externa by the terminal division of the a. carotis communis in 92 % (46 animals). The same origin was described by Nejedlý (1965) and Popesko et al. (1990). But we found except this typical arrangement the

Until now the origin of the bilateral a. cerebri rostralis et caudalis and a. cerebelli rostralis et caudalis was described at the same level from the a. basilaris and the a. carotis interna (Nejedlý, 1965; Popesko et al., 1990). We found that in 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra. In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than

These all arteries were divided into the r. rostralis and r. caudalis (Nejedlý, 1965; Popesko et al., 1990). In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r. rostralis,

The bilateral aa. cerebri rostrales are fused together. The same arrangement was described by Popesko et al. (1990). The a. communicans rostralis as a connection between bilateral aa.

The effect of various chemical substances (Cantu **&** Hegsted**, 1970**) on the brain nerve tissue damaged by ischemia as well as various pathological and pathophysiological changes induced by the total cerebral ischemia in rabbits and other laboratory animals are the subject

The place of origin of the truncus brachiocephalicus and the a. subclavia sinistra are most commonly used to induce the total cerebral ischemia by ligation (Hossmann 1998; Iwama et al., 2000; Pluta, 1987). Harukuni and Bhardwaj (2006) present also the ligation of the truncus brachiocephalicus and the a. subclavia sinistra as a possible way to induce the total cerebral ischemia. The question is, whether this method of induction of the total cerebral ischemia is

the place of fusion of the a. vertebralis dextra et sinistra to the a. basilaris.

origin of the a. carotis interna from the a. occipitalis in 8 % (4 animals).

independent branches of the arcus aortae.

**3.2.3 A. vertebralis** 

**3.2.4 A. carotis interna** 

**3.2.5 The arteries of the brain** 

the a. cerebelli caudalis dextra.

cerebri rostrales was described by Nejedlý (1965).

of many studies (Ishiyama et al., 2010).

r. medius and r. caudalis.

**4. Conclusion** 

correct.


**Part 2** 

**Pathophysiology of Ischemic** 

**or Anoxic Damage** 

 sevoflurane anesthesia during global cerebral ischemia and reperfusion in rabbits. *Journal of neurosurgical anesthesiology*, Vol. 22, No. 3, pp. 207-213, ISSN 0898-4921


## **Part 2**

**Pathophysiology of Ischemic or Anoxic Damage** 

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**5** 

*Canada* 

**Cerebral Ischemia Induced Proteomic** 

Willard J. Costain1, Arsalan S. Haqqani2, Ingrid Rasquinha1,

*1Glycosyltransferases and Neuroglycomics, Institute for Biological Sciences,* 

*2Proteomics, Institute for Biological Sciences, National Research Council, Ottawa, ON,* 

The synapse is the focal point for neuronal communication and neuron-glia interactions. Synaptic structure and function are intimately related and many of the proteins that provide structure to the synapse also regulate synaptic function (Abe et al. 2004, Couchman 2003, Ehlers 2002, Passafaro et al. 2003). The synaptic structure - function relationship is highly apparent during pathological conditions. This is exemplified in neurodegenerative disorders, such as Alzheimer's disease, where synaptic function is positively correlated with neuronal function (Gasic & Nicotera 2003) and viability (Deisseroth et al. 2003), with

Maintenance of synaptic structure and functionality is a process that is highly energy dependent. Studies of synaptosomal morphology and metabolism have indicated that the synapse is highly susceptible to ischemic damage (Pastuszko et al. 1982, Rafalowska et al. 1980, Sulkowski et al. 2002, Enright et al. 2007, Zhang & Murphy 2007). This exceptional requirement for energy necessitates the localization of numerous mitochondria proximal to the synaptic bouton and mitochondria are a commonly observed feature in synaptosomal preparations (Costain et al. 2008). Synaptosomal metabolic activity (Rafalowska et al. 1980) and the rate of neurotransmitter re-uptake (Pastuszko et al. 1982) are decreased following acute hypoxia. Furthermore, Sulkowski *et al* (2002) observed that ischemia decreases

synaptosomal oxygen consumption and metabolic capacity for a period of 24 hours.

Cerebral ischemia induces a marked depletion in synaptic vesicle content and increases the number of damaged mitochondria (Sulkowski et al. 2002, Costain et al. 2008). Importantly, ischemia-induced alterations in synaptosomal morphology are indistinguishable from that of brain slices (Sulkowski et al. 2002). Ischemia-like conditions (hypoxic stress or excitotoxicity) induce dramatic and rapidly reversible structural/morphological changes in dendritic spines (Hasbani et al. 2001, Mattson et al. 1998, Park et al. 1996, Enright et al. 2007,

synaptic pathology preceding cell death (Gasic & Nicotera 2003).

**1. Introduction** 

**Alterations: Consequences for** 

**the Synapse and Organelles** 

Marie-Soleil Giguere2 and Jacqueline Slinn3

*3Cerebrovascular Research, Institute for Biological Sciences,* 

*National Research Council, Ottawa, ON,* 

*National Research Council, Ottawa, ON,* 

Willard J. Costain1, Arsalan S. Haqqani2, Ingrid Rasquinha1, Marie-Soleil Giguere2 and Jacqueline Slinn3 *1Glycosyltransferases and Neuroglycomics, Institute for Biological Sciences, National Research Council, Ottawa, ON, 2Proteomics, Institute for Biological Sciences, National Research Council, Ottawa, ON, 3Cerebrovascular Research, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada* 

### **1. Introduction**

The synapse is the focal point for neuronal communication and neuron-glia interactions. Synaptic structure and function are intimately related and many of the proteins that provide structure to the synapse also regulate synaptic function (Abe et al. 2004, Couchman 2003, Ehlers 2002, Passafaro et al. 2003). The synaptic structure - function relationship is highly apparent during pathological conditions. This is exemplified in neurodegenerative disorders, such as Alzheimer's disease, where synaptic function is positively correlated with neuronal function (Gasic & Nicotera 2003) and viability (Deisseroth et al. 2003), with synaptic pathology preceding cell death (Gasic & Nicotera 2003).

Maintenance of synaptic structure and functionality is a process that is highly energy dependent. Studies of synaptosomal morphology and metabolism have indicated that the synapse is highly susceptible to ischemic damage (Pastuszko et al. 1982, Rafalowska et al. 1980, Sulkowski et al. 2002, Enright et al. 2007, Zhang & Murphy 2007). This exceptional requirement for energy necessitates the localization of numerous mitochondria proximal to the synaptic bouton and mitochondria are a commonly observed feature in synaptosomal preparations (Costain et al. 2008). Synaptosomal metabolic activity (Rafalowska et al. 1980) and the rate of neurotransmitter re-uptake (Pastuszko et al. 1982) are decreased following acute hypoxia. Furthermore, Sulkowski *et al* (2002) observed that ischemia decreases synaptosomal oxygen consumption and metabolic capacity for a period of 24 hours.

Cerebral ischemia induces a marked depletion in synaptic vesicle content and increases the number of damaged mitochondria (Sulkowski et al. 2002, Costain et al. 2008). Importantly, ischemia-induced alterations in synaptosomal morphology are indistinguishable from that of brain slices (Sulkowski et al. 2002). Ischemia-like conditions (hypoxic stress or excitotoxicity) induce dramatic and rapidly reversible structural/morphological changes in dendritic spines (Hasbani et al. 2001, Mattson et al. 1998, Park et al. 1996, Enright et al. 2007,

MacManus 2002), and more recently ischemia induced autophagy has become an active area of interest (Liu et al. 2010). While necrotic cell death is well known to occur in cerebral infarcts, the recent identification of programed necrosis, or 'necroptosis', has reinvigorated

Fig. 1. Outline of the ischemic synaptosomal proteomic analysis procedure. Focal ischemia was performed 20 hours prior to the isolation of the ipsilateral (ischemic) and contralateral (non-ischemic) mouse brain hemispheres. Synaptosomes were isolated from the entire

While cell death mechanisms are typically viewed as pathways involving multiple proteins and organelles, neuronal pathology can also be examined from an organelle centric perspective. Organelle dysfunction can precipitate the initiation of cell death pathways, rather than simply being relay points for signaling events. The essence of this distinction is the difference between extrinsic activation of cell death and internal/intrinsic activation. The primary route of activation of cell death following cerebral ischemia is through intrinsic pathways involving the mitochondria, lysosomes endoplasmic reticulum, and nucleus (Yamashima & Oikawa 2009, Chen et al. 2010, Ankarcrona et al. 1995). Thus, a strategy that involves examining the interaction between organelles during pathological conditions may enable the identification of new targets that can be exploited as therapies for cerebral

forebrain hemispheres and processed as described in the diagram.

ischemia.

research in ischemia-induced necrotic cell death.

Zhang & Murphy 2007). The structural (Park et al. 1996) and biochemical alterations (Martone et al. 1999) at the synapse rapidly return to normal following cessation of ischemic stressors. This initial period of apparent recovery is followed by a period of morphological and biochemical alterations that persist for upwards of 24 hours (Martone et al. 1999). Similarly, dysregulation of synaptic adhesion is observed prior to the onset of neuronal cell death and continues thereafter (Costain et al. 2008). These studies indicate that the synapse is highly responsive to ischemia and is an important modulator of post-ischemic neuronal fate. Signals triggered at the synapse propagate toward the cell body and instigate delayed post-ischemic neuronal death in a process termed as *synaptic apoptosis* (Mattson et al. 1998).

Synaptically localized signals, either anti- or pro-apoptotic, can be propagated to the cell body in both an anterograde and retrograde manner (Mattson & Duan 1999). Apoptotic stimuli have been shown to induce caspase-3 activation, mitochondrial membrane depolarization and phospholipid asymmetry in isolated synaptosomes (Mattson et al. 1998). Similarly, trophic factor withdrawal increases axonal caspase-3 activity, but not within the neuronal soma (Mattson & Duan 1999). In hippocampal neurons, apoptotic signals initiated at the dendrites have been shown to subsequently spread toward the cell body (Mattson & Duan 1999). Synaptic apoptosis may be a mechanism that is necessary for synaptic remodeling under non-pathological conditions as well as contributing to or initiating neuronal apoptosis during pathological conditions. Pro-apoptotic proteins have been found to play a role in non-pathological processes such as neurogenesis, neurite outgrowth and synaptic plasticity (Mattson & Gleichmann 2005). This suggests that signals triggered at the synapse may propagate toward the cell body and instigate postischemic neuronal death.

Cellular protein levels are determined by the balance between the rates of synthesis and degradation, and cerebral ischemia has a pronounced effect on these processes. The transcriptional response to cerebral ischemia has been studied using high throughput methods under a variety of experimental conditions (Gilbert et al. 2003, MacManus et al. 2004). Similarly, cerebral ischemia-induced protein degradation has been examined for a variety of individual proteins. Activation of a variety of proteases, such as members of the caspase, calpain and cathepsin families, is a well-described consequence of cerebral ischemia (Vanderklish & Bahr 2000, Kagedal et al. 2001). Complicating this is the observation that cerebral ischemia causes proteosomal (DeGracia et al. 2002), lysosomal (Costain et al. 2010), mitochondrial (Costain et al. 2010) and endoplasmic reticulum dysfunction (Ge et al. 2007). Thus, it is almost impossible to predict post-ischemic cellular protein levels from gene expression data alone. When focusing on a subcellular structure, such as the synapse, an additional mechanism will impact protein levels. Intracellular transport mechanisms can target a protein to a specific region or be involved in sequestering proteins away from their original location (Zhao et al. 2005, Vanderklish & Bahr 2000). As a result of these factors, the best approach for determining post-ischemic synaptic protein levels is to perform a direct assessment using proteomic methodologies.

An understanding of the cell death processes that are precipitated by exposure to cerebral ischemia is necessary for designing rational therapeutic intervention. Considering that cell death can be mediated by multiple inter-related mechanisms, it is perhaps unsurprising that the majority of single target small molecules have failed in clinical trials for stroke (Ginsberg 2008). The role of apoptotic cell death in cerebral ischemia has long been studied (Hou &

Zhang & Murphy 2007). The structural (Park et al. 1996) and biochemical alterations (Martone et al. 1999) at the synapse rapidly return to normal following cessation of ischemic stressors. This initial period of apparent recovery is followed by a period of morphological and biochemical alterations that persist for upwards of 24 hours (Martone et al. 1999). Similarly, dysregulation of synaptic adhesion is observed prior to the onset of neuronal cell death and continues thereafter (Costain et al. 2008). These studies indicate that the synapse is highly responsive to ischemia and is an important modulator of post-ischemic neuronal fate. Signals triggered at the synapse propagate toward the cell body and instigate delayed post-ischemic neuronal death in a process termed as *synaptic apoptosis* (Mattson et al.

Synaptically localized signals, either anti- or pro-apoptotic, can be propagated to the cell body in both an anterograde and retrograde manner (Mattson & Duan 1999). Apoptotic stimuli have been shown to induce caspase-3 activation, mitochondrial membrane depolarization and phospholipid asymmetry in isolated synaptosomes (Mattson et al. 1998). Similarly, trophic factor withdrawal increases axonal caspase-3 activity, but not within the neuronal soma (Mattson & Duan 1999). In hippocampal neurons, apoptotic signals initiated at the dendrites have been shown to subsequently spread toward the cell body (Mattson & Duan 1999). Synaptic apoptosis may be a mechanism that is necessary for synaptic remodeling under non-pathological conditions as well as contributing to or initiating neuronal apoptosis during pathological conditions. Pro-apoptotic proteins have been found to play a role in non-pathological processes such as neurogenesis, neurite outgrowth and synaptic plasticity (Mattson & Gleichmann 2005). This suggests that signals triggered at the synapse may propagate toward the cell body and instigate post-

Cellular protein levels are determined by the balance between the rates of synthesis and degradation, and cerebral ischemia has a pronounced effect on these processes. The transcriptional response to cerebral ischemia has been studied using high throughput methods under a variety of experimental conditions (Gilbert et al. 2003, MacManus et al. 2004). Similarly, cerebral ischemia-induced protein degradation has been examined for a variety of individual proteins. Activation of a variety of proteases, such as members of the caspase, calpain and cathepsin families, is a well-described consequence of cerebral ischemia (Vanderklish & Bahr 2000, Kagedal et al. 2001). Complicating this is the observation that cerebral ischemia causes proteosomal (DeGracia et al. 2002), lysosomal (Costain et al. 2010), mitochondrial (Costain et al. 2010) and endoplasmic reticulum dysfunction (Ge et al. 2007). Thus, it is almost impossible to predict post-ischemic cellular protein levels from gene expression data alone. When focusing on a subcellular structure, such as the synapse, an additional mechanism will impact protein levels. Intracellular transport mechanisms can target a protein to a specific region or be involved in sequestering proteins away from their original location (Zhao et al. 2005, Vanderklish & Bahr 2000). As a result of these factors, the best approach for determining post-ischemic synaptic protein levels is to perform a direct

An understanding of the cell death processes that are precipitated by exposure to cerebral ischemia is necessary for designing rational therapeutic intervention. Considering that cell death can be mediated by multiple inter-related mechanisms, it is perhaps unsurprising that the majority of single target small molecules have failed in clinical trials for stroke (Ginsberg 2008). The role of apoptotic cell death in cerebral ischemia has long been studied (Hou &

1998).

ischemic neuronal death.

assessment using proteomic methodologies.

MacManus 2002), and more recently ischemia induced autophagy has become an active area of interest (Liu et al. 2010). While necrotic cell death is well known to occur in cerebral infarcts, the recent identification of programed necrosis, or 'necroptosis', has reinvigorated research in ischemia-induced necrotic cell death.

Fig. 1. Outline of the ischemic synaptosomal proteomic analysis procedure. Focal ischemia was performed 20 hours prior to the isolation of the ipsilateral (ischemic) and contralateral (non-ischemic) mouse brain hemispheres. Synaptosomes were isolated from the entire forebrain hemispheres and processed as described in the diagram.

While cell death mechanisms are typically viewed as pathways involving multiple proteins and organelles, neuronal pathology can also be examined from an organelle centric perspective. Organelle dysfunction can precipitate the initiation of cell death pathways, rather than simply being relay points for signaling events. The essence of this distinction is the difference between extrinsic activation of cell death and internal/intrinsic activation. The primary route of activation of cell death following cerebral ischemia is through intrinsic pathways involving the mitochondria, lysosomes endoplasmic reticulum, and nucleus (Yamashima & Oikawa 2009, Chen et al. 2010, Ankarcrona et al. 1995). Thus, a strategy that involves examining the interaction between organelles during pathological conditions may enable the identification of new targets that can be exploited as therapies for cerebral ischemia.

Fig. 2. NanoLC-MS analysis of contralateral (control) and ipsilateral (stroke) samples. Shown are images representing the nanoLC-MS data from each sample, where each spot represents a peptide ion. MatchRx software was used to extract peptide data, align the control and stroke datasets and correct retention time variations. Shown are the merged images before and after MatchRx-dependent correction. More than 5000 ions were detected per sample.

were dissolved in an appropriate volume of denaturing buffer (50 mM Tris-HCl, pH 8.5, 0.1% SDS) to a final protein concentration of 2 mg/mL. One hundred µg of each protein was transferred to a fresh tube. The proteins were reduced using dithiothrietol (4 mM, 10 min at 95 C), alkylated using iodoacetamide (10 mM, 20 min at room temperature in dark), and digested using 5 µg of MS-grade trypsin gold (Promega, 12-18 h at 37 C). The digested peptides were diluted 10-fold in 10 mM KH2PO4, pH 3.0, 25% acetonitrile and loaded onto a cation exchange column (POROS® 50 HS, 50-µm particle size, 4.0 mm x 15 mm, Applied Biosystems) for separation. Five fractions were eluted using step-gradient of 0-350 mM KCl. Each fraction was evaporated to dryness and dissolved in 5% acetronitrile, 1% acetic acid for

analysis by mass spectrometry (MS).

The effects of cerebral ischemia are often, by necessity, described from a highly reductionist point of view, with most studies focusing on specific signaling pathways or individual molecules. Conversely, genomic and proteomic datasets offer the opportunity to expand the scope of understanding and enable the interpretation of systematic responses. While there is a wealth of data available describing neuronal proteins localized in synaptosomes as well as pre-synaptic and post-synaptic preparations, to date studies on the effects of cerebral ischemia on the synaptic proteome are limited (Costain et al. 2008). The aim of this chapter is to integrate new and existing genomic and proteomic datasets to provide a comprehensive understanding of the effect of cerebral ischemia on the function of neuronal organelles, as well as their role in mediating cell death and / or neuroprotection.

### **2. Materials and methods**

### **2.1 Animal care**

A local committee for the Canadian Council on Animal Care approved all procedures using mice. The C57B mice were purchased from Charles River Canada (St-Constant, PQ). Under temporary isoflurane anesthesia, the mice (20-23 g) were subjected to occlusion of the left middle cerebral artery (MCAO) using an intraluminal filament as previously described (Costain et al. 2008). After 1 hr of ischemia, the animals were briefly reanesthetized, the filament withdrawn and wounds sutured. After 20 hrs of reperfusion, mice were briefly anesthetized with isoflurane and the brain rapidly excised and dissected on ice.

### **2.2 Synaptosome preparation**

Contralateral (CT) and ischemic (IS) hemispheres from one mouse were manually homogenized in 2 ml of HM buffer (0.32 M sucrose, 1 mM EDTA, 0.25 mM DTT, 1 U/ml RNasin (Promega)) using a dounce homogenizer, 800 rpm 13 strokes at 4 °C. The homogenates were centrifuged (1000 g for 10 min at 4 C) and the supernatant retained. The pellet was homogenized and centrifuged (as before) a second time. The first and second supernatants were transferred to 2 ml polycarbonate tubes and centrifuged at 20,000 g for 20 min at 4 C. The resultant pellet was resuspended in 2 ml HM buffer using a dounce homogenizer. Discontinuous sucrose:percoll gradients were prepared by layering 2 ml each, in order, of 25% (percoll in HM buffer), 15%, 10% and 3% into 10 ml polycarbonate centrifuge tubes. One ml of the sample was then layered on top of a gradient and centrifuged at 32,000 g for 5 min at 4 C. Five fractions were collected following centrifugation: F1 - 3% percoll (cytoplasm), F2 - interface between 3% and 10% percoll (myelin), F3 - interface between 10% and 15% percoll (small synaptosomes, myelin & mitochondria), F4 - interphase between 15% and 25% percoll (intact synaptosomes), and F5 pellet (mitochondria). Fractions F1 - F4 were made up to 3 ml with HM buffer and centrifuged at 12,000 g for 20 min at 4 C. The supernatant was removed and the pellet washed with 1 x PBS, twice (each time spinning at 12000 x g for 10min). The final pellet was resuspended in either HM buffer or 50 mM Tris pH 8.5, 0.1% SDS. The physical and biochemical characteristics of the synaptosome preparation used here are described in further detail in Costain *et al*. (2008).

### **2.3 Protein preparation, digestion and ion exchange chromatography**

Proteins from each synaptosome sample were precipitated by adding 10 volumes of cold acetone and incubating at 1 h at -20 C followed by centrifugation at 5000g for 5 min. Pellets

The effects of cerebral ischemia are often, by necessity, described from a highly reductionist point of view, with most studies focusing on specific signaling pathways or individual molecules. Conversely, genomic and proteomic datasets offer the opportunity to expand the scope of understanding and enable the interpretation of systematic responses. While there is a wealth of data available describing neuronal proteins localized in synaptosomes as well as pre-synaptic and post-synaptic preparations, to date studies on the effects of cerebral ischemia on the synaptic proteome are limited (Costain et al. 2008). The aim of this chapter is to integrate new and existing genomic and proteomic datasets to provide a comprehensive understanding of the effect of cerebral ischemia on the function of neuronal

A local committee for the Canadian Council on Animal Care approved all procedures using mice. The C57B mice were purchased from Charles River Canada (St-Constant, PQ). Under temporary isoflurane anesthesia, the mice (20-23 g) were subjected to occlusion of the left middle cerebral artery (MCAO) using an intraluminal filament as previously described (Costain et al. 2008). After 1 hr of ischemia, the animals were briefly reanesthetized, the filament withdrawn and wounds sutured. After 20 hrs of reperfusion, mice were briefly

Contralateral (CT) and ischemic (IS) hemispheres from one mouse were manually homogenized in 2 ml of HM buffer (0.32 M sucrose, 1 mM EDTA, 0.25 mM DTT, 1 U/ml RNasin (Promega)) using a dounce homogenizer, 800 rpm 13 strokes at 4 °C. The homogenates were centrifuged (1000 g for 10 min at 4 C) and the supernatant retained. The pellet was homogenized and centrifuged (as before) a second time. The first and second supernatants were transferred to 2 ml polycarbonate tubes and centrifuged at 20,000 g for 20 min at 4 C. The resultant pellet was resuspended in 2 ml HM buffer using a dounce homogenizer. Discontinuous sucrose:percoll gradients were prepared by layering 2 ml each, in order, of 25% (percoll in HM buffer), 15%, 10% and 3% into 10 ml polycarbonate centrifuge tubes. One ml of the sample was then layered on top of a gradient and centrifuged at 32,000 g for 5 min at 4 C. Five fractions were collected following centrifugation: F1 - 3% percoll (cytoplasm), F2 - interface between 3% and 10% percoll (myelin), F3 - interface between 10% and 15% percoll (small synaptosomes, myelin & mitochondria), F4 - interphase between 15% and 25% percoll (intact synaptosomes), and F5 pellet (mitochondria). Fractions F1 - F4 were made up to 3 ml with HM buffer and centrifuged at 12,000 g for 20 min at 4 C. The supernatant was removed and the pellet washed with 1 x PBS, twice (each time spinning at 12000 x g for 10min). The final pellet was resuspended in either HM buffer or 50 mM Tris pH 8.5, 0.1% SDS. The physical and biochemical characteristics of the synaptosome preparation used here are described in

organelles, as well as their role in mediating cell death and / or neuroprotection.

anesthetized with isoflurane and the brain rapidly excised and dissected on ice.

**2.3 Protein preparation, digestion and ion exchange chromatography** 

Proteins from each synaptosome sample were precipitated by adding 10 volumes of cold acetone and incubating at 1 h at -20 C followed by centrifugation at 5000g for 5 min. Pellets

**2. Materials and methods** 

**2.2 Synaptosome preparation** 

further detail in Costain *et al*. (2008).

**2.1 Animal care** 

Fig. 2. NanoLC-MS analysis of contralateral (control) and ipsilateral (stroke) samples. Shown are images representing the nanoLC-MS data from each sample, where each spot represents a peptide ion. MatchRx software was used to extract peptide data, align the control and stroke datasets and correct retention time variations. Shown are the merged images before and after MatchRx-dependent correction. More than 5000 ions were detected per sample.

were dissolved in an appropriate volume of denaturing buffer (50 mM Tris-HCl, pH 8.5, 0.1% SDS) to a final protein concentration of 2 mg/mL. One hundred µg of each protein was transferred to a fresh tube. The proteins were reduced using dithiothrietol (4 mM, 10 min at 95 C), alkylated using iodoacetamide (10 mM, 20 min at room temperature in dark), and digested using 5 µg of MS-grade trypsin gold (Promega, 12-18 h at 37 C). The digested peptides were diluted 10-fold in 10 mM KH2PO4, pH 3.0, 25% acetonitrile and loaded onto a cation exchange column (POROS® 50 HS, 50-µm particle size, 4.0 mm x 15 mm, Applied Biosystems) for separation. Five fractions were eluted using step-gradient of 0-350 mM KCl. Each fraction was evaporated to dryness and dissolved in 5% acetronitrile, 1% acetic acid for analysis by mass spectrometry (MS).

al. 1993). The initial database utilized was a composite of forward and reverse Uniprot-Swiss-Prot *Mus musculus* protein database (Aug 2011 containing 16,390 sequences). Unmatched peptides were subsequently searched against the remaining Uniprot-Swiss-Prot database (Aug 2011 containing 531,473 sequences). Searches were performed with a specified trypsin enzymatic cleavage with one possible missed cleavage. The false-positive rates (FPR) in database searching by Mascot were calculated as described earlier (Peng et al. 2003): FPR = (2 × Nrev)/(Nrev + Nfwd), where Nrev is the number of peptides identified (after filtering) from the reverse-database, and Nfwd is the number of peptides identified (after filtering) from the forward database. To maximize the number of peptides and keep the FPR < 0.5%, ion scores > 20, parent ion tolerance of < 0.1 Da, fragment ion tolerance of < 0.2 Da, and minimal number of missed cleavages were chosen. As an independent statistical measure of peptide identification, Peptide Prophet probabilities were also measured. All identified peptides had p ≥ 0.90. The MS/MS spectrum of each differentially expressed

Fig. 4. Examples of differentially expressed proteins on nanoLC-MS images. Shown are merged images representing nanoLC-MS data of control (green image) and stroke (red image) samples. The two down-regulated peptides (green) are from 3-Oxoacid CoA Transferase 1 (Q9D0K2, Oxct1) and the four up-regulated peptides (red) are from glial high affinity glutamate transporter (P43006, Slc1a2). Peptides were identified by targeted nanoLC-MS/MS. In each image, *inset* shows a close-up image of the indicated peptide and the fold-change in stroke sample relative to control. For detailed list see **Tables 1** and **2**.

An outline of the experimental procedure is provided in **Fig. 1**. Following cerebral ischemia, the ipsilateral and contralateral hemispheres were separated and synaptosomal proteins isolated. The proteins from each sample were digested into sequenceable peptides, separated into 4 cation exchange chromatography fractions and analyzed by nanoLC-MS to quantify the level of each peptide. Each cation exchange fraction contained more than 12,000 peptide peaks as identified by nanoLC-MS analysis (**Fig. 2**). MatchRx software was thus used to identify quantitative differences in the nanoLC-MS runs between all the ipsilateral and contralateral fractions. The software extracts peptide-peak intensities and enables correction of retention time variations amongst multiple nanoLC-MS runs (**Fig. 2**), thus allowing accurate peptide alignment and quantitative comparison of the samples. Statistical

**3.1 Label-free proteomic analysis of ischemic synaptosomes** 

peptide pair was manually examined and confirmed.

**3. Results** 

Fig. 3. Scatter plot showing relative expression of peptides in stroke and control synaptosomes. Peptide intensities were extracted from nanoLC-MS runs using MatchRx and ratio-intensity plots of stroke-vs-control samples after global median normalization were plotted. Peptides showing up (red) or down (green) regulation by 1.5-fold in stroke relative to control samples and showing a *p* <0.05 were considered differentially expressed. These corresponded to ~27% of the peptides and were used for targeted identification using nanoLC-MS/MS.

### **2.4 MS analysis and protein identification**

A hybrid quadrupole time-of-flight MS (Q-TOF Ultima, Waters, Millford, MA) with an electrospray ionization source (ESI) and with an online reverse-phase nanoflow liquid chromatography column (nanoLC, 0.075 mm × 150 mm PepMap C18 capillary column, Dionex/LC-Packings, San Francisco, CA) was used for all analyses. Samples were separated on the nanoLC column using a gradient of 5-75% acetonitrile and 0.2% formic acid in 90 min, at 350 nL/min supplied by a CapLC HPLC pump (Waters). Five percent of each sample was first analyzed by nanoLC-MS in a survey (MS-only) mode for quantitation using MatchRx software as described recently (Haqqani et al. 2008). Briefly, each scan was background-subtracted, Savitzky-Golay-based smoothed, and centroided using Masslynx software v4.0 (Waters) and exported as an mzXML file (Pedrioli et al. 2004). Using MatchRx software, isotopic distribution pattern, charge state, and quantitative abundance of peptides in each nanoLC-MS run were determined. The peptides were then aligned across multiple nanoLC-MS runs through a neighboring-peak finding algorithm (Haqqani et al. 2008) followed by quantitatively comparing the levels of each peptide in the ipsilateral and contralateral fractions to identify differentially expressed peptides. Peptides showing consistent log2 fold-change of >1.5 or <-1.5 among biological replicates and showing significant difference from mean expression levels (*p* < 0.05, Wilcoxon matched pairs test) were considered differentially expressed. Images of each run were also generated to visually validate the differentially expressed peptides using MatchRx (**Fig. 4**). To sequence the differentially expressed peptides, they were included in a 'include list'. Another 5% of each sample was then re-injected into the mass spectrometer, and only the peptides included in the 'include list' were sequenced in MS/MS mode (targeted nanoLC-MS/MS). All MS/MS spectra were obtained on 2+, 3+, and 4+ ions. Peak lists were submitted to a probabilitybased search engine, Mascot version 2.2.0 (Matrix Science Ltd., London, U.K.) (Hirosawa et al. 1993). The initial database utilized was a composite of forward and reverse Uniprot-Swiss-Prot *Mus musculus* protein database (Aug 2011 containing 16,390 sequences). Unmatched peptides were subsequently searched against the remaining Uniprot-Swiss-Prot database (Aug 2011 containing 531,473 sequences). Searches were performed with a specified trypsin enzymatic cleavage with one possible missed cleavage. The false-positive rates (FPR) in database searching by Mascot were calculated as described earlier (Peng et al. 2003): FPR = (2 × Nrev)/(Nrev + Nfwd), where Nrev is the number of peptides identified (after filtering) from the reverse-database, and Nfwd is the number of peptides identified (after filtering) from the forward database. To maximize the number of peptides and keep the FPR < 0.5%, ion scores > 20, parent ion tolerance of < 0.1 Da, fragment ion tolerance of < 0.2 Da, and minimal number of missed cleavages were chosen. As an independent statistical measure of peptide identification, Peptide Prophet probabilities were also measured. All identified peptides had p ≥ 0.90. The MS/MS spectrum of each differentially expressed peptide pair was manually examined and confirmed.

Fig. 4. Examples of differentially expressed proteins on nanoLC-MS images. Shown are merged images representing nanoLC-MS data of control (green image) and stroke (red image) samples. The two down-regulated peptides (green) are from 3-Oxoacid CoA Transferase 1 (Q9D0K2, Oxct1) and the four up-regulated peptides (red) are from glial high affinity glutamate transporter (P43006, Slc1a2). Peptides were identified by targeted nanoLC-MS/MS. In each image, *inset* shows a close-up image of the indicated peptide and the fold-change in stroke sample relative to control. For detailed list see **Tables 1** and **2**.

### **3. Results**

90 Advances in the Preclinical Study of Ischemic Stroke

Fig. 3. Scatter plot showing relative expression of peptides in stroke and control

nanoLC-MS/MS.

**2.4 MS analysis and protein identification** 

synaptosomes. Peptide intensities were extracted from nanoLC-MS runs using MatchRx and ratio-intensity plots of stroke-vs-control samples after global median normalization were plotted. Peptides showing up (red) or down (green) regulation by 1.5-fold in stroke relative to control samples and showing a *p* <0.05 were considered differentially expressed. These corresponded to ~27% of the peptides and were used for targeted identification using

A hybrid quadrupole time-of-flight MS (Q-TOF Ultima, Waters, Millford, MA) with an electrospray ionization source (ESI) and with an online reverse-phase nanoflow liquid chromatography column (nanoLC, 0.075 mm × 150 mm PepMap C18 capillary column, Dionex/LC-Packings, San Francisco, CA) was used for all analyses. Samples were separated on the nanoLC column using a gradient of 5-75% acetonitrile and 0.2% formic acid in 90 min, at 350 nL/min supplied by a CapLC HPLC pump (Waters). Five percent of each sample was first analyzed by nanoLC-MS in a survey (MS-only) mode for quantitation using MatchRx software as described recently (Haqqani et al. 2008). Briefly, each scan was background-subtracted, Savitzky-Golay-based smoothed, and centroided using Masslynx software v4.0 (Waters) and exported as an mzXML file (Pedrioli et al. 2004). Using MatchRx software, isotopic distribution pattern, charge state, and quantitative abundance of peptides in each nanoLC-MS run were determined. The peptides were then aligned across multiple nanoLC-MS runs through a neighboring-peak finding algorithm (Haqqani et al. 2008) followed by quantitatively comparing the levels of each peptide in the ipsilateral and contralateral fractions to identify differentially expressed peptides. Peptides showing consistent log2 fold-change of >1.5 or <-1.5 among biological replicates and showing significant difference from mean expression levels (*p* < 0.05, Wilcoxon matched pairs test) were considered differentially expressed. Images of each run were also generated to visually validate the differentially expressed peptides using MatchRx (**Fig. 4**). To sequence the differentially expressed peptides, they were included in a 'include list'. Another 5% of each sample was then re-injected into the mass spectrometer, and only the peptides included in the 'include list' were sequenced in MS/MS mode (targeted nanoLC-MS/MS). All MS/MS spectra were obtained on 2+, 3+, and 4+ ions. Peak lists were submitted to a probabilitybased search engine, Mascot version 2.2.0 (Matrix Science Ltd., London, U.K.) (Hirosawa et

### **3.1 Label-free proteomic analysis of ischemic synaptosomes**

An outline of the experimental procedure is provided in **Fig. 1**. Following cerebral ischemia, the ipsilateral and contralateral hemispheres were separated and synaptosomal proteins isolated. The proteins from each sample were digested into sequenceable peptides, separated into 4 cation exchange chromatography fractions and analyzed by nanoLC-MS to quantify the level of each peptide. Each cation exchange fraction contained more than 12,000 peptide peaks as identified by nanoLC-MS analysis (**Fig. 2**). MatchRx software was thus used to identify quantitative differences in the nanoLC-MS runs between all the ipsilateral and contralateral fractions. The software extracts peptide-peak intensities and enables correction of retention time variations amongst multiple nanoLC-MS runs (**Fig. 2**), thus allowing accurate peptide alignment and quantitative comparison of the samples. Statistical

departures from zero, indicating that there was no correlation between expression and protein size / mascot score or error and mascot score. A runs test indicated that there was no departure from linearity in the regression analysis of error versus mascot score.

proteins in any of the analyses.

and **2**).

Furthermore, no significant differences were detected between the up and down regulated

The differentially expressed peaks were then sequenced by re-injecting each fraction and analyzing with targeted nanoLC-MS/MS using an include list containing the masses and retention times of each targeted peak. The sequenced peptides were used to generate a list of ischemia-responsive proteins. A total of 371 proteins were identified as responsive to cerebral ischemia (hereafter referred to as either the IS dataset or IS proteins) (**Tables 1** and **2**). Two-thirds (68%) of these proteins were up-regulated and the remaining were downregulated. Examples of up- and down-regulated proteins are shown in **Fig. 4**. A series of linear regression analyses were performed to ensure that the expression data was free of systematic bias. The results of the linear regression analyses indicate that the expression values were not correlated to protein mass (**Fig. 5A**) or Mascot score (**Fig. 5B**). Similarly, the measurement error was not correlated to Mascot score (**Fig. 5C**). These results indicate that

Fig. 6. (A) Venn diagram showing the number of differentially expressed proteins identified by label-free proteomics, ICAT proteomics (Costain et al. 2010) and both methods. (B) Venn diagram showing the overlap between the IS dataset using label-free proteomics (this study, 371 proteins) and the known synaptosomal proteins (1582 proteins) in the literature (Cheng et al. 2006, Morciano et al. 2005, Phillips et al. 2005, Schrimpf et al. 2005, Witzmann et al.

To validate the purity of our synaptosomal preps, the IS proteins were searched against a known collection of synaptic protein datasets from recent literature that had been identified by proteomics. More than 80% of the IS proteins were found in these datasets (**Fig. 6B**), demonstrating that our preps are consistent with other synaptosomal datasets. However, the IS proteins corresponded to only about 23% of all the known synaptic proteins (**Fig. 6B**), which is also consistent with the fact that < 30% of the peaks were found as differentially expressed (**Fig. 3**). Literature mining through PubMed search identified that 56 proteins are known to be associated with middle cerebral artery occlusion model of ischemia (**Tables 1**

2005, Jordan et al. 2004, Peng et al. 2004, Stevens et al. 2003, Li et al. 2004).

the observed expression and error values are due to biological effects.

analyses were carried out to identify differentially expressed peaks, resulting in the determination that 27% of the peaks showed differential expression (≥ 1.5 fold difference) between the two hemispheres (**Fig. 3**). As the variability between two biological synaptosomal preps was found to be about 10% (unpublished data), the observed differences were primarily attributable to the effects of cerebral ischemia and much less due to biological variability.

Fig. 5. Linear regression analysis of bias in label-free proteomics data. Expression data were plotted against protein mass and mascot score in panels A and B, respectively. Error values were plotted against mascot score in panel C. Linear regression analyses were performed on the up (red)- and down (green)-regulated proteins independently. Regression lines are plotted in panels B and C. Analyses of the regression slopes did not detect significant

analyses were carried out to identify differentially expressed peaks, resulting in the determination that 27% of the peaks showed differential expression (≥ 1.5 fold difference) between the two hemispheres (**Fig. 3**). As the variability between two biological synaptosomal preps was found to be about 10% (unpublished data), the observed differences were primarily attributable to the effects of cerebral ischemia and much less due

Fig. 5. Linear regression analysis of bias in label-free proteomics data. Expression data were plotted against protein mass and mascot score in panels A and B, respectively. Error values were plotted against mascot score in panel C. Linear regression analyses were performed on the up (red)- and down (green)-regulated proteins independently. Regression lines are plotted in panels B and C. Analyses of the regression slopes did not detect significant

to biological variability.

departures from zero, indicating that there was no correlation between expression and protein size / mascot score or error and mascot score. A runs test indicated that there was no departure from linearity in the regression analysis of error versus mascot score. Furthermore, no significant differences were detected between the up and down regulated proteins in any of the analyses.

The differentially expressed peaks were then sequenced by re-injecting each fraction and analyzing with targeted nanoLC-MS/MS using an include list containing the masses and retention times of each targeted peak. The sequenced peptides were used to generate a list of ischemia-responsive proteins. A total of 371 proteins were identified as responsive to cerebral ischemia (hereafter referred to as either the IS dataset or IS proteins) (**Tables 1** and **2**). Two-thirds (68%) of these proteins were up-regulated and the remaining were downregulated. Examples of up- and down-regulated proteins are shown in **Fig. 4**. A series of linear regression analyses were performed to ensure that the expression data was free of systematic bias. The results of the linear regression analyses indicate that the expression values were not correlated to protein mass (**Fig. 5A**) or Mascot score (**Fig. 5B**). Similarly, the measurement error was not correlated to Mascot score (**Fig. 5C**). These results indicate that the observed expression and error values are due to biological effects.

Fig. 6. (A) Venn diagram showing the number of differentially expressed proteins identified by label-free proteomics, ICAT proteomics (Costain et al. 2010) and both methods. (B) Venn diagram showing the overlap between the IS dataset using label-free proteomics (this study, 371 proteins) and the known synaptosomal proteins (1582 proteins) in the literature (Cheng et al. 2006, Morciano et al. 2005, Phillips et al. 2005, Schrimpf et al. 2005, Witzmann et al. 2005, Jordan et al. 2004, Peng et al. 2004, Stevens et al. 2003, Li et al. 2004).

To validate the purity of our synaptosomal preps, the IS proteins were searched against a known collection of synaptic protein datasets from recent literature that had been identified by proteomics. More than 80% of the IS proteins were found in these datasets (**Fig. 6B**), demonstrating that our preps are consistent with other synaptosomal datasets. However, the IS proteins corresponded to only about 23% of all the known synaptic proteins (**Fig. 6B**), which is also consistent with the fact that < 30% of the peaks were found as differentially expressed (**Fig. 3**). Literature mining through PubMed search identified that 56 proteins are known to be associated with middle cerebral artery occlusion model of ischemia (**Tables 1** and **2**).

Accession Symbol Exprs Mito synDB MCAO

P20152 Vim 1.9 ± 0.4 Yes Yes 9

Q08460 Kcnma1 1.9 ± 0.5 Yes 5

P60879 Snap25 1.9 ± 0.5 Yes 8

P14873 Map1b 1.8 ± 0.8 Yes 4 Q01279 Egfr 1.8 ± 0.6 5

P17742 Ppia 1.7 ± 0.3 Yes Yes 1

P14231 Atp1b2 1.7 ± 0.6 Yes

Q9D6M3 Slc25a22 1.7 ± 0.4 Yes Yes O55131 Sept7 1.7 ± 0.7 Yes P62761 Vsnl1 1.7 ± 0.6 Yes Q9D051 Pdhb 1.7 ± 0.3 Yes Yes P14824 Anxa6 1.6 ± 0.3 Yes Yes

E9Q6J4 Ceacam3 1.7 ± 0.4

Q9QY06 Myo9b 1.7 ± 0.3

P35803 Gpm6b 2 ± 0.8 Yes

Q61792 Lasp1 2 ± 0.8 Yes

Q99P72 Rtn4 1.9 ± 0.5 Yes P62874 Gnb1 1.9 ± 0.5 Yes Yes

Q9CPP6 Ndufa5 1.9 ± 0.3 Yes Yes

Q9CR62 Slc25a11 1.9 ± 0.3 Yes Yes Q8BVE3 Atp6v1h 1.9 ± 0.4 Yes Yes P14094 Atp1b1 1.9 ± 0.9 Yes

P18872 Gnao1 1.9 ± 0.8 Yes

Q8VDD5 Myh9 1.8 ± 0.4 Yes Q80Z24 Negr1 1.8 ± 0.5 Yes Q925N0 Sfxn5 1.8 ± 0.3 Yes Yes Q9UPR5 SLC8A2 1.8 ± 0.5 Yes Yes Q8BFR5 Tufm 1.8 ± 0.4 Yes Yes Q68FD5 Cltc 1.8 ± 0.7 Yes Yes P17426 Ap2a1 1.8 ± 0.4 Yes P12960 Cntn1 1.8 ± 0.5 Yes Q9CR68 Uqcrfs1 1.8 ± 0.3 Yes Yes Q91VD9 Ndufs1 1.8 ± 0.3 Yes Yes Q62425 Ndufa4 1.8 ± 0.2 Yes Yes Q8CAA7 Pgm2l1 1.8 ± 0.5 Yes P50516 Atp6v1a 1.8 ± 0.3 Yes Yes Q9Z2I0 Letm1 1.8 ± 0.2 Yes Yes Q96PV0 Syngap1 1.7 ± 0.5 Yes Q80TJ1 Cadps 1.7 ± 0.5 Yes

Q8VEA4 Chchd4 1.9 ± 0.5 Yes

Q9ERS2 Ndufa13 1.8 ± 0.5 Yes

Q09666 Ahnak 2 ± 0.5

Q8BIZ0 Pcdh20 1.9 ± 0.2

NP\_444473 PRSS1 1.9 ± 0.7

P57776-1 Eef1d 1.9 ± 0.4


94 Advances in the Preclinical Study of Ischemic Stroke

Accession Symbol Exprs Mito synDB MCAO

O08709 Prdx6 2.6 ± 0.6 Yes Yes 2

P16858 Gapdh 2.4 ± 0.6 Yes Yes 5

P15105 Glul 2.3 ± 0.5 Yes Yes 8

Q61207 Psap 2.3 ± 0.5 Yes Yes 3

P10637 Mapt 2.2 ± 0.7 Yes 4

P97493 Txn2 2.1 ± 0.4 Yes 9

P09671 Sod2 2 ± 0.6 Yes Yes 3

Q9Z2W1 Stk25 2 ± 0.5 1 P48318 Gad1 2 ± 0.6 Yes Yes 5 P06745 Gpi 2 ± 0.2 Yes 1

P13595 Ncam1 2 ± 0.8 Yes 6 Q8R4N0 Clybl 2 ± 0.2 Yes 2

Q9CR67 Tmem33 3.3 ± 0.5 Yes Q06185 Atp5i 3.3 ± 0.7 Yes Yes O54774 Ap3d1 3.3 ± 0.4 Yes P41216 Acsl1 2.8 ± 0.8 Yes Yes XP\_618960 2.7 ± 0.3 Yes Yes Q5NCP0 Rnf43 2.6 ± 0.9 Yes Yes

Q8BWF0 Aldh5a1 2.5 ± 0.2 Yes Yes Q64487 Ptprd 2.5 ± 0.6 Yes Q68FF7 Slain1 2.5 ± 0.5 Yes Q8K2B3 Sdha 2.4 ± 0.4 Yes Yes

P20108 Prdx3 2.4 ± 0.5 Yes Yes

Q9QYB8 Add2 2.3 ± 0.4 Yes

Q9QYA2 Tomm40 2.3 ± 0.6 Yes Yes Q8VE33 Gdap1l1 2.3 ± 0.3 Yes Yes P11881 Itpr1 2.3 ± 0.4 Yes

Q9CR61 Ndufb7 2.2 ± 0.1 Yes Yes Q8BH59 Slc25a12 2.1 ± 0.8 Yes Yes Q7TPR4 Actn1 2.1 ± 0.5 Yes Yes

Q9Z1G4 Atp6v0a1 2.1 ± 0.8 Yes

NP\_001074599 Ogdhl 2.1 ± 0.5 Yes Yes Q8BMS1 Hadha 2.1 ± 0.7 Yes Yes Q9JLZ3 Auh 2 ± 0.4 Yes Yes

Q3TRM8 Hk3 2 ± 0.4 Yes

Q60931 Vdac3 2 ± 0.8 Yes Yes Q8BMF4 Dlat 2 ± 0.6 Yes Yes

O55143 Atp2a2 2 ± 0.7 Yes Yes O08599 Stxbp1 2 ± 0.4 Yes Yes Q9DCX2 Atp5h 2 ± 0.5 Yes Yes P35486 Pdha1 2 ± 0.7 Yes Yes

P11531 Dmd 2.4 ± 0.5

Q921L8 Galnt11 2.2 ± 0.4

P42228 Stat4 2.1 ± 0.1

P81066 Irx2 2 ± 0.6


Accession Symbol Exprs Mito synDB MCAO

P54227 Stmn1 1.5 ± 0.3 Yes 3 P31648 Slc6a1 1.4 ± 0.5 Yes 1

Q9R1T4 Sept6 1.5 ± 0.3 Yes P61922 Abat 1.5 ± 0.3 Yes Yes

P06151 Ldha 1.5 ± 0.2 Yes Yes

P28652 Camk2b 1.4 ± 0.3 Yes Q61548 Snap91 1.4 ± 0.2 Yes Yes P48962 Slc25a4 1.4 ± 0.2 Yes Yes P68254 Ywhaq 1.4 ± 0.2 Yes Yes P50518 Atp6v1e1 1.4 ± 0.1 Yes Yes Q8VHW2 Cacng8 1.4 ± 0.4 Yes O55125 Nipsnap1 1.4 ± 0.3 Yes Yes O70566 Diaph2 1.4 ± 0.2 Yes Yes

Q9ES97 Rtn3 1.4 ± 0.3 Yes

Q60930 Vdac2 1.4 ± 0.5 Yes Yes Q02053 Uba1 1.4 ± 0.2 Yes

P10126 Eef1a1 1.4 ± 0.6 Yes Yes Q60932 Vdac1 1.4 ± 0.3 Yes Yes

P62835 Rap1a 1.4 ± 0.2 Yes Yes Q9D0F9 Pgm1 1.3 ± 0.2 Yes

P47753 Capza1 1.3 ± 0.4 Yes P52480 Pkm2 1.3 ± 0.4 Yes Yes Q9QZD8 Slc25a10 1.3 ± 0.4 Yes Yes

Q5SUA5 Myo1g 1.3 ± 0.2 Yes Yes Q9D0S9 Hint2 1.3 ± 0.3 Yes Yes Q62261 Sptbn1 1.3 ± 0.2 Yes Q8BVI4 Qdpr 1.3 ± 0.2 Yes Yes O70443 Gnaz 1.3 ± 0.2 Yes Yes P18760 Cfl1 1.3 ± 0.3 Yes Yes

P35802 Gpm6a 1.3 ± 0.4 Yes

O08749 Dld 1.3 ± 0.4 Yes Yes

O88737 Bsn 1.3 ± 0.3 Yes

Q9D6R2 Idh3a 1.3 ± 0.2 Yes Yes 1

P38647 Hspa9 1.3 ± 0.4 Yes Yes 1

P50114 S100b 1.4 ± 0.2 Yes 7 O08553 Dpysl2 1.4 ± 0.4 Yes Yes 2

Q9R0P9 Uchl1 1.4 ± 0.2 Yes 2

P06837 Gap43 1.4 ± 0.3 Yes 14

P62204 Calm1 1.3 ± 0.2 Yes 25

Q9EQF6 Dpysl5 1.3 ± 0.5 Yes 1

Q61206 Pafah1b2 1.5 ± 0.4

P20936 RASA1 1.4 ± 0.2

Q62315 Jarid2 1.3 ± 0.2 Q03963 Eif2ak2 1.3 ± 0.2

P70268 Pkn1 1.3 ± 0.3


96 Advances in the Preclinical Study of Ischemic Stroke

Accession Symbol Exprs Mito synDB MCAO

P63038 Hspd1 1.7 ± 0.7 Yes Yes 10

P99029 Prdx5 1.6 ± 0.5 Yes Yes 2

P23818 Gria1 1.6 ± 0.4 Yes 5

P60710 Actb 1.6 ± 0.4 Yes Yes 12 P31650 Slc6a11 1.6 ± 0.7 Yes 1

Q99KI0 Aco2 1.6 ± 0.5 Yes Yes 5

Q99104 Myo5a 1.5 ± 0.5 Yes 1

Q64133 Maoa 1.5 ± 0.3 Yes Yes 3 P97807 Fh 1.5 ± 0.2 Yes Yes 1

Q9CQ69 Uqcrq 1.5 ± 0.4 Yes Yes P46096 Syt1 1.5 ± 0.4 Yes Q8CAQ8 Immt 1.5 ± 0.3 Yes Yes

Q9Z2I9 Sucla2 1.5 ± 0.2 Yes Yes

O94925 GLS 1.5 ± 0.7 Yes Yes

Q91WF3 Adcy4 1.5 ± 0.3

Q63810 Ppp3r1 1.5 ± 0.3

Q61644 Pacsin1 1.7 ± 0.3 Yes Q9D2G2 Dlst 1.7 ± 0.3 Yes Yes Q9DBL1 Acadsb 1.7 ± 0.4 Yes Yes P17182 Eno1 1.7 ± 0.5 Yes

Q9ULD0 OGDHL 1.7 ± 0.3 Yes Yes Q8BKZ9 Pdhx 1.6 ± 0.3 Yes Yes P62748 Hpcal1 1.6 ± 0.2 Yes P14211 Calr 1.6 ± 0.4 Yes

P40142 Tkt 1.6 ± 0.3 Yes

Q8VEM8 Slc25a3 1.6 ± 0.4 Yes Yes

O35526 Stx1a 1.6 ± 0.5 Yes P51830 Adcy9 1.6 ± 0.4 Yes

P53994 Rab2a 1.6 ± 0.4 Yes Yes

Q9Z2Q6 Sept5 1.6 ± 0.7 Yes Yes

P46460 Nsf 1.6 ± 0.5 Yes

Q8R404 Qil1 1.6 ± 0.1 Yes Yes O09111 Ndufb11 1.5 ± 0.7 Yes Yes Q9DC69 Ndufa9 1.5 ± 0.3 Yes Yes O88741 Gdap1 1.5 ± 0.5 Yes P07477 PRSS1 1.5 ± 0.4 Yes P17751 Tpi1 1.5 ± 0.3 Yes Yes Q91V61 Sfxn3 1.5 ± 0.3 Yes Yes P57780 Actn4 1.5 ± 0.5 Yes Yes P97300 Nptn 1.5 ± 0.4 Yes Q99LC3 Ndufa10 1.5 ± 0.1 Yes Yes P63017 Hspa8 1.5 ± 0.5 Yes Yes

Q6IFX4 Krt39 1.6 ± 0.5 Yes

P62482 Kcnab2 1.6 ± 0.5

XP\_927453 1.6 ± 0.2


Accession Symbol Exprs Mito synDB MCAO

P08228 Sod1 0.9 ± 0.2 Yes Yes 10

Q62277 Syp 0.9 ± 0.2 Yes 30

Q8K183 Pdxk 0.8 ± 0.2 2

P0CG49 Ubb 0.7 ± 0 Yes Yes 17

P05202 Got2 0.3 ± 1.6 Yes Yes 1 Table 1. List of up-regulated proteins in the IS dataset. The expression (Exprs) of each proteins is provided (mean ± SD, n = 3), as well as their presence in the mitochondria (Mito) or synapse (SynDB). The number of citations in PubMed that have associated each proteins

To further characterize the IS dataset, the proteins were categorized into biological processes and subcellular localizations using a combination of Panther, Uniprot and other datasets (Taylor et al. 2003). Categorization by biological processes showed that the majority of the IS proteins are involved in transport, signal transduction, intracellular trafficking and carbohydrate metabolism (**Fig. 7A**). Additionally, up-regulated proteins were involved in the processes of immunity/defense, cell adhesion and neurogenesis. Subcellular classification mainly categorized the proteins into mitochondrial, cell membrane, cytoplasmic and membrane/cytoplasmic localizations (**Fig. 7B**). Consistent with our previous report (Costain et al. 2010), approximately half (51%) of the IS dataset were mitochondrial proteins (**Fig. 7B**), whereas < 25% of known synaptosomal proteins are mitochondrial. These changes were mainly due to mitochondrial oxidoreductases involved in electron transport and proteins involved in the TCA cycle, suggesting severe deficits in the capacity of the mitochondria to produce energy. About 20% of the identified ischemic

Q9H4G0 EPB41L1 0.9 ± 0.2 Yes

P62137 Ppp1ca 0.9 ± 0.3 Yes

Q9QZ83 Actg1 0.9 ± 0.2 Yes Yes

P08556 Nras 0.8 ± 0.2 Yes Yes

Q9DCS9 Ndufb10 0.8 ± 0.1 Yes Yes Q62420 Sh3gl2 0.8 ± 0.2 Yes

Q9CWZ7 Napg 0.7 ± 0.1 Yes Yes Q99JY0 Hadhb 0.7 ± 0.1 Yes Yes Q8K0T0 Rtn1 0.7 ± 0.2 Yes

P84091 Ap2m1 0.7 ± 0.1 Yes Yes

Q76MZ3 Ppp2r1a 0.6 ± 0 Yes Yes Q8BGZ1 Hpcal4 0.6 ± 0.2 Yes

P48771 Cox7a2 0.8 ± 0.3 Yes

Q6GQS1 Slc25a23 0.8 ± 0.3 Yes

A8E4K7 Pcdhb8 0.8 ± 0.1

Q61194 Pik3c2a 0.8 ± 0.3

O35728 Cyp4a14 0.8 ± 0.1

P70335 Rock1 0.7 ± 0.2 O09112 Dusp8 0.7 ± 0.1 Q3UK37 0.7 ± 0.1 Q8BLF1 Nceh1 0.7 ± 0.2

B9EJA4 Clasp2 0.6 ± 0.1

with stoke (MCAO) are also provided.



Accession Symbol Exprs Mito synDB MCAO

Q61879 Myh10 1.3 ± 0.3 Yes P62814 Atp6v1b2 1.3 ± 0.2 Yes P07901 Hsp90aa1 1.3 ± 0.3 Yes Yes P63328 Ppp3ca 1.3 ± 0.3 Yes Yes P56382 Atp5e 1.2 ± 0.2 Yes Yes P99028 Uqcrh 1.2 ± 0 Yes Yes P05201 Got1 1.2 ± 0.2 Yes Yes P16277 Blk 1.2 ± 0.3 Yes Q9CQA3 Sdhb 1.2 ± 0.3 Yes Yes

Q9WV55 Vapa 1.2 ± 0.3 Yes P28663 Napb 1.2 ± 0.2 Yes O35658 C1qbp 1.1 ± 0.2 Yes Yes Q9CZW5 Tomm70a 1.1 ± 0.2 Yes Yes

Q80XN0 Bdh1 1.1 ± 0.2 Yes Yes

Q8K314 Atp2b1 1.1 ± 0.3 Yes Q640R3 Hepacam 1.1 ± 0.1 Yes Q5SWU9 Acaca 1.1 ± 0.1 Yes Yes

Q9JI46 Nudt3 1 ± 0.2 Yes Q6PCP5 Mff 1 ± 0.1 Yes Yes

Q80Y86 Mapk15 1 ± 0.2 Yes Q9CQ54 Ndufc2 1 ± 0.2 Yes Yes P00405 Mtco2 1 ± 0.3 Yes Yes

Q9R111 Gda 1 ± 0.2 Yes Q9CQI3 Gmfb 0.9 ± 0.3 Yes

Q8BM92 Cdh7 0.9 ± 0.1 Yes P56564 Slc1a3 0.8 ± 0.2 Yes Yes

Q9Z0J4 Nos1 1.1 ± 0.3 Yes Yes 66

P34884 Mif 1.1 ± 0.2 Yes 3

Q00690 Sele 1.1 ± 0 26

P43006 Slc1a2 1 ± 0.2 Yes 10

P48320 Gad2 0.9 ± 0.1 Yes 1 Q61735 Cd47 0.9 ± 0.1 1

P07146 Prss2 1.2 ± 0.2

A2AJ76 Hmcn2 1 ± 0.3

O70283 Wnt2b 1 ± 0.2 P70295 Aup1 1 ± 0.2

P63082 Atp6v0c 1 ± 0.2

XP\_922613 Spnb5 1 ± 0.3

XP\_922643 0.9 ± 0.1

NP\_082646 Pot1b 0.9 ± 0.2 Q9JKR6 Hyou1 0.9 ± 0.2

Q9D8W7 Ociad2 1 ± 0.2 Yes

O35857 Timm44 0.9 ± 0.2 Yes

O54983 Crym 0.9 ± 0.1 Yes

Q9CQH3 Ndufb5 0.9 ± 0.2 Yes


Table 1. List of up-regulated proteins in the IS dataset. The expression (Exprs) of each proteins is provided (mean ± SD, n = 3), as well as their presence in the mitochondria (Mito) or synapse (SynDB). The number of citations in PubMed that have associated each proteins with stoke (MCAO) are also provided.

To further characterize the IS dataset, the proteins were categorized into biological processes and subcellular localizations using a combination of Panther, Uniprot and other datasets (Taylor et al. 2003). Categorization by biological processes showed that the majority of the IS proteins are involved in transport, signal transduction, intracellular trafficking and carbohydrate metabolism (**Fig. 7A**). Additionally, up-regulated proteins were involved in the processes of immunity/defense, cell adhesion and neurogenesis. Subcellular classification mainly categorized the proteins into mitochondrial, cell membrane, cytoplasmic and membrane/cytoplasmic localizations (**Fig. 7B**). Consistent with our previous report (Costain et al. 2010), approximately half (51%) of the IS dataset were mitochondrial proteins (**Fig. 7B**), whereas < 25% of known synaptosomal proteins are mitochondrial. These changes were mainly due to mitochondrial oxidoreductases involved in electron transport and proteins involved in the TCA cycle, suggesting severe deficits in the capacity of the mitochondria to produce energy. About 20% of the identified ischemic

Accession Symbol Exprs Mito SynDB MCAO

Q9DBF1 Aldh7a1 -1.6 ± 0.4 Yes Yes 8

Q2EMV9 Parp14 -1.5 ± 0.3 Yes Yes 1

BAE40217 Tubb5 -1.3 ± 0.3 Yes 5

P17427 Ap2a2 -1.6 ± 0.3 Yes Yes

P39053 Dnm1 -1.5 ± 0.2 Yes Yes P70404 Idh3g -1.5 ± 0.1 Yes Yes P99024 Tubb5 -1.5 ± 0.3 Yes Yes P12787 Cox5a -1.5 ± 0.3 Yes Yes Q8CI94 Pygb -1.5 ± 0.6 Yes XP\_001006010 -1.5 ± 0.1 Yes Yes Q9QXV0 Pcsk1n -1.5 ± 0.3 Yes P68368 Tuba4a -1.5 ± 0.3 Yes

P05064 Aldoa -1.5 ± 0.3 Yes NP\_031573 Sirpa -1.4 ± 0.4 Yes O88342 Wdr1 -1.4 ± 0.4 Yes Yes Q9D0M3 Cyc1 -1.4 ± 0.2 Yes

Q69ZK9 Nlgn2 -1.4 ± 0.2 Yes P56480 Atp5b -1.4 ± 0.2 Yes Q8CIV2 ORF61 -1.4 ± 0.3 Yes Yes Q3UM45 Ppp1r7 -1.4 ± 0.5 Yes Yes O55100 Syngr1 -1.4 ± 0.2 Yes Yes P97797 Sirpa -1.3 ± 0.3 Yes Yes

AAA40509 Tubb4 -1.3 ± 0.4 Yes

Q8CHC4 Synj1 -1.3 ± 0.4 Yes Yes P14152 Mdh1 -1.3 ± 0.2 Yes Yes

P23116 Eif3a -1.2 ± 0.1 Yes O43837 IDH3B -1.2 ± 0.4 Yes Yes Q9CZU6 Cs -1.2 ± 0.3 Yes XP\_889898 -1.1 ± 0.3 Yes Yes Q6NXI6 Rprd2 -1.1 ± 0.2 Yes Yes NP\_001013813 Gm5468 -1.1 ± 0.1 Yes Yes P11627 L1cam -1.1 ± 0.3 Yes Yes O77784 IDH3B -1.1 ± 0.4 Yes Yes Q9CPU4 Mgst3 -1.1 ± 0.1 Yes Yes Q8R570 Snap47 -1.1 ± 0.3 Yes Q8TCB6 OR51E1 -1.1 ± 0.2 Yes NP\_082221 Csl -1.1 ± 0.4 Yes Yes Q9WUM4 Coro1c -1.1 ± 0.1 Yes Yes P10649 Gstm1 -1.1 ± 0.2 Yes

Q9DB20 Atp5o -1 ± 0.2 Yes

Q9QWI6 Srcin1 -1.4 ± 0.5

P51174 Acadl -1.3 ± 0.3 Q9CZ13 Uqcrc1 -1.3 ± 0.4

Q9WUM5 Suclg1 -1.3 ± 0.3

P17710 Hk1 -1.2 ± 0.2


100 Advances in the Preclinical Study of Ischemic Stroke

Accession Symbol Exprs Mito SynDB MCAO

Q920I9 Wdr7 -3.3 ± 0.2 Yes Q8R1B4 Eif3c -3.3 ± 1.3 Yes P42356 PI4KA -2.9 ± 0.6 Yes Q8R071 Itpka -2.7 ± 0.5 Yes

P19783 Cox4i1 -2.3 ± 0.8 Yes Q4U256 Ank3 -2.2 ± 0.6 Yes Q9DBG3 Ap2b1 -2.2 ± 0.5 Yes Yes

Q3V1U8 Elmod1 -2.1 ± 0.5 Yes Yes P58281 Opa1 -2.1 ± 0.3 Yes Q64516 Gyk -2.1 ± 0.4 Yes

P54285 Cacnb3 -2 ± 0.7 Yes Yes Q8VD37 Sgip1 -2 ± 0.5 Yes P49615 Cdk5 -2 ± 0.4 Yes Yes

Q9DB77 Uqcrc2 -2 ± 0.6 Yes Q9DCT2 Ndufs3 -2 ± 0.3 Yes Yes P21803 Fgfr2 -1.9 ± 0 Yes Q7TQD2 Tppp -1.9 ± 0.5 Yes Yes Q64521 Gpd2 -1.9 ± 0.6 Yes Yes P08249 Mdh2 -1.9 ± 0.2 Yes Q91WS0 Cisd1 -1.9 ± 0.7 Yes

Q03265 Atp5a1 -1.9 ± 0.2 Yes Yes

Q04447 Ckb -1.8 ± 0.3 Yes Yes P61161 Actr2 -1.8 ± 0.2 Yes Yes

Q9JHU4 Dync1h1 -1.7 ± 0.3 Yes Yes Q91VR2 Atp5c1 -1.7 ± 0.4 Yes Yes P14618 PKM2 -1.7 ± 0.5 Yes

Q9D0K2 Oxct1 -1.6 ± 0.5 Yes Yes Q61330 Cntn2 -1.6 ± 0.7 Yes Yes Q91XV3 Basp1 -1.6 ± 0.5 Yes Q3V1L4 Nt5c2 -1.6 ± 0.3 Yes Yes

P61982 Ywhag -1.6 ± 0.3 Yes Yes Q80ZF8 Bai3 -1.6 ± 0.4 Yes

Q9CPQ1 Cox6c -1.8 ± 0.5 Yes

Q9D6J6 Ndufv2 -1.8 ± 0.5 Yes

Q62159 Rhoc -2 ± 0.6 Yes Yes 2

P67778 Phb -2.3 ± 0.4 Yes

P51881 Slc25a5 -2.1 ± 0.5 Yes

Q99LY9 Ndufs5 -2 ± 0.6

Q5DU25 Iqsec2 -1.9 ± 0.4

Q5NVN0 PKM2 -1.8 ± 0.4 Q6PIC6 Atp1a3 -1.7 ± 0.4 Q8QZT1 Acat1 -1.7 ± 0.6 O08539 Bin1 -1.7 ± 0.7

O88935 Syn1 -1.6 ± 0.4

Q8BG39 Sv2b -1.6 ± 0.4


**A B** 

Fig. 7. Classification of differentially expressed proteins after targeted identification using nanoLC-MS/MS. (A) Protein classification by biological processes using the Panther classification system. (B) Protein classification by subcellular localizations using Panther,

Graphical and statistical analyses of the Gene Ontology (GO) annotations were performed using the BiNGO 2.4.4 plugin for Cytoscape 2.8.2. This analysis involves gathering the GO meta data associated with the IS proteins, and determining if any annotations are statistically over-represented for each annotation category. BiNGO makes use of the hierarchical structure of the GO database to produce a graphical representation of the significantly over-represented ontologies, enabling the depiction of the parent-child relationships of the annotations. **Fig. 8** is the graphical representation of the relative positions of the terms in the GO hierarchy, with the degree of significance indicated according to color (white nodes are not significant). The size of each node (area) is proportional to the number of proteins annotated to each node. The major, significant *cellular component* ontologies (**Fig. 8A**) are cytoplasm, mitochondrion, cytoskeleton, plasma membrane, cytoplasmic membrane-bounded vesicle, Golgi apparatus, endoplasmic reticulum and vacuole. This list indicates that cerebral ischemia induces dramatic alterations in the proteomes of organelles that are involved in regulating cell death/survival processes. This is further supported by the *molecular function* ontologies (**Fig. 8C**) that are significantly enriched in terms such as kinase activity, transporter activity, antioxidant activity, electron carrier activity and nucleotide binding. Additionally, the enriched *biological process* ontologies (**Fig. 8B**) are dominated by a variety of metabolic processes, as well as transport, cellular component organization,

Uniprot and a dataset from Taylor et al. (2003).

**3.2 Statistical analysis of Gene Ontology meta data** 


Table 2. List of down-regulated proteins in the IS dataset. The expression (Exprs) of each proteins is provided (mean ± SD, n = 3), as well as their presence in the mitochondria (Mito) or synapse (SynDB). The number of citations in PubMed that have associated each proteins with stoke (MCAO) are also provided.

proteins were classified as membrane/cytoplasmic, i.e., they exist in both cell membranes and cytoplasm. These 73 proteins consist of membrane trafficking proteins, transfer/carrier proteins, calcium-binding proteins, cytoskeletal proteins and transporters and are known to be involved in the processes of vesicle trafficking and synaptic transmission. Of the remaining identified ischemic proteins, 11% were classified as membrane-only and 9% as cytoplasm-only (**Fig. 7B**). The membrane-only proteins consist of transporters, adhesion molecules and cytokine receptors, which are known to be involved in the processes of cell adhesion, cell communication, signal transduction, neurogenesis and transport. The cytoplasm-only proteins consist of cytoskeletal proteins, enzymes (e.g., hydrolases, kinases, phosphatases) and G-proteins and are involved in the processes of signal transduction, cell structure maintenance and cell motility.

Fig. 7. Classification of differentially expressed proteins after targeted identification using nanoLC-MS/MS. (A) Protein classification by biological processes using the Panther classification system. (B) Protein classification by subcellular localizations using Panther, Uniprot and a dataset from Taylor et al. (2003).

### **3.2 Statistical analysis of Gene Ontology meta data**

102 Advances in the Preclinical Study of Ischemic Stroke

Accession Symbol Exprs Mito SynDB MCAO

Q8BK30 Ndufv3 -1 ± 0.2 Yes 1

P30275 Ckmt1 -1 ± 0.2 Yes Yes 1

Q3TMW1 Ccdc102a -0.9 ± 0.2 Yes 4

Q8BH44 Coro2b -0.7 ± 0.1 Yes Yes 6

Table 2. List of down-regulated proteins in the IS dataset. The expression (Exprs) of each proteins is provided (mean ± SD, n = 3), as well as their presence in the mitochondria (Mito) or synapse (SynDB). The number of citations in PubMed that have associated each proteins

proteins were classified as membrane/cytoplasmic, i.e., they exist in both cell membranes and cytoplasm. These 73 proteins consist of membrane trafficking proteins, transfer/carrier proteins, calcium-binding proteins, cytoskeletal proteins and transporters and are known to be involved in the processes of vesicle trafficking and synaptic transmission. Of the remaining identified ischemic proteins, 11% were classified as membrane-only and 9% as cytoplasm-only (**Fig. 7B**). The membrane-only proteins consist of transporters, adhesion molecules and cytokine receptors, which are known to be involved in the processes of cell adhesion, cell communication, signal transduction, neurogenesis and transport. The cytoplasm-only proteins consist of cytoskeletal proteins, enzymes (e.g., hydrolases, kinases, phosphatases) and G-proteins and are involved in the processes of signal transduction, cell

Q9R0N5 Syt5 -1 ± 0.3 Yes Yes Q9QXY2 Srcin1 -1 ± 0.2 Yes Q9CQY6 LOC675054 -1 ± 0.2 Yes Yes Q3TC72 Fahd2 -1 ± 0.4 Yes Yes

Q9EQ20 Aldh6a1 -1 ± 0.3 Yes

Q8CHU3 Epn2 -1 ± 0.3 Yes Yes O35643 Ap1b1 -0.9 ± 0.2 Yes Yes O35874 Slc1a4 -0.9 ± 0.2 Yes P56391 Cox6b1 -0.9 ± 0.1 Yes Yes A2AGT5 Ckap5 -0.9 ± 0.2 Yes Yes P19536 Cox5b -0.9 ± 0.1 Yes Q60597 Ogdh -0.9 ± 0.3 Yes

NP\_570954 IDH3B -0.9 ± 0.4 Yes P61205 Arf3 -0.9 ± 0.2 Yes Yes Q9DC70 Ndufs7 -0.9 ± 0.3 Yes Yes Q61124 Cln3 -0.8 ± 0.2 Yes Yes

P63040 Cplx1 -0.8 ± 0.1 Yes Yes

Q9CXZ1 Ndufs4 -0.7 ± 0.1 Yes P49025 Cit -0.7 ± 0.1 Yes Yes Q7TQF7 Amph -0.7 ± 0.2 Yes Yes P48678 Lmna -0.7 ± 0.1 Yes Yes O89053 Coro1a -0.6 ± 0.1 Yes Q8BUV3 Gphn -0.6 ± 0.1 Yes

Q99LC5 Etfa -0.8 ± 0.1

P35831 Ptpn12 -0.6 ± 0.1

with stoke (MCAO) are also provided.

structure maintenance and cell motility.

Graphical and statistical analyses of the Gene Ontology (GO) annotations were performed using the BiNGO 2.4.4 plugin for Cytoscape 2.8.2. This analysis involves gathering the GO meta data associated with the IS proteins, and determining if any annotations are statistically over-represented for each annotation category. BiNGO makes use of the hierarchical structure of the GO database to produce a graphical representation of the significantly over-represented ontologies, enabling the depiction of the parent-child relationships of the annotations. **Fig. 8** is the graphical representation of the relative positions of the terms in the GO hierarchy, with the degree of significance indicated according to color (white nodes are not significant). The size of each node (area) is proportional to the number of proteins annotated to each node. The major, significant *cellular component* ontologies (**Fig. 8A**) are cytoplasm, mitochondrion, cytoskeleton, plasma membrane, cytoplasmic membrane-bounded vesicle, Golgi apparatus, endoplasmic reticulum and vacuole. This list indicates that cerebral ischemia induces dramatic alterations in the proteomes of organelles that are involved in regulating cell death/survival processes. This is further supported by the *molecular function* ontologies (**Fig. 8C**) that are significantly enriched in terms such as kinase activity, transporter activity, antioxidant activity, electron carrier activity and nucleotide binding. Additionally, the enriched *biological process* ontologies (**Fig. 8B**) are dominated by a variety of metabolic processes, as well as transport, cellular component organization,

Fig. 9. Identification of therapeutic targets using a network of interactions between the IS dataset and key proteins involved in cell death mechanisms. Networks were constructed with MiMI from regulators of cell death pathways for apoptosis (Map3k14, Endog, Aifm1, Apaf1, Cycs, Capn1, Casp8, Casp10), autophagy (Aup1, Map1lc3a, Atg3, Atg5, Atg7, Atg10, Atg12), necroptosis (Aifm1, Aifm2, Parp1, Ripk1) and ER stress (ERAD; Ern1, Ern2, Hspa5, Atf6, Eif2ak3). These networks were joined with an ischemia network constructed of interacting proteins in the IS dataset (diamonds). The expression of the ischemic synaptic proteins is mapped onto the nodes according to the scale provided, with red and green representing up- and down-regulation, respectively. Key proteins (highlighted with bold node borders) for joining the cell death networks to the ischemia network (Hsp90aa1, Tuba4a, Vim) as well as highly integrated proteins

(Ywhag and Taf1) were identified.

cellular homeostasis, response to stress and response to external stimulus. Taken together, the gene ontology information paints a picture of a dynamic proteome that is responding to highly stressful injury.

Fig. 8. Statistical analysis of Gene Ontology meta data. Statistical analysis of the Gene Ontology classification of the IS dataset was performed using the BiNGO plugin in Cytoscape. Graphical representations of significantly enriched ontologies are presented for cellular component (A), biological process (B) and molecular function (C).

cellular homeostasis, response to stress and response to external stimulus. Taken together, the gene ontology information paints a picture of a dynamic proteome that is responding

Fig. 8. Statistical analysis of Gene Ontology meta data. Statistical analysis of the Gene Ontology classification of the IS dataset was performed using the BiNGO plugin in

cellular component (A), biological process (B) and molecular function (C).

Cytoscape. Graphical representations of significantly enriched ontologies are presented for

to highly stressful injury.

Fig. 9. Identification of therapeutic targets using a network of interactions between the IS dataset and key proteins involved in cell death mechanisms. Networks were constructed with MiMI from regulators of cell death pathways for apoptosis (Map3k14, Endog, Aifm1, Apaf1, Cycs, Capn1, Casp8, Casp10), autophagy (Aup1, Map1lc3a, Atg3, Atg5, Atg7, Atg10, Atg12), necroptosis (Aifm1, Aifm2, Parp1, Ripk1) and ER stress (ERAD; Ern1, Ern2, Hspa5, Atf6, Eif2ak3). These networks were joined with an ischemia network constructed of interacting proteins in the IS dataset (diamonds). The expression of the ischemic synaptic proteins is mapped onto the nodes according to the scale provided, with red and green representing up- and down-regulation, respectively. Key proteins (highlighted with bold node borders) for joining the cell death networks to the ischemia network (Hsp90aa1, Tuba4a, Vim) as well as highly integrated proteins (Ywhag and Taf1) were identified.

nanoLC-MS per sample >12,000 <4,000 *p<0.0001* 

Peptides per protein 3.9 1.2 *p<0.0001*  Protein coverage (%) 15% 4.4% *p<0.0001*  Peptide scores 71 32 *p<0.0001* 

In an effort to identify proteins that are important in mediating ischemia-induced cell death, interaction networks were constructed using the MiMI plugin in Cytoscape. Key proteins involved in initiating the four major active cell death mechanisms were identified and stringent interaction networks were individually constructed. Additionally, a network of interactions among the proteins in the IS dataset was constructed, which resulted in the generation of a network consisting of 203 proteins (IS network). The cell death networks were merged with the IS network, resulting in the identification of a number of proteins that are likely to be important mediators of synaptic pathology and cell death (**Fig. 9**). This analysis revealed a strong degree of association between apoptosis and necroptosis, but little direct interaction with autophagy. In comparison, ER associated degradation (ERAD) was associated with both necroptosis and autophagy. **Fig. 9** indicates that the IS network is connected to apoptosis primarily through the vimentin (Vim) protein, whereas the necroptosis and ERAD networks are connected to the IS network in large part due to Hsp90aa1. In comparison, the autophagy network was connected to the IS network through Tuba4a. Additionally, the 14-3-3 protein Ywhag was revealed to be a highly connected protein within the IS network and the transcription factor Taf1 is a point of convergence for

As mentioned, many of the IS proteins are involved in a group of activities that are of obvious importance to cellular metabolism and signaling. These functionalities are highly important in maintaining cellular homeostasis as well as in deciding cellular fate during injurious conditions. **Fig. 10** is a graphical representation of the interactions among the proteins involved in the process of oxidative phosphorylation. The proteins segregated into three groups with related functions: cytochrome-C oxidase activity, NADH dehydrogenase (ubiquinone) activity and hydrogen ion transmembrane transporter activity. The expression of the proteins reveals that the Cox proteins were universally down-regulated, whereas proteins in the other groups exhibited more varied effects. **Fig. 11A** is a network of the proteins involved in glycolysis – gluconeogenesis. The figure demonstrates that there is widespread up-regulation of glycolytic proteins, with down-regulation of only two proteins. Similarly, **Fig. 11B** indicates that there is widespread up-regulation of proteins with antioxidant activity. Interestingly, two of the proteins identified in **Fig. 9**, Hsp90aa1 and Taf1, are also involved in the antioxidant response. The results depicted in **Fig. 11** clearly demonstrate that the ischemic synaptosomal proteome is actively engaged in an attempt to counteract the effects of cerebral ischemia, namely energy depletion and oxidative damage.

Quantitative reproducibility (mean coefficient of variance) 10% 9% *ns* 

Total unique proteins identified 371 41

Number of cysteine-free proteins 13 N/A Table 3. Statistical comparison of label-free and ICAT based nanoLC-MS proteomics.

Mean number of ions detected by

**3.4 Protein interaction analysis** 

the ERAD, autophagy and necroptosis pathways.

Label-free ICAT *t-test* 

### **3.3 Comparison with ischemic-responsive proteins identified by ICAT**

We recently identified 41 ischemia-responsive synaptosomal proteins at 20 h using ICATbased nanoLC-MS/MS proteomics. While the ICAT-based method exhibited a high quantitative reproducibility, quantitative accuracy, and a wide dynamic range (Costain et al. 2010), it did not provide a very comprehensive analysis of the proteins. A comparison between the label-free and ICAT methods is shown in **Table 3**. While both methods have high quantitative reproducibility, the label-free method identified about 5-times more proteins and peptides in a synaptosome prep than the ICAT method. In addition, the number of peptides per protein and proteome coverage was significantly higher using the label-free method. The label-free method also showed higher peptide scores and identified several cysteine-free proteins. The label-free method identified 32 of the proteins that were also identified by ICAT (**Fig. 6A**) and the majority (80%) of their expressions values (i.e., fold change in response to MCAO) were in agreement between the two methods. Thus, while both methods are quantitatively comparable, the label-free approach provided a much more comprehensive coverage of the proteome and addressed some of the limitations of ICAT, including the detection of cysteine-free proteins.

Fig. 10. Cerebral ischemia disrupts synaptosomal oxidative phosphorylation. An interaction network was constructed from proteins involved in oxidative phosphorylation using MiMI. The network segregated into proteins from 3 groups: complex I (NADH dehydrogenase (ubiquinone), complex IV (cytochrome-C oxidase) and complex V (ATP synthesis).


Table 3. Statistical comparison of label-free and ICAT based nanoLC-MS proteomics.

### **3.4 Protein interaction analysis**

106 Advances in the Preclinical Study of Ischemic Stroke

We recently identified 41 ischemia-responsive synaptosomal proteins at 20 h using ICATbased nanoLC-MS/MS proteomics. While the ICAT-based method exhibited a high quantitative reproducibility, quantitative accuracy, and a wide dynamic range (Costain et al. 2010), it did not provide a very comprehensive analysis of the proteins. A comparison between the label-free and ICAT methods is shown in **Table 3**. While both methods have high quantitative reproducibility, the label-free method identified about 5-times more proteins and peptides in a synaptosome prep than the ICAT method. In addition, the number of peptides per protein and proteome coverage was significantly higher using the label-free method. The label-free method also showed higher peptide scores and identified several cysteine-free proteins. The label-free method identified 32 of the proteins that were also identified by ICAT (**Fig. 6A**) and the majority (80%) of their expressions values (i.e., fold change in response to MCAO) were in agreement between the two methods. Thus, while both methods are quantitatively comparable, the label-free approach provided a much more comprehensive coverage of the proteome and addressed some of the limitations of ICAT,

Fig. 10. Cerebral ischemia disrupts synaptosomal oxidative phosphorylation. An interaction network was constructed from proteins involved in oxidative phosphorylation using MiMI. The network segregated into proteins from 3 groups: complex I (NADH dehydrogenase (ubiquinone), complex IV (cytochrome-C oxidase) and complex V (ATP synthesis).

**3.3 Comparison with ischemic-responsive proteins identified by ICAT** 

including the detection of cysteine-free proteins.

In an effort to identify proteins that are important in mediating ischemia-induced cell death, interaction networks were constructed using the MiMI plugin in Cytoscape. Key proteins involved in initiating the four major active cell death mechanisms were identified and stringent interaction networks were individually constructed. Additionally, a network of interactions among the proteins in the IS dataset was constructed, which resulted in the generation of a network consisting of 203 proteins (IS network). The cell death networks were merged with the IS network, resulting in the identification of a number of proteins that are likely to be important mediators of synaptic pathology and cell death (**Fig. 9**). This analysis revealed a strong degree of association between apoptosis and necroptosis, but little direct interaction with autophagy. In comparison, ER associated degradation (ERAD) was associated with both necroptosis and autophagy. **Fig. 9** indicates that the IS network is connected to apoptosis primarily through the vimentin (Vim) protein, whereas the necroptosis and ERAD networks are connected to the IS network in large part due to Hsp90aa1. In comparison, the autophagy network was connected to the IS network through Tuba4a. Additionally, the 14-3-3 protein Ywhag was revealed to be a highly connected protein within the IS network and the transcription factor Taf1 is a point of convergence for the ERAD, autophagy and necroptosis pathways.

As mentioned, many of the IS proteins are involved in a group of activities that are of obvious importance to cellular metabolism and signaling. These functionalities are highly important in maintaining cellular homeostasis as well as in deciding cellular fate during injurious conditions. **Fig. 10** is a graphical representation of the interactions among the proteins involved in the process of oxidative phosphorylation. The proteins segregated into three groups with related functions: cytochrome-C oxidase activity, NADH dehydrogenase (ubiquinone) activity and hydrogen ion transmembrane transporter activity. The expression of the proteins reveals that the Cox proteins were universally down-regulated, whereas proteins in the other groups exhibited more varied effects. **Fig. 11A** is a network of the proteins involved in glycolysis – gluconeogenesis. The figure demonstrates that there is widespread up-regulation of glycolytic proteins, with down-regulation of only two proteins. Similarly, **Fig. 11B** indicates that there is widespread up-regulation of proteins with antioxidant activity. Interestingly, two of the proteins identified in **Fig. 9**, Hsp90aa1 and Taf1, are also involved in the antioxidant response. The results depicted in **Fig. 11** clearly demonstrate that the ischemic synaptosomal proteome is actively engaged in an attempt to counteract the effects of cerebral ischemia, namely energy depletion and oxidative damage.

Fig. 12. Interaction network of ischemia regulated protein kinases. The network shows the regulated kinases (diamonds) interact with a subset of 16 other proteins in the IS dataset.

Actively regulated cell death processes rely on a variety of cellular signaling cascades, typically involving specific protein kinases. We found that the expression of 24 protein kinases were altered in post-ischemic synaptosomes and constructed an interaction network from this subset of proteins (**Fig. 12**). The resultant kinase network included 17 of the 24 kinases as well as 16 non-kinase synaptosomal proteins that were in the IS dataset. Interestingly, three of the key proteins identified in **Fig. 9** were present in the kinase network (Hsp90aa1, Vim and Taf1), supporting a broad role for these proteins in ischemic pathology. Interestingly, a group of proteins constituting the pyruvate dehydroxylase complex (Pdhb, Pdha1, Pdhx, Dld and Dlat), which links glycolysis and the tricarboxylic acid (TCA) cycle, were up-regulated, while citrate synthase (Cs) was down-regulated. The figure confirms that a wide variety of signaling pathways are affected by cerebral ischemia.

The aim of this study is to characterize the effects of cerebral ischemia on the synaptic proteome and to examine the functional consequences of these alterations with particular emphasis on their relevance to neuronal cell death processes. As outlined, cellular protein levels are determined by the balance between synthesis and degradation, which is affected by a multitude of factors including: transcription, translation, proteases activity, and posttranslational modifications. Ischemic conditions complicate the matter by causing dysfunction in the proteosome (DeGracia et al. 2002) and endoplasmic reticulum (Ge et al. 2007). Thus, gene expression data alone is a poor indicator of post-ischemic cellular protein levels. Furthermore focusing on the synapse adds the prospect of intracellular transport

**4. Discussion** 

Fig. 11. Interaction network analysis demonstrates cellular responses to energy depletion and oxidative stress. Panel A is a network of proteins involved in glycolysis gluconeogenesis, showing the up-regulation of proteins involved in this process. Panel B is a network showing the up-regulation of proteins with anti-oxidative properties.

Fig. 12. Interaction network of ischemia regulated protein kinases. The network shows the regulated kinases (diamonds) interact with a subset of 16 other proteins in the IS dataset.

Actively regulated cell death processes rely on a variety of cellular signaling cascades, typically involving specific protein kinases. We found that the expression of 24 protein kinases were altered in post-ischemic synaptosomes and constructed an interaction network from this subset of proteins (**Fig. 12**). The resultant kinase network included 17 of the 24 kinases as well as 16 non-kinase synaptosomal proteins that were in the IS dataset. Interestingly, three of the key proteins identified in **Fig. 9** were present in the kinase network (Hsp90aa1, Vim and Taf1), supporting a broad role for these proteins in ischemic pathology. Interestingly, a group of proteins constituting the pyruvate dehydroxylase complex (Pdhb, Pdha1, Pdhx, Dld and Dlat), which links glycolysis and the tricarboxylic acid (TCA) cycle, were up-regulated, while citrate synthase (Cs) was down-regulated. The figure confirms that a wide variety of signaling pathways are affected by cerebral ischemia.

### **4. Discussion**

108 Advances in the Preclinical Study of Ischemic Stroke

Fig. 11. Interaction network analysis demonstrates cellular responses to energy depletion

gluconeogenesis, showing the up-regulation of proteins involved in this process. Panel B is a

and oxidative stress. Panel A is a network of proteins involved in glycolysis -

network showing the up-regulation of proteins with anti-oxidative properties.

The aim of this study is to characterize the effects of cerebral ischemia on the synaptic proteome and to examine the functional consequences of these alterations with particular emphasis on their relevance to neuronal cell death processes. As outlined, cellular protein levels are determined by the balance between synthesis and degradation, which is affected by a multitude of factors including: transcription, translation, proteases activity, and posttranslational modifications. Ischemic conditions complicate the matter by causing dysfunction in the proteosome (DeGracia et al. 2002) and endoplasmic reticulum (Ge et al. 2007). Thus, gene expression data alone is a poor indicator of post-ischemic cellular protein levels. Furthermore focusing on the synapse adds the prospect of intracellular transport

BiNGO and MiMI are valuable analysis tools that are integrated into the Cytoscape framework. The GO networks created by BiNGO identify the statistically overrepresented ontologies associated with a given gene or protein dataset. This enables rapid identification and characterization of ontologies (biological process, molecular function, cellular location) that are specific to a given dataset, as well as the hierarchical nature of the ontologies. MiMI, on the other hand, gathers interaction information from various public databases and constructs an interaction network based on a list of proteins of interest. In these interaction networks, lines drawn between entities (proteins) can represent a variety of interactions, such as binding, phosphorylation, or other biologically relevant modifications. Such analyses allow for the identification of intermediary proteins that are important to the network, but are not directly identified by either biochemical analysis or literature mining. Importantly, interaction networks can be used to identify highly integrated 'hubs', which

Cell death can occur either in an unregulated or a regulated manner. Apoptosis is a wellstudied regulated cell death mechanism, and awareness of regulated necrosis (necroptosis) has been increasing (Ankarcrona et al. 1995, Baines 2010, Hitomi et al. 2008). Additionally, autophagy and ER associated degradation (ERAD) are regulated processes that are vital to cell fate decisions during injurious conditions (Liu et al. 2010, Petrovski et al. 2011). Importantly, Liu et al. (2010) recently reported that cerebral ischemia induces protein aggregation, leading to multiple organelle damage that is likely to be responsible for delayed neuronal death. We constructed an IS protein interaction network that enabled the identification of five proteins that appear to be critical in linking the consequences of synaptic ischemia to regulated cell death processes (**Fig. 9**). Of the proteins identified, Vim appeared to provide the strongest association with apoptosis, whereas Hsp90aa1 was the protein that provided a link to necroptosis and ERAD. Although Vim is an intermediate filament protein expressed in glia that may not be expected to be found in the synapse, it is common to find proteins such as Vim and Gfap in synaptosomal preparations (Costain et al. 2008) and is likely to be due to the intimate association between glia and synaptic structures. Nonetheless, up-regulation of Vim following cerebral ischemia has been frequently reported, and is thought to represent the activation of astrocytes and the reactive gliosis process. Furthermore, genetic ablation of Vim has been shown to counteract neuronal pathology, indicating that Vim is relevant to ischemic synaptosomal function (Pekny & Pekna 2004). Similarly, up-regulation of heat shock proteins, such as Hsp27 and Hsp70, in response to cerebral ischemia is a well-documented finding (Franklin et al. 2005, Currie & Plumier 1998). The Hsp90 family of molecular chaperones are involved in a variety of cellular processes, such as signal transduction, protein folding and protein degradation. Hsp90aa1 is the inducible cytoplasmic form of Hsp90 and aids in the folding of a wide variety of proteins. While other ischemia-responsive heat shock proteins that are associated with MCAO were identified in the present study (**Table 1** and **2**; Hspd1, Hspa9), Hsp90aa1 has not previously been associated with cerebral ischemia and is therefore a good candidate

are likely to represent key factors in a given biological process or pathology.

for further examination of its role in ischemia-induced necroptosis and ERAD.

The IS network analysis indicated that autophagy was associated with the IS dataset though the cytoskeletal protein Tuba4a (**Fig. 9**). Tuba4a has previously been identified as a synaptic protein, but has not been associated with MCAO. Alterations in cellular cytoskeletal proteins, such as Map2, are known to occur following exposure to ischemic injury (Kharlamov et al. 2009) and the observed reduction in Tuba4a expression is consistent with disruption of cytoskeletal structures. Reduced expression in other tubulin/tubulin related

mechanisms and localized translation further blurring the association between transcription and synaptic protein levels (Zhao et al. 2005, Vanderklish & Bahr 2000, Havik et al. 2003). As a result of these factors, the best approach for determining post-ischemic synaptic protein levels is to perform a direct assessment using proteomic methodologies.

### **4.1 Cerebral ischemia-induced alterations in the synaptic proteome**

Here, we determined the proteomic response of the mouse brain synapse to cerebral ischemia by performing an analysis of mouse brain synaptosomes. Using a label-free nanoLC-MS/MS method, we identified 371 synaptosomal proteins that were altered 20 hrs after cerebral ischemia (**Table 3**), representing ≈ 27% of the total peaks detected. Linear regression analysis was used to exclude the possible influence of systemic bias on expression due to protein size and MASCOT score (**Fig. 5**). The purity of the synaptosomal prep was validated by the determination that > 80% of the IS dataset were previously characterized as being localized in the synapse (**Fig. 6**). Consistent with our previous study, the majority of the IS proteins were localized in the mitochondria (Costain et al. 2010), and the localization of the remaining proteins was consistent with the synaptic compartment. Furthermore, literature mining of PubMed indicated that only ≈ 15% of the IS proteins were associated with the MCAO model of cerebral ischemia. A statistical analysis of the ontological classification of the IS dataset revealed a number of important details (**Fig. 8**). Firstly, the significant enrichment of proteins located in the mitochondria, endoplasmic reticulum and Golgi apparatus indicate the importance of these structures in mediating post-ischemic neuronal function. Secondly, significantly enriched molecular function ontologies include kinase, transport, antioxidant and electron carrier activity. These functions are related to metabolism, and are consistent with tissues responding to catastrophic energy depletion. Lastly, the significantly enriched biological process ontologies were also associated with metabolism and stress responses. The present results, obtained using label-free nanoLC-MS/MS, are largely in agreement with our previous ICAT-based proteomics study (**Table 3**). The consistency between these datasets reflects the reproducibility and purity of synaptosomal preparations, as well as the proteomic methodologies. Furthermore, the label-free method produced a more comprehensive dataset than the ICAT method, exhibiting greater protein coverage, peptide scores and the ability to identify cysteine-free proteins.

### **4.2 Interaction network analysis**

In an effort to place the observed alterations in the IS dataset into biological context, a variety of protein interaction analyses were performed. Interaction networks can be constructed from a variety of data types, such as protein-protein, protein-DNA and genetic interactions. The value of examining such interactions is that the overall biochemical function of proteins or DNA is a product of the interactions in which they participate. Thus, analyzing functional interactions enables the construction of signaling pathways or interaction networks that are capable of modeling a system or interpreting systematic responses to a given perturbation. In the present study, we used our observed systematic alterations in synaptosomal protein expression, and protein-protein interaction analyses to aid in interpreting the net effect of ischemia on the biological system (synapses and neurons). Furthermore, we integrated our observations in IS synaptosomes with key proteins in cell death pathways that are highly predictive of cell fate. Lastly, we focused on certain subsets of proteins to produce interaction networks that provided insight into the key biological processes.

mechanisms and localized translation further blurring the association between transcription and synaptic protein levels (Zhao et al. 2005, Vanderklish & Bahr 2000, Havik et al. 2003). As a result of these factors, the best approach for determining post-ischemic synaptic protein

Here, we determined the proteomic response of the mouse brain synapse to cerebral ischemia by performing an analysis of mouse brain synaptosomes. Using a label-free nanoLC-MS/MS method, we identified 371 synaptosomal proteins that were altered 20 hrs after cerebral ischemia (**Table 3**), representing ≈ 27% of the total peaks detected. Linear regression analysis was used to exclude the possible influence of systemic bias on expression due to protein size and MASCOT score (**Fig. 5**). The purity of the synaptosomal prep was validated by the determination that > 80% of the IS dataset were previously characterized as being localized in the synapse (**Fig. 6**). Consistent with our previous study, the majority of the IS proteins were localized in the mitochondria (Costain et al. 2010), and the localization of the remaining proteins was consistent with the synaptic compartment. Furthermore, literature mining of PubMed indicated that only ≈ 15% of the IS proteins were associated with the MCAO model of cerebral ischemia. A statistical analysis of the ontological classification of the IS dataset revealed a number of important details (**Fig. 8**). Firstly, the significant enrichment of proteins located in the mitochondria, endoplasmic reticulum and Golgi apparatus indicate the importance of these structures in mediating post-ischemic neuronal function. Secondly, significantly enriched molecular function ontologies include kinase, transport, antioxidant and electron carrier activity. These functions are related to metabolism, and are consistent with tissues responding to catastrophic energy depletion. Lastly, the significantly enriched biological process ontologies were also associated with metabolism and stress responses. The present results, obtained using label-free nanoLC-MS/MS, are largely in agreement with our previous ICAT-based proteomics study (**Table 3**). The consistency between these datasets reflects the reproducibility and purity of synaptosomal preparations, as well as the proteomic methodologies. Furthermore, the label-free method produced a more comprehensive dataset than the ICAT method, exhibiting greater protein coverage, peptide scores and the ability to

In an effort to place the observed alterations in the IS dataset into biological context, a variety of protein interaction analyses were performed. Interaction networks can be constructed from a variety of data types, such as protein-protein, protein-DNA and genetic interactions. The value of examining such interactions is that the overall biochemical function of proteins or DNA is a product of the interactions in which they participate. Thus, analyzing functional interactions enables the construction of signaling pathways or interaction networks that are capable of modeling a system or interpreting systematic responses to a given perturbation. In the present study, we used our observed systematic alterations in synaptosomal protein expression, and protein-protein interaction analyses to aid in interpreting the net effect of ischemia on the biological system (synapses and neurons). Furthermore, we integrated our observations in IS synaptosomes with key proteins in cell death pathways that are highly predictive of cell fate. Lastly, we focused on certain subsets of proteins to produce interaction networks that provided insight into the

levels is to perform a direct assessment using proteomic methodologies.

**4.1 Cerebral ischemia-induced alterations in the synaptic proteome** 

identify cysteine-free proteins.

key biological processes.

**4.2 Interaction network analysis** 

BiNGO and MiMI are valuable analysis tools that are integrated into the Cytoscape framework. The GO networks created by BiNGO identify the statistically overrepresented ontologies associated with a given gene or protein dataset. This enables rapid identification and characterization of ontologies (biological process, molecular function, cellular location) that are specific to a given dataset, as well as the hierarchical nature of the ontologies. MiMI, on the other hand, gathers interaction information from various public databases and constructs an interaction network based on a list of proteins of interest. In these interaction networks, lines drawn between entities (proteins) can represent a variety of interactions, such as binding, phosphorylation, or other biologically relevant modifications. Such analyses allow for the identification of intermediary proteins that are important to the network, but are not directly identified by either biochemical analysis or literature mining. Importantly, interaction networks can be used to identify highly integrated 'hubs', which are likely to represent key factors in a given biological process or pathology.

Cell death can occur either in an unregulated or a regulated manner. Apoptosis is a wellstudied regulated cell death mechanism, and awareness of regulated necrosis (necroptosis) has been increasing (Ankarcrona et al. 1995, Baines 2010, Hitomi et al. 2008). Additionally, autophagy and ER associated degradation (ERAD) are regulated processes that are vital to cell fate decisions during injurious conditions (Liu et al. 2010, Petrovski et al. 2011). Importantly, Liu et al. (2010) recently reported that cerebral ischemia induces protein aggregation, leading to multiple organelle damage that is likely to be responsible for delayed neuronal death. We constructed an IS protein interaction network that enabled the identification of five proteins that appear to be critical in linking the consequences of synaptic ischemia to regulated cell death processes (**Fig. 9**). Of the proteins identified, Vim appeared to provide the strongest association with apoptosis, whereas Hsp90aa1 was the protein that provided a link to necroptosis and ERAD. Although Vim is an intermediate filament protein expressed in glia that may not be expected to be found in the synapse, it is common to find proteins such as Vim and Gfap in synaptosomal preparations (Costain et al. 2008) and is likely to be due to the intimate association between glia and synaptic structures. Nonetheless, up-regulation of Vim following cerebral ischemia has been frequently reported, and is thought to represent the activation of astrocytes and the reactive gliosis process. Furthermore, genetic ablation of Vim has been shown to counteract neuronal pathology, indicating that Vim is relevant to ischemic synaptosomal function (Pekny & Pekna 2004). Similarly, up-regulation of heat shock proteins, such as Hsp27 and Hsp70, in response to cerebral ischemia is a well-documented finding (Franklin et al. 2005, Currie & Plumier 1998). The Hsp90 family of molecular chaperones are involved in a variety of cellular processes, such as signal transduction, protein folding and protein degradation. Hsp90aa1 is the inducible cytoplasmic form of Hsp90 and aids in the folding of a wide variety of proteins. While other ischemia-responsive heat shock proteins that are associated with MCAO were identified in the present study (**Table 1** and **2**; Hspd1, Hspa9), Hsp90aa1 has not previously been associated with cerebral ischemia and is therefore a good candidate for further examination of its role in ischemia-induced necroptosis and ERAD.

The IS network analysis indicated that autophagy was associated with the IS dataset though the cytoskeletal protein Tuba4a (**Fig. 9**). Tuba4a has previously been identified as a synaptic protein, but has not been associated with MCAO. Alterations in cellular cytoskeletal proteins, such as Map2, are known to occur following exposure to ischemic injury (Kharlamov et al. 2009) and the observed reduction in Tuba4a expression is consistent with disruption of cytoskeletal structures. Reduced expression in other tubulin/tubulin related

the effects cerebral ischemia on metabolic function, as well as to identify five proteins that are integral to cell death pathways and are potential targets for therapeutic intervention. This report also confirms that cerebral ischemia induces marked aberrations in synaptic mitochondria, lysosomes, endoplasmic reticulum and golgi apparatus, thereby emphasizing

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**6. References** 

proteins (Tubb4, Tubb5, Tppp) was also observed, indicating an autophagy-mediated failure in the microtubule system and collapse of the synaptic structure function relationship.

Another interesting finding to arise from the IS network analysis is the prominence of the transcription factor Taf1 in connecting the autophagy, ERAD and necroptosis sub-networks. While Taf1 is involved in basal transcription, it has recently been found to be an important factor in certain neurodegenerative conditions (Davidson et al. 2009, Sako et al. 2011). Similarly, the down-regulated protein Ywhag was a highly integrated protein within the IS network, whereas the related protein Ywhaq was up-regulated with fewer interconnections (**Fig. 9**). There is growing interest in Ywhag as a mediator of neuroprotection in cerebral ischemia (Dong et al. 2010) and the observed decrease in its expression is consistent with the activation of cell death pathways. Additionally, up-regulation of Ywhaq has been observed in amyotrophic lateral sclerosis patients (Malaspina et al. 2000), and has been found to be necessary for autophagy (Wang et al. 2010). These findings suggest that the 14-3-3 proteins are likely to be playing an important role in mediating post-ischemic neuronal cell death and are good targets for therapeutic intervention in stroke.

The role of mitochondria in the pathology of ischemic neuronal death (apoptotic and necrotic) is well established (Iijima 2006, Tsujimoto & Shimizu 2007). Cerebral ischemia results in a sustained increase in intracellular Ca++ that is buffered by the mitochondria. The increased Ca++ levels disrupt the mitochondrial membrane potential and induce the formation of the permeability transition pore, thereby activating the intrinsic apoptosis pathway (Tsujimoto & Shimizu 2007). In the synaptosome, hypoxia induces mitochondrial membrane potential disruption (Aldinucci et al. 2007) and Brown *et al* (2006) have demonstrated that synaptic mitochondria are more sensitive to Ca++ overload than non-synaptic mitochondria. Our present findings confirm our previous observation that widespread alterations in protein expression occur in post-ischemic synaptic mitochondria (Costain et al. 2010). An interaction network was constructed from the large number of IS proteins that are involved in oxidative phosphorylation (**Fig. 10**). This interaction network clearly demonstrates that cerebral ischemia induces an imbalance in oxidative phosphorylation, with down-regulation of complex IV components and more variable effects on complex I and V. While oxidative phosphorylation is clearly disrupted, there is evidence that the cells are attempting to compensate by upregulating glycolytic enzymes (**Fig. 11A**) as well as proteins with anti-oxidative activity (**Fig. 11B**). Unfortunately, glycolysis cannot produce the same amount of ATP that is derived from oxidative phosphorylation.

A wide variety of protein kinases are involved in mediating regulated cell death, and we identified a subset of 24 protein kinases within the IS dataset. An interaction network was constructed from the IS kinases subset (**Fig. 12**) and the kinase network independently identified three of the key proteins singled out in the IS network analysis (**Fig. 9**), supporting the contention that these proteins are involved in post-ischemic cell signaling pathways. Hsp90aa1, Taf1 and Vim were essential for connecting the four cell death mechanisms to the IS dataset, and their association with alterations in protein kinase levels further confirms their importance in mediating synaptic pathology.

### **5. Conclusion**

In closing, this study has demonstrated that the synapse is highly responsive to cerebral ischemia and is highly informative about cerebral ischemia-induced cell death mechanisms at the organelle level. We have used interaction network analyses of the IS dataset to clarify the effects cerebral ischemia on metabolic function, as well as to identify five proteins that are integral to cell death pathways and are potential targets for therapeutic intervention. This report also confirms that cerebral ischemia induces marked aberrations in synaptic mitochondria, lysosomes, endoplasmic reticulum and golgi apparatus, thereby emphasizing the interplay between organelles during oxidative damage.

### **6. References**

112 Advances in the Preclinical Study of Ischemic Stroke

proteins (Tubb4, Tubb5, Tppp) was also observed, indicating an autophagy-mediated failure in the microtubule system and collapse of the synaptic structure function relationship. Another interesting finding to arise from the IS network analysis is the prominence of the transcription factor Taf1 in connecting the autophagy, ERAD and necroptosis sub-networks. While Taf1 is involved in basal transcription, it has recently been found to be an important factor in certain neurodegenerative conditions (Davidson et al. 2009, Sako et al. 2011). Similarly, the down-regulated protein Ywhag was a highly integrated protein within the IS network, whereas the related protein Ywhaq was up-regulated with fewer interconnections (**Fig. 9**). There is growing interest in Ywhag as a mediator of neuroprotection in cerebral ischemia (Dong et al. 2010) and the observed decrease in its expression is consistent with the activation of cell death pathways. Additionally, up-regulation of Ywhaq has been observed in amyotrophic lateral sclerosis patients (Malaspina et al. 2000), and has been found to be necessary for autophagy (Wang et al. 2010). These findings suggest that the 14-3-3 proteins are likely to be playing an important role in mediating post-ischemic neuronal cell death

The role of mitochondria in the pathology of ischemic neuronal death (apoptotic and necrotic) is well established (Iijima 2006, Tsujimoto & Shimizu 2007). Cerebral ischemia results in a sustained increase in intracellular Ca++ that is buffered by the mitochondria. The increased Ca++ levels disrupt the mitochondrial membrane potential and induce the formation of the permeability transition pore, thereby activating the intrinsic apoptosis pathway (Tsujimoto & Shimizu 2007). In the synaptosome, hypoxia induces mitochondrial membrane potential disruption (Aldinucci et al. 2007) and Brown *et al* (2006) have demonstrated that synaptic mitochondria are more sensitive to Ca++ overload than non-synaptic mitochondria. Our present findings confirm our previous observation that widespread alterations in protein expression occur in post-ischemic synaptic mitochondria (Costain et al. 2010). An interaction network was constructed from the large number of IS proteins that are involved in oxidative phosphorylation (**Fig. 10**). This interaction network clearly demonstrates that cerebral ischemia induces an imbalance in oxidative phosphorylation, with down-regulation of complex IV components and more variable effects on complex I and V. While oxidative phosphorylation is clearly disrupted, there is evidence that the cells are attempting to compensate by upregulating glycolytic enzymes (**Fig. 11A**) as well as proteins with anti-oxidative activity (**Fig. 11B**). Unfortunately, glycolysis cannot produce the same amount of ATP that is derived from

A wide variety of protein kinases are involved in mediating regulated cell death, and we identified a subset of 24 protein kinases within the IS dataset. An interaction network was constructed from the IS kinases subset (**Fig. 12**) and the kinase network independently identified three of the key proteins singled out in the IS network analysis (**Fig. 9**), supporting the contention that these proteins are involved in post-ischemic cell signaling pathways. Hsp90aa1, Taf1 and Vim were essential for connecting the four cell death mechanisms to the IS dataset, and their association with alterations in protein kinase levels

In closing, this study has demonstrated that the synapse is highly responsive to cerebral ischemia and is highly informative about cerebral ischemia-induced cell death mechanisms at the organelle level. We have used interaction network analyses of the IS dataset to clarify

further confirms their importance in mediating synaptic pathology.

and are good targets for therapeutic intervention in stroke.

oxidative phosphorylation.

**5. Conclusion** 


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**6** 

*Canada* 

**Molecular Pathways** 

*University of Manitoba* 

**Delayed Neuronal Death in Ischemic Stroke:** 

Ischemic stroke is caused by a loss of blood flow and deficiency in glucose and oxygen to the brain. The lack of sufficient glucose and oxygen results in varying degrees of tissue damage and cell death following stroke. Reperfusion of blood flow after ischemia often compounds tissue damage that is sustained during the initial drop in local blood

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There are major differences in the physiology and biochemistry of cell death between the core and penumbra, which suggests that different mechanisms of cell death are at work in

During a stroke, the ischemic core suffers a drop in energy. ATP levels in the core fall to a level only 15% of typical basal values within one or two minutes (Katsura et al., 1993; Lipton, 1999; Lipton & Whittingham, 1982; Martin et al., 1994) and do not recover by much after reperfusion (Sun et al., 1995). The core rapidly loses ion transporter functions and undergoes anoxic depolarization (Balestrino, 1995). Homeostasis of potassium, calcium, and sodium ions is lost (Harris & Symon, 1984). After reperfusion, extracellular K+ typically returns to control levels for six hours and then stays slightly elevated above normal (Gido et al., 1997). There is some restoration of K+ transporter functions, despite widespread cell

**1. Introduction** 

availability.

date.

**2. Events in stroke** 

**2.1 Core of ischemic infarct** 

these two regions.

Victor Li, Xiaoying Bi, Paul Szelemej and Jiming Kong


## **Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways**

Victor Li, Xiaoying Bi, Paul Szelemej and Jiming Kong *University of Manitoba Canada* 

### **1. Introduction**

116 Advances in the Preclinical Study of Ischemic Stroke

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Ischemic stroke is caused by a loss of blood flow and deficiency in glucose and oxygen to the brain. The lack of sufficient glucose and oxygen results in varying degrees of tissue damage and cell death following stroke. Reperfusion of blood flow after ischemia often compounds tissue damage that is sustained during the initial drop in local blood availability.

The size and position of the affected region depends on which vessel is occluded. A complete loss of blood flow is rare, as rich networks of nearby blood vessels often compensate for reduced flow. The centre of the ischemic region, the core, is characterized by acute and mostly necrotic cell death resulting from severe anoxia and hypoglycemia. The region enveloping the core is known as the penumbra, which experiences a milder ischemic insult. The penumbra should be targeted for treatment strategies; it is usually much larger than the core and has a longer window of opportunity during which neurons can be prevented from dying. Many studies elucidate the molecular pathways of delayed neuronal death. This chapter presents the pathways and strategies that have been investigated to date.

### **2. Events in stroke**

There are major differences in the physiology and biochemistry of cell death between the core and penumbra, which suggests that different mechanisms of cell death are at work in these two regions.

### **2.1 Core of ischemic infarct**

During a stroke, the ischemic core suffers a drop in energy. ATP levels in the core fall to a level only 15% of typical basal values within one or two minutes (Katsura et al., 1993; Lipton, 1999; Lipton & Whittingham, 1982; Martin et al., 1994) and do not recover by much after reperfusion (Sun et al., 1995). The core rapidly loses ion transporter functions and undergoes anoxic depolarization (Balestrino, 1995). Homeostasis of potassium, calcium, and sodium ions is lost (Harris & Symon, 1984). After reperfusion, extracellular K+ typically returns to control levels for six hours and then stays slightly elevated above normal (Gido et al., 1997). There is some restoration of K+ transporter functions, despite widespread cell

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 119

elevated, and then rise during reperfusion due to a variety of metabolic and inflammatory mechanisms (Beckman et al., 1990; Chambers et al., 1985; Clemens et al., 1997; Kuehl et al., 1980; Zhu et al., 2004b). Arachidonic acid metabolites are a source of oxidative stress following reperfusion. Mitochondrial dysfunction, COX-2 activation, endothelial and neural production of NO, and conversion of xanthine dehydrogenase to xanthine oxidase play

Oxidative stress can cause lipid peroxidation, sulfhydryl oxidation, proteolysis, and destruction of nuclear material. Excessive free radicals can activate p53, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and activator protein-1, to drive their expression of pro-apoptotic genes. Free radicals and oxidative stress are involved in lysosomal dysfunction and autophagic cell death (Pivtoraiko et al., 2009). Free radicals can disrupt the electron transport chain in mitochondria, which results in autoxidation of flavoproteins and ubisemiquinone and an increase in superoxide generation (Zhu et al.,

Neuronal mitochondria release cell-death factors and free radicals into the cytosol. Mitochondria have roles in apoptotic and necrosis-like cell death. ATP depletion, Ca2+ overload, and free radical damage causes the opening of mitochondrial permeability transition pores (Friberg & Wieloch, 2002) and releases cytochrome *c*, high temperature requirement protein A2 (HtrA2/Omi), second mitochondria-derived activator of caspases

The Bcl-2 family of proteins that regulate apoptosis includes both pro-apoptotic and antiapoptotic proteins that counteract one another and regulate mitochondrial outer membrane permeability (MOMP) and release of mitochondrial apoptotic factors. After ischemic damage, Bcl-2 and Bcl-xL are inhibited, allowing Bax and Bak to act, which form channels in the mitochondria and release cytochrome *c*, HtrA2/Omi, and Smac/DIABLO and other cell-

Neurotrophins play a role in immediate protection of neurons and in long-term cellular remodeling and regeneration. Following ischemia, neurotrophic factors are upregulated (Ferrer et al., 1998; Takeda et al., 1993; Tsukahara et al., 1994), although local levels of brainderived neurotrophic factor (BDNF) levels drop below baseline in vulnerable cell types (Kokaia et al., 1996). Other growth factors with neuroprotective effects include transforming growth factor-beta (TGF-β), acidic fibroblast growth factor (FGF1), and vascular endothelial growth factor (VEGF). Many mechanisms for the protective action of nerve growth factors that are induced or reduced post-ischemia have been proposed. FGF1 inhibits excitotoxicity by preventing or delaying the rise of Ca2+ during ischemia (Mitani et al., 1992). BDNF protects against excitotoxicity by preventing the decrease in protein kinase C that follows ischemia (Tremblay et al., 1999). The induction of BDNF is attributed to the reduction of free radicals (Mattson et al., 1995) and acts by upregulating antioxidant enzymes such as superoxide dismutases (SOD) and glutathione reductase. The function of BDNF depends on

Maintenance of protein synthesis after ischemia is mediated by tyrosine kinase systems activated by neurotrophins or other growth factors. Lack of neurotrophic action in neurons

(Smac/DIABLO), apoptosis-inducing factor (AIF), and endonuclease G (EndoG).

phosphorylating other cellular components (Dugan et al., 1997).

significant roles in producing free radicals.

**2.5 Mitochondria-mediated death pathways** 

2004b).

death-inducing factors.

**2.6 Neurotrophins** 

injury and cell death within the core. Other responses to ischemia-induced energy loss include reduction or a complete halting of protein synthesis due to translation initiation factor (IF) inactivation (White et al., 2000) and insufficient GTP for ribosomal function. Permanent absence of protein synthesis continuing beyond reperfusion results in necrotic cell death. Recovery of protein synthesis is necessary for cell survival. Necrosis in the core is accompanied by glutamate release and excitotoxic cell damage to neighbouring regions.

### **2.2 Penumbra of ischemic infarct**

Blood flow within the penumbra can vary substantially, subjecting cells to a wide range of stresses. Ischemic injury within the penumbra is variable in whether it results in cell death and in which molecular mechanisms are involved. Many of these mechanisms induce cell death in a delayed manner in neurons, which allows them to be saved if some neuroprotection is provided. This delay allows for therapeutic treatment, since the majority of stroke patients present many hours after suffering a stroke.

Penumbral ischemia is milder than in the core; levels of ATP in the penumbra drop to an average of 50-70% of normal levels. Protein synthesis can be stalled following massive Ca2+ influx, which can inactivate eIF-2a by preventing activation of eIF-2 and guanine nucleotide exchange factor during the initiation of translation (Kumar et al., 2001). Protein synthesis resumes after reperfusion and has a role in determining the extent of delayed neuronal death.

### **2.3 Excitotoxicity**

Penumbral cells are subject to excessive excitatory amino acid release from depolarized nearby cells in the ischemic core. Glutamate is the major excitatory neurotransmitter in the brain and the key mediator of intracellular communication, plasticity, growth and differentiation. The glutamate receptors implicated in excitotoxicity include the NMDA, AMPA, kainate, and other metabotropic glutamate receptors (Prass & Dirnagl, 1998). While present in synapses at micromolar concentrations, ischemia-induced depolarization causes a much larger release that triggers a chain reaction of depolarization and effects glutamate release in surrounding neurons (Paschen, 1996). The overstimulated neurons release Ca2+ into their cytosol, halting protein synthesis and activating cyclooxygenase-2 (COX-2), increased nitric oxide (NO) production, phospholipases, calpains, cathepsins, and calcineurin (Ferrer, 2006; White et al., 2000). Degradation of calpain substrates such as spectrin and eIF4G then follows (White et al., 2000), while cathepsin activation may increase lysosomal activity and lead to autophagic cell death (Yamashima et al., 1998). Membranes are degraded by hyperactivated phospholipases, which produce free arachidonic acid that is metabolized during reperfusion to produce peroxidative derivatives that then act as free radicals.

Excitotoxicity describes the damaging effects resulting from excessive excitatory neurotransmitter release. It is implicated in necrotic, apoptotic, and necroptotic cell death (Choi, 1996; Li et al., 2008).

### **2.4 Oxidative stress**

Oxidative damage by free radical generation mediates cell damage in ischemia (Gilgun-Sherki et al., 2002). It is involved in excitotoxicity, apoptosis, autophagic cell death, and inflammation. Penumbral free radical levels increase during early ischemia, remain elevated, and then rise during reperfusion due to a variety of metabolic and inflammatory mechanisms (Beckman et al., 1990; Chambers et al., 1985; Clemens et al., 1997; Kuehl et al., 1980; Zhu et al., 2004b). Arachidonic acid metabolites are a source of oxidative stress following reperfusion. Mitochondrial dysfunction, COX-2 activation, endothelial and neural production of NO, and conversion of xanthine dehydrogenase to xanthine oxidase play significant roles in producing free radicals.

Oxidative stress can cause lipid peroxidation, sulfhydryl oxidation, proteolysis, and destruction of nuclear material. Excessive free radicals can activate p53, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and activator protein-1, to drive their expression of pro-apoptotic genes. Free radicals and oxidative stress are involved in lysosomal dysfunction and autophagic cell death (Pivtoraiko et al., 2009). Free radicals can disrupt the electron transport chain in mitochondria, which results in autoxidation of flavoproteins and ubisemiquinone and an increase in superoxide generation (Zhu et al., 2004b).

### **2.5 Mitochondria-mediated death pathways**

Neuronal mitochondria release cell-death factors and free radicals into the cytosol. Mitochondria have roles in apoptotic and necrosis-like cell death. ATP depletion, Ca2+ overload, and free radical damage causes the opening of mitochondrial permeability transition pores (Friberg & Wieloch, 2002) and releases cytochrome *c*, high temperature requirement protein A2 (HtrA2/Omi), second mitochondria-derived activator of caspases (Smac/DIABLO), apoptosis-inducing factor (AIF), and endonuclease G (EndoG).

The Bcl-2 family of proteins that regulate apoptosis includes both pro-apoptotic and antiapoptotic proteins that counteract one another and regulate mitochondrial outer membrane permeability (MOMP) and release of mitochondrial apoptotic factors. After ischemic damage, Bcl-2 and Bcl-xL are inhibited, allowing Bax and Bak to act, which form channels in the mitochondria and release cytochrome *c*, HtrA2/Omi, and Smac/DIABLO and other celldeath-inducing factors.

### **2.6 Neurotrophins**

118 Advances in the Preclinical Study of Ischemic Stroke

injury and cell death within the core. Other responses to ischemia-induced energy loss include reduction or a complete halting of protein synthesis due to translation initiation factor (IF) inactivation (White et al., 2000) and insufficient GTP for ribosomal function. Permanent absence of protein synthesis continuing beyond reperfusion results in necrotic cell death. Recovery of protein synthesis is necessary for cell survival. Necrosis in the core is accompanied by glutamate release and excitotoxic cell damage to neighbouring regions.

Blood flow within the penumbra can vary substantially, subjecting cells to a wide range of stresses. Ischemic injury within the penumbra is variable in whether it results in cell death and in which molecular mechanisms are involved. Many of these mechanisms induce cell death in a delayed manner in neurons, which allows them to be saved if some neuroprotection is provided. This delay allows for therapeutic treatment, since the majority

Penumbral ischemia is milder than in the core; levels of ATP in the penumbra drop to an average of 50-70% of normal levels. Protein synthesis can be stalled following massive Ca2+ influx, which can inactivate eIF-2a by preventing activation of eIF-2 and guanine nucleotide exchange factor during the initiation of translation (Kumar et al., 2001). Protein synthesis resumes after reperfusion and has a role in determining the extent of delayed neuronal

Penumbral cells are subject to excessive excitatory amino acid release from depolarized nearby cells in the ischemic core. Glutamate is the major excitatory neurotransmitter in the brain and the key mediator of intracellular communication, plasticity, growth and differentiation. The glutamate receptors implicated in excitotoxicity include the NMDA, AMPA, kainate, and other metabotropic glutamate receptors (Prass & Dirnagl, 1998). While present in synapses at micromolar concentrations, ischemia-induced depolarization causes a much larger release that triggers a chain reaction of depolarization and effects glutamate release in surrounding neurons (Paschen, 1996). The overstimulated neurons release Ca2+ into their cytosol, halting protein synthesis and activating cyclooxygenase-2 (COX-2), increased nitric oxide (NO) production, phospholipases, calpains, cathepsins, and calcineurin (Ferrer, 2006; White et al., 2000). Degradation of calpain substrates such as spectrin and eIF4G then follows (White et al., 2000), while cathepsin activation may increase lysosomal activity and lead to autophagic cell death (Yamashima et al., 1998). Membranes are degraded by hyperactivated phospholipases, which produce free arachidonic acid that is metabolized during reperfusion to produce peroxidative derivatives that then act as free

Excitotoxicity describes the damaging effects resulting from excessive excitatory neurotransmitter release. It is implicated in necrotic, apoptotic, and necroptotic cell death

Oxidative damage by free radical generation mediates cell damage in ischemia (Gilgun-Sherki et al., 2002). It is involved in excitotoxicity, apoptosis, autophagic cell death, and inflammation. Penumbral free radical levels increase during early ischemia, remain

**2.2 Penumbra of ischemic infarct** 

death.

radicals.

(Choi, 1996; Li et al., 2008).

**2.4 Oxidative stress** 

**2.3 Excitotoxicity** 

of stroke patients present many hours after suffering a stroke.

Neurotrophins play a role in immediate protection of neurons and in long-term cellular remodeling and regeneration. Following ischemia, neurotrophic factors are upregulated (Ferrer et al., 1998; Takeda et al., 1993; Tsukahara et al., 1994), although local levels of brainderived neurotrophic factor (BDNF) levels drop below baseline in vulnerable cell types (Kokaia et al., 1996). Other growth factors with neuroprotective effects include transforming growth factor-beta (TGF-β), acidic fibroblast growth factor (FGF1), and vascular endothelial growth factor (VEGF). Many mechanisms for the protective action of nerve growth factors that are induced or reduced post-ischemia have been proposed. FGF1 inhibits excitotoxicity by preventing or delaying the rise of Ca2+ during ischemia (Mitani et al., 1992). BDNF protects against excitotoxicity by preventing the decrease in protein kinase C that follows ischemia (Tremblay et al., 1999). The induction of BDNF is attributed to the reduction of free radicals (Mattson et al., 1995) and acts by upregulating antioxidant enzymes such as superoxide dismutases (SOD) and glutathione reductase. The function of BDNF depends on phosphorylating other cellular components (Dugan et al., 1997).

Maintenance of protein synthesis after ischemia is mediated by tyrosine kinase systems activated by neurotrophins or other growth factors. Lack of neurotrophic action in neurons

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 121

1997). DNA fragments are of random size. This form of necrotic cell death is present during

Autophagy is a regulated catabolic process involving the degradation of a cell's own cytoplasmic macromolecules and organelles through digestion by the lysosomal system. Autophagy can be triggered by defective cell machinery. Through the formation of autophagolysosomes, a cell is capable of degrading the constituents, effectively recycling macronutrients and reducing the cell's metabolic requirements. The role of autophagy in cell homeostasis is undisputed. Autophagocytosis as a unique mechanism of programmed cell death is a controversial concept. There is evidence that autophagy is a separate mechanism of cell death and not merely an adaptive response to nutrient limitation (Cho & Toledo-Pereyra, 2008). It is unclear if the observed autophagic processes and mechanisms associated with cell death are the effectors of cell death or merely an overshoot of their initially

The autophagic cell death process is distinct from apoptosis and necrosis; it is characterized by autophagic degradation of cellular components prior to nuclear destruction (Bursch et al., 2000a; Schwartz et al., 1993). The most representative morphological feature is the formation of numerous autophagosomes in the cytosol with a condensed nucleus (Bursch et al., 2000b). Evidence suggests that autophagy contributes to the neuronal degeneration following cerebral ischemia. Autophagy occurs in both neonatal and adult mouse cortices and hippocampi after ischemic injury. Increased autophagosomal marker LC3-II levels are detected as early as 8 h after ischemia; more pronunciation occurs at 24 h and 72 h after hypoxic ischemia (Koike et al., 2008; Zhu et al., 2005). Damaged neurons show features of autophagic cell death, such as increased lysosomal cysteine proteinases, formation of cytoplasmic autophagic vacuoles, and the induction of GFP-LC3 immunofluorescence, during cerebral hypoxia or ischemia in adult mice (Adhami et al., 2006; Nitatori et al., 1995). Inhibition of autophagy provides neuroprotection in situations where most other

A pathway for autophagous cell death has been proposed that relies on the runaway activation of beclin 1. Beclin 1 is a primary inducer of autophagy and the first identified mammalian autophagy gene product (Aita et al., 1999). Beclin 1 was originally isolated as a Bcl-2-interacting protein (Liang et al., 1999). Bcl-2 inhibits beclin 1 and beclin-1-dependent autophagy in yeast and mammalian cells. Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy (Pattingre et al., 2005). The pharmacological BH3 mimetic ABT-737 can inhibit the interaction between beclin 1 and Bcl-2 or Bcl-xL and also stimulate autophagy (Maiuri et al., 2007a; Maiuri et al., 2007b). Beclin 1 is regulated through binding with Bcl-2 proteins. Bcl-2 downregulation may result in excessive autophagy causing cell death. Autophagy regulation through Bcl-2 is attributed to its expression at the ER membrane, suggesting that signalling events originating from the ER are crucial for autophagy (Rodriguez et al., 2011). ER stress triggers autophagy; this is regulated by UPR stress sensors (Kouroku et al., 2007; Ogata et al., 2006). Stimuli that increase cytosolic calcium can activate ER stress and autophagy, which can be blocked by Bcl-2 (Hoyer-Hansen et al., 2007). ER and

pharmacological treatments are ineffective (Puyal & Clarke, 2009).

a stroke; it is found within the ischemic core during severe acute ischemic damage.

**3.2 Autophagy** 

beneficial intentions.

**3.2.1 Autophagy pathways** 

results in a failure to restore protein synthesis (Hu & Wieloch, 1994). TGF-β may provide neuroprotection in ischemia by moderating transcription factors and cell-death pathways. TGF-β controls the activation of mitogen-activated protein kinases (MAPK) (Friguls et al., 2002) and inhibits Bad and caspase-3 to reduce cell death (Buisson et al., 2003; Zhu et al., 2002). These effects may be mediated by NF-κB (Zhu et al., 2004a).

VEGF promotes angiogenesis, vascular permeability, and endothelial proliferation. VEGF is implicated in neurogenesis (Storkebaum et al., 2004). VEGF is up-regulated between six and 24 hours after stroke in the penumbra (Marti et al., 2000; Plate et al., 1999). In the penumbra, VEGF modulates the PI3K/Akt/NF-κB signalling pathway and inhibits caspase-3 activity to reduce apoptosis (Sun & Guo, 2005).

### **2.7 Heat shock proteins**

Heat shock proteins (Hsp) are involved in proper protein folding and are expressed following heat and oxidative stresses. During the first minutes of stroke, Hsp70 and Hsp90 mRNA expression rises and persists in the penumbra (Ikeda et al., 1994; Kawagoe et al., 1992; Kinouchi et al., 1993; Woodburn et al., 1993), with upregulated protein expression following a few hours later. Cell survival is positively correlated with Hsp70 production, since overexpression of Hsp70 protects against infarction in rats (Mestril et al., 1996). Lower or reduced expression of Hsp70 positively correlates with neuronal death.

Expression of Hsps inhibits the activation of the transcription factor NF-κB (Schell et al., 2005), which primarily serves a detrimental function in ischemia. Hsp70 inhibits apoptosis through interacting with key proteins in various cell-death pathways. Hsp70 prevents activation of caspase-8 and caspase-9 (Matsumori et al., 2006). Hsp70 protects cells after caspase-3 activation by blocking activation of phospholipase A-2 in the cell nucleus (Jaattela et al., 1998). Hsps may work by preserving proper protein conformation in neurons suffering ischemia (Lipton, 1999).

Ubiquitin decreases after ischemia. Expression then recovers except in vulnerable neurons destined to die (Deshpande et al., 1992; Magnusson & Wieloch, 1989). This phenomenon may be involved in cell damage, possibly by allowing the accumulation of denatured proteins (Lipton, 1999).

### **3. Modes of neuronal cell death**

Mechanisms of cell death vary along a continuum between regulated, programmed cell death and unregulated, necrotic cell death. One on end of the spectrum, apoptotic cell death is tightly regulated and normally involved in tissue maintenance. Necrosis, at the opposite end of the spectrum, is unregulated and results from injury. Between these extremes are pathways with varying semblance to either necrosis or apoptosis, including necrosis-like cell death, necroptosis, and autophagic cell death.

### **3.1 Necrosis**

Classical necrosis lacks regulation, order, and energy dependence. It is caused by physical or chemical insult. It is characterized by an acute loss of osmotic homeostasis and an early decline in plasma membrane integrity and ATP levels, resulting in burst cells and inflammation from the scattered cell contents. DNA cleavage occurs late in cell death through a mechanism dependent on serine proteases (Bicknell & Cohen, 1995; Dong et al., 1997). DNA fragments are of random size. This form of necrotic cell death is present during a stroke; it is found within the ischemic core during severe acute ischemic damage.

### **3.2 Autophagy**

120 Advances in the Preclinical Study of Ischemic Stroke

results in a failure to restore protein synthesis (Hu & Wieloch, 1994). TGF-β may provide neuroprotection in ischemia by moderating transcription factors and cell-death pathways. TGF-β controls the activation of mitogen-activated protein kinases (MAPK) (Friguls et al., 2002) and inhibits Bad and caspase-3 to reduce cell death (Buisson et al., 2003; Zhu et al.,

VEGF promotes angiogenesis, vascular permeability, and endothelial proliferation. VEGF is implicated in neurogenesis (Storkebaum et al., 2004). VEGF is up-regulated between six and 24 hours after stroke in the penumbra (Marti et al., 2000; Plate et al., 1999). In the penumbra, VEGF modulates the PI3K/Akt/NF-κB signalling pathway and inhibits caspase-3 activity to

Heat shock proteins (Hsp) are involved in proper protein folding and are expressed following heat and oxidative stresses. During the first minutes of stroke, Hsp70 and Hsp90 mRNA expression rises and persists in the penumbra (Ikeda et al., 1994; Kawagoe et al., 1992; Kinouchi et al., 1993; Woodburn et al., 1993), with upregulated protein expression following a few hours later. Cell survival is positively correlated with Hsp70 production, since overexpression of Hsp70 protects against infarction in rats (Mestril et al., 1996). Lower

Expression of Hsps inhibits the activation of the transcription factor NF-κB (Schell et al., 2005), which primarily serves a detrimental function in ischemia. Hsp70 inhibits apoptosis through interacting with key proteins in various cell-death pathways. Hsp70 prevents activation of caspase-8 and caspase-9 (Matsumori et al., 2006). Hsp70 protects cells after caspase-3 activation by blocking activation of phospholipase A-2 in the cell nucleus (Jaattela et al., 1998). Hsps may work by preserving proper protein conformation in neurons

Ubiquitin decreases after ischemia. Expression then recovers except in vulnerable neurons destined to die (Deshpande et al., 1992; Magnusson & Wieloch, 1989). This phenomenon may be involved in cell damage, possibly by allowing the accumulation of denatured

Mechanisms of cell death vary along a continuum between regulated, programmed cell death and unregulated, necrotic cell death. One on end of the spectrum, apoptotic cell death is tightly regulated and normally involved in tissue maintenance. Necrosis, at the opposite end of the spectrum, is unregulated and results from injury. Between these extremes are pathways with varying semblance to either necrosis or apoptosis, including necrosis-like cell

Classical necrosis lacks regulation, order, and energy dependence. It is caused by physical or chemical insult. It is characterized by an acute loss of osmotic homeostasis and an early decline in plasma membrane integrity and ATP levels, resulting in burst cells and inflammation from the scattered cell contents. DNA cleavage occurs late in cell death through a mechanism dependent on serine proteases (Bicknell & Cohen, 1995; Dong et al.,

2002). These effects may be mediated by NF-κB (Zhu et al., 2004a).

or reduced expression of Hsp70 positively correlates with neuronal death.

reduce apoptosis (Sun & Guo, 2005).

suffering ischemia (Lipton, 1999).

**3. Modes of neuronal cell death** 

death, necroptosis, and autophagic cell death.

proteins (Lipton, 1999).

**3.1 Necrosis** 

**2.7 Heat shock proteins** 

Autophagy is a regulated catabolic process involving the degradation of a cell's own cytoplasmic macromolecules and organelles through digestion by the lysosomal system. Autophagy can be triggered by defective cell machinery. Through the formation of autophagolysosomes, a cell is capable of degrading the constituents, effectively recycling macronutrients and reducing the cell's metabolic requirements. The role of autophagy in cell homeostasis is undisputed. Autophagocytosis as a unique mechanism of programmed cell death is a controversial concept. There is evidence that autophagy is a separate mechanism of cell death and not merely an adaptive response to nutrient limitation (Cho & Toledo-Pereyra, 2008). It is unclear if the observed autophagic processes and mechanisms associated with cell death are the effectors of cell death or merely an overshoot of their initially beneficial intentions.

The autophagic cell death process is distinct from apoptosis and necrosis; it is characterized by autophagic degradation of cellular components prior to nuclear destruction (Bursch et al., 2000a; Schwartz et al., 1993). The most representative morphological feature is the formation of numerous autophagosomes in the cytosol with a condensed nucleus (Bursch et al., 2000b). Evidence suggests that autophagy contributes to the neuronal degeneration following cerebral ischemia. Autophagy occurs in both neonatal and adult mouse cortices and hippocampi after ischemic injury. Increased autophagosomal marker LC3-II levels are detected as early as 8 h after ischemia; more pronunciation occurs at 24 h and 72 h after hypoxic ischemia (Koike et al., 2008; Zhu et al., 2005). Damaged neurons show features of autophagic cell death, such as increased lysosomal cysteine proteinases, formation of cytoplasmic autophagic vacuoles, and the induction of GFP-LC3 immunofluorescence, during cerebral hypoxia or ischemia in adult mice (Adhami et al., 2006; Nitatori et al., 1995). Inhibition of autophagy provides neuroprotection in situations where most other pharmacological treatments are ineffective (Puyal & Clarke, 2009).

### **3.2.1 Autophagy pathways**

A pathway for autophagous cell death has been proposed that relies on the runaway activation of beclin 1. Beclin 1 is a primary inducer of autophagy and the first identified mammalian autophagy gene product (Aita et al., 1999). Beclin 1 was originally isolated as a Bcl-2-interacting protein (Liang et al., 1999). Bcl-2 inhibits beclin 1 and beclin-1-dependent autophagy in yeast and mammalian cells. Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy (Pattingre et al., 2005). The pharmacological BH3 mimetic ABT-737 can inhibit the interaction between beclin 1 and Bcl-2 or Bcl-xL and also stimulate autophagy (Maiuri et al., 2007a; Maiuri et al., 2007b). Beclin 1 is regulated through binding with Bcl-2 proteins. Bcl-2 downregulation may result in excessive autophagy causing cell death. Autophagy regulation through Bcl-2 is attributed to its expression at the ER membrane, suggesting that signalling events originating from the ER are crucial for autophagy (Rodriguez et al., 2011). ER stress triggers autophagy; this is regulated by UPR stress sensors (Kouroku et al., 2007; Ogata et al., 2006). Stimuli that increase cytosolic calcium can activate ER stress and autophagy, which can be blocked by Bcl-2 (Hoyer-Hansen et al., 2007). ER and

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 123

apoptosis can be carried out through a variety of discrete pathways, which can be categorized as either intrinsic or extrinsic. In neurons, intrinsic pathways can be triggered by intracellular damage that is caused by free radicals or excitotoxicity; extrinsic death pathways can be activated by tumour necrosis factors (TNF) or lack of neurotrophins and other growth factors. Once activated, both intrinsic and extrinsic pathways can trigger

The caspase family of proteases is the most common and best understood mediators of apoptosis. In humans, at least seven caspases are implicated in apoptosis, including the initiator caspases 2, 8, 9, and 10, and the executioner caspases 3, 6, and 7 (Kroemer & Martin, 2005). Activated initiator caspases are able to cleave themselves and downstream targets, causing a cascade of caspase activation culminating at the executioner caspases, which have cell structures as their substrates and directly induce apoptosis. Caspase-activated deoxyribonuclease (CAD) causes the characteristic laddered DNA fragmentation observed when its inhibitor, ICAD, is cleaved by executioner caspases (Liu et al., 1997; Liu et al.,

Following cerebral ischemia, the caspase cascade can be initiated early on through cell-death receptors or by mitochondrially mediated pathways (Ashkenazi & Dixit, 1998). The two mechanisms are not necessarily mutually exclusive and can be activated sequentially

The Fas receptor, a primary death receptor in ischemia-induced apoptosis (Ferrer & Planas, 2003), belongs to the TNF receptor (TNFR) family and is specific for the Fas ligand (FasL) expressed on T cells. Activation of the Fas receptor causes formation of the cell-deathinducing signalling complex (DISC), which activates caspase-8 through the Fas-associated death domain (FADD). Caspase-8 can then activate caspase-3 to bring about apoptosis or activate the mitochondrial death pathway by cleaving Bid, a promoter for mitochondrial apoptosis-induced channel (MAC) formation (Planas et al., 1997). TNFR1 is also a member of the TNFR family, which induces apoptosis through a similar mechanism (Stanger et al., 1995). The upstream activators of the TNFRs in stroke models are increased during

The mitochondrial pathway is activated by inducing MOMP through the formation of the MAC, which is thought to be an oligomerized product of the Bcl-2 proteins Bax and Bak (Dejean et al., 2010; Martinez-Caballero et al., 2009). Regulation of pore formation is carried out by the Bcl-2 family, which includes anti-apoptotic proteins Bcl-2 and Bcl-xL and proapoptotic proteins Bid (which is cleaved to become the active tBid), Bim, and Bad (Gross et al., 1999; Imazu et al., 1999; Susin et al., 1996; Yang et al., 1997). Upon formation, the MAC allows cytochrome *c* release to the cytoplasm, where it interacts with Apaf-1 and dATP to form apoptosomes that cleave and activate caspase-9 (Zou et al., 1997). Caspase-9 then activates executioner caspases 3, 6, and 7 to bring about apoptosis. Smac/DIABLO and HtrA2/Omi are also released from the mitochondria along with cytochrome *c* (Du et al., 2000; Suzuki et al., 2001; Verhagen et al., 2000). Both promote apoptosis by respectively removing inhibitor of apoptosis protein (IAP)'s and X-linked inhibitor of apoptosis protein (XIAP)'s inhibition of caspase-3 and caspase-9 (Suzuki et al., 2001). The action of Smac/DIABLO is inhibited by Bcl-2 and Bcl-xL, which gives some degree of control over

caspase-dependent or caspase-independent cell death.

depending upon cell type and insult stimuli.

inflammation and include FasL and TNF-α.

apoptosis even after the activation of the MAC.

**3.3.1 Apoptotic pathways** 

1999).

oxidative stresses, which are common in cerebral ischemia, are critical triggers of autophagy in neurons.

BH3-only proteins regulate autophagy under different settings, possibly by disrupting the interaction between beclin 1 and Bcl-2 or Bcl-xL via their BH3 domains (Bellot et al., 2009; Maiuri et al., 2007a). Prolonged expression or acute overexpression of BNIP3 beyond an autophagic survival threshold may result in autophagic cell death. Prolonged exposure to hypoxia of several apoptosis-competent cancer lines induces autophagy and cell death in a BNIP3-dependent manner. Beclin 1 liberation from Bcl-2 or Bcl-xL may be one of the mechanisms through which BH3-only members promote autophagy (Azad et al., 2008; Chinnadurai et al., 2008). BNIP3 may induce autophagy as a consequence of mitochondrial injury, as a loss of MPT induces autophagy (Elmore et al., 2001). Our lab has found a unique caspase-independent cell-death pathway that features the mitochondrial localization of BNIP3 followed by EndoG and AIF release from mitochondria and translocation into the nuclei, which results in cell death. Autophagy may play a part in this pathway by affecting mitochondrial stabilization or acting as a parallel cell-death-inducing pathway. Beclin 1 levels positively correlate with BNIP3 expression following ischemia. The increase in both proteins is accompanied by increased autophagic cell death that is inhibited by the autophagy inhibitor 3-methyladenine and by knockdown of BNIP3 with miRNA.

Autophagy and apoptosis can be triggered by upstream signals, often resulting in a mixed phenotype of both cell-death patterns. Neurons can switch between responses in a mutually exclusive manner. Both mechanisms are capable of inhibiting the other. Caspase inhibitors may arrest apoptosis but also promote autophagic cell death (Yu et al., 2004). Calpainmediated cleavage of Beclin 1 can switch autophagy to apoptosis (Yousefi et al., 2006). Pathways linking the apoptotic and autophagic machineries have been deciphered at the molecular level (Maiuri et al., 2007c; Rubinsztein et al., 2005).

### **3.3 Apoptosis**

Apoptosis is involved in cell development, differentiation, proliferation, homoeostasis, regulation, immune function, and removal of defective and harmful cells. In stroke, it is a mechanism of delayed neuronal death in response to ischemic injury. Key apoptotic proteins are activated and upregulated after cerebral ischemia, while inhibition of these proteins protects neurons from death (Chen et al., 1998).

Regulated apoptotic pathways activate cascades leading to cell suicide without the leakage of harmful cell contents. Main players in regulation include proteins from the Bcl-2 family, Smac/DIABLO (Du et al., 2000; Verhagen et al., 2000), HtrA2/Omi (Suzuki et al., 2001) and apoptotic protease-activating factor (Apaf-1) (Manfredi & Beal, 2000; Tatton & Olanow, 1999; Yuan & Yankner, 2000). Typical hallmarks of apoptosis include cell shrinkage and rounding, pyknosis and karyorhexis with DNA laddering on gel electrophoresis, membrane blebbing, and gradual disintegration of the cell into membrane-enclosed apoptotic bodies (Choi, 1996; Love, 2003; Zhang et al., 2004). Organelle structures, particularly mitochondria, are mostly preserved because apoptosis is an energy-consuming process (Friberg & Wieloch, 2002).

Coded proteins that are inactivated by covalent modifications or interactions with other anti-apoptotic regulatory molecules are necessary for pro-apoptotic signalling. Cell death stimuli are able to bring about cellular changes that remove the covalent modifications and block binding of anti-apoptotic regulators, thereby effecting apoptosis. In neurons, apoptosis can be carried out through a variety of discrete pathways, which can be categorized as either intrinsic or extrinsic. In neurons, intrinsic pathways can be triggered by intracellular damage that is caused by free radicals or excitotoxicity; extrinsic death pathways can be activated by tumour necrosis factors (TNF) or lack of neurotrophins and other growth factors. Once activated, both intrinsic and extrinsic pathways can trigger caspase-dependent or caspase-independent cell death.

### **3.3.1 Apoptotic pathways**

122 Advances in the Preclinical Study of Ischemic Stroke

oxidative stresses, which are common in cerebral ischemia, are critical triggers of autophagy

BH3-only proteins regulate autophagy under different settings, possibly by disrupting the interaction between beclin 1 and Bcl-2 or Bcl-xL via their BH3 domains (Bellot et al., 2009; Maiuri et al., 2007a). Prolonged expression or acute overexpression of BNIP3 beyond an autophagic survival threshold may result in autophagic cell death. Prolonged exposure to hypoxia of several apoptosis-competent cancer lines induces autophagy and cell death in a BNIP3-dependent manner. Beclin 1 liberation from Bcl-2 or Bcl-xL may be one of the mechanisms through which BH3-only members promote autophagy (Azad et al., 2008; Chinnadurai et al., 2008). BNIP3 may induce autophagy as a consequence of mitochondrial injury, as a loss of MPT induces autophagy (Elmore et al., 2001). Our lab has found a unique caspase-independent cell-death pathway that features the mitochondrial localization of BNIP3 followed by EndoG and AIF release from mitochondria and translocation into the nuclei, which results in cell death. Autophagy may play a part in this pathway by affecting mitochondrial stabilization or acting as a parallel cell-death-inducing pathway. Beclin 1 levels positively correlate with BNIP3 expression following ischemia. The increase in both proteins is accompanied by increased autophagic cell death that is inhibited by the

autophagy inhibitor 3-methyladenine and by knockdown of BNIP3 with miRNA.

molecular level (Maiuri et al., 2007c; Rubinsztein et al., 2005).

proteins protects neurons from death (Chen et al., 1998).

Autophagy and apoptosis can be triggered by upstream signals, often resulting in a mixed phenotype of both cell-death patterns. Neurons can switch between responses in a mutually exclusive manner. Both mechanisms are capable of inhibiting the other. Caspase inhibitors may arrest apoptosis but also promote autophagic cell death (Yu et al., 2004). Calpainmediated cleavage of Beclin 1 can switch autophagy to apoptosis (Yousefi et al., 2006). Pathways linking the apoptotic and autophagic machineries have been deciphered at the

Apoptosis is involved in cell development, differentiation, proliferation, homoeostasis, regulation, immune function, and removal of defective and harmful cells. In stroke, it is a mechanism of delayed neuronal death in response to ischemic injury. Key apoptotic proteins are activated and upregulated after cerebral ischemia, while inhibition of these

Regulated apoptotic pathways activate cascades leading to cell suicide without the leakage of harmful cell contents. Main players in regulation include proteins from the Bcl-2 family, Smac/DIABLO (Du et al., 2000; Verhagen et al., 2000), HtrA2/Omi (Suzuki et al., 2001) and apoptotic protease-activating factor (Apaf-1) (Manfredi & Beal, 2000; Tatton & Olanow, 1999; Yuan & Yankner, 2000). Typical hallmarks of apoptosis include cell shrinkage and rounding, pyknosis and karyorhexis with DNA laddering on gel electrophoresis, membrane blebbing, and gradual disintegration of the cell into membrane-enclosed apoptotic bodies (Choi, 1996; Love, 2003; Zhang et al., 2004). Organelle structures, particularly mitochondria, are mostly preserved because apoptosis is an energy-consuming process (Friberg & Wieloch,

Coded proteins that are inactivated by covalent modifications or interactions with other anti-apoptotic regulatory molecules are necessary for pro-apoptotic signalling. Cell death stimuli are able to bring about cellular changes that remove the covalent modifications and block binding of anti-apoptotic regulators, thereby effecting apoptosis. In neurons,

in neurons.

**3.3 Apoptosis** 

2002).

The caspase family of proteases is the most common and best understood mediators of apoptosis. In humans, at least seven caspases are implicated in apoptosis, including the initiator caspases 2, 8, 9, and 10, and the executioner caspases 3, 6, and 7 (Kroemer & Martin, 2005). Activated initiator caspases are able to cleave themselves and downstream targets, causing a cascade of caspase activation culminating at the executioner caspases, which have cell structures as their substrates and directly induce apoptosis. Caspase-activated deoxyribonuclease (CAD) causes the characteristic laddered DNA fragmentation observed when its inhibitor, ICAD, is cleaved by executioner caspases (Liu et al., 1997; Liu et al., 1999).

Following cerebral ischemia, the caspase cascade can be initiated early on through cell-death receptors or by mitochondrially mediated pathways (Ashkenazi & Dixit, 1998). The two mechanisms are not necessarily mutually exclusive and can be activated sequentially depending upon cell type and insult stimuli.

The Fas receptor, a primary death receptor in ischemia-induced apoptosis (Ferrer & Planas, 2003), belongs to the TNF receptor (TNFR) family and is specific for the Fas ligand (FasL) expressed on T cells. Activation of the Fas receptor causes formation of the cell-deathinducing signalling complex (DISC), which activates caspase-8 through the Fas-associated death domain (FADD). Caspase-8 can then activate caspase-3 to bring about apoptosis or activate the mitochondrial death pathway by cleaving Bid, a promoter for mitochondrial apoptosis-induced channel (MAC) formation (Planas et al., 1997). TNFR1 is also a member of the TNFR family, which induces apoptosis through a similar mechanism (Stanger et al., 1995). The upstream activators of the TNFRs in stroke models are increased during inflammation and include FasL and TNF-α.

The mitochondrial pathway is activated by inducing MOMP through the formation of the MAC, which is thought to be an oligomerized product of the Bcl-2 proteins Bax and Bak (Dejean et al., 2010; Martinez-Caballero et al., 2009). Regulation of pore formation is carried out by the Bcl-2 family, which includes anti-apoptotic proteins Bcl-2 and Bcl-xL and proapoptotic proteins Bid (which is cleaved to become the active tBid), Bim, and Bad (Gross et al., 1999; Imazu et al., 1999; Susin et al., 1996; Yang et al., 1997). Upon formation, the MAC allows cytochrome *c* release to the cytoplasm, where it interacts with Apaf-1 and dATP to form apoptosomes that cleave and activate caspase-9 (Zou et al., 1997). Caspase-9 then activates executioner caspases 3, 6, and 7 to bring about apoptosis. Smac/DIABLO and HtrA2/Omi are also released from the mitochondria along with cytochrome *c* (Du et al., 2000; Suzuki et al., 2001; Verhagen et al., 2000). Both promote apoptosis by respectively removing inhibitor of apoptosis protein (IAP)'s and X-linked inhibitor of apoptosis protein (XIAP)'s inhibition of caspase-3 and caspase-9 (Suzuki et al., 2001). The action of Smac/DIABLO is inhibited by Bcl-2 and Bcl-xL, which gives some degree of control over apoptosis even after the activation of the MAC.

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 125

EndoG efflux. The pro-apoptotic protein BNIP3 causes caspase-independent cell death in

The BNIP3 protein has four domains: a PEST domain that targets BNIP3 for degradation, a putative Bcl-2 homology 3 (BH3) domain that is homologous to those on other members of the Bcl-2 family, a CD domain that is conserved from *C. elegans* to humans, and a C-terminal transmembrane domain that is necessary for its mitochondrial localization and for its celldeath-inducing activity (Chen et al., 1999; Farooq et al., 2001; Yasuda et al., 1998). The BH3 domain is often necessary for Bcl-2 proteins to mediate cell death. BNIP3 possesses a BH3 domain that is not necessary for its cell-death-inducing ability in vivo and in vitro (Cizeau et al., 2000; Ray et al., 2000). The mechanism may operate independently of interaction with

BNIP3 is not detectable in normal neurons but is inducible under hypoxia in a variety of cells and tissues (Bruick, 2000; Guo et al., 2001; Sowter et al., 2001). The BNIP3 promoter contains a functional HIF-1 response element (HRE) that is activated by either hypoxia or forced expression of HIF-1α (Bruick, 2000). HIF-1α accumulation and subsequent activation

HIF-1 is a basic helix-loop-helix PAS domain (BHLH-PAS) transcription factor that normally regulates homeostatic responses to hypoxia in cells (Greijer & van der Wall, 2004). HIF-1 is composed of HIF-1α and HIF-1β; HIF-1 requires heterodimerization of both to function. HIF-1β is constitutively expressed, while HIF-1α expression and stability is dependent on intracellular oxygen levels. Under hypoxia, HIF-1α stabilizes and binds to HIF-1β in order to

Our work shows that hypoxia increases both BNIP3 and HIF-1α levels in neurons and that knockdown of HIF-1α expression is able to protect cells from hypoxia-induced death (Z. Zhang et al., 2007). Delayed neuronal death is also reduced when cortical neuron cultures are given a dominant-negative form of HIF-1α (HIFdn) via a herpes amplicon (Halterman et al., 1999). Our proposed pathway and other major caspase-independent pathways are

The root of ischemic damage can be traced to a loss of adequate oxygen and glucose due to interrupted blood flow. While this is a singular event responsible for most, if not all, subsequent neuronal death, it is unrealistic to design treatments that are able to restore blood flow in the few seconds to minutes before any damage occurs. Rather, treatment must focus on either prophylactic manipulations of these mechanisms or downstream pathways

**3.4.1 The BNIP3-activated and EndoG and AIF-mediated neuronal death pathway**  BNIP3 is part of a unique subfamily of death-inducing mitochondrial proteins that includes BNIP3, NIX, BNIP3h and a Caenorhabditis elegans ortholog, ceBNIP3. Expression of BNIP3 can induce death of various cells (Chen et al., 1997), including neurons (Zhang et al., 2007a; Zhang et al., 2007b; Zhang et al., 2007c). Cell death mediated by BNIP3 is characterized through cell transfection studies by early permeabilization of the plasma membrane and damage to the mitochondria without release of cytochrome *c* or activation of caspases (Cizeau et al., 2000; Ray et al., 2000). BNIP3 triggers mPTP opening, decreases mitochondrial membrane potential, and increases generation of ROS once it localizes to the mitochondrial

hypoxia and stroke through the action of EndoG (Zhang et al., 2007b).

outer membrane (Vande Velde et al., 2000).

of BNIP3 is induced by oxidative stress (Zhang et al., 2007a).

form HIF-1, which activates genes with HREs in their promoters.

the Bcl-2 family.

shown in figure 1.

**4. Therapy** 

The mitochondrial permeability transition pore (mPTP) is activated by excess Ca2+ levels, loss of voltage between inner and outer mitochondrial membranes, and high levels of free radicals. It is regulated by Bcl-2 proteins and is capable of releasing cytochrome *c* to bring about caspase-dependent apoptosis. The mPTP is often associated with excitotoxicity, which provides the requisite levels of Ca2+ needed to induce the mPTP to open (Ichas & Mazat, 1998; Martin, 2011). The mPTP is associated with cytochrome *c* release and various reactive oxygen species (ROS). It is involved in oxidative-stress-mediated apoptosis (Baumgartner et al., 2009).

### **3.4 Necrosis-like cell death**

Despite the prevalence of apoptosis in delayed neuronal death, there is another cell-death pathway capable of inducing cell death independently of caspase activation (Kim et al., 2005a; Kroemer & Martin, 2005; Lang-Rollin et al., 2003; Le et al., 2002; Lockshin & Zakeri, 2002). Because it is with features of necrosis, the caspase-independent cell death is also known as necrosis-like cell death (Vande Velde, et al. 2000). Preventing caspase activation by using broad caspase inhibitors such as zVAD-fmk or testing with caspase 3 or 9 knockouts provides only minor protection against cell death after brain ischemia (Himi et al., 1998; Kim et al., 2005b; Le et al., 2002). Dying neurons in the penumbra exhibit 50 kbp DNA fragments, which is atypical of the caspase-dependent chromatin fragmentation that usually results in fragments of 200-1000 bp (MacManus et al., 1997). These findings indicate that caspase-independent, or necrosis-like, cell-death pathways are probably involved in delayed neuronal death. AIF and EndoG may be important players in necrosis-like celldeath pathways (Cande et al., 2002; van Loo et al., 2001).

AIF is a mitochondrial protein localized in the inner mitochondrial membrane, where it is an oxidoreductase. Upon mitochondrial permeabilization, AIF is released into the cytoplasm and subsequently translocates into the nucleus, where it contributes to chromatin condensation and fragmentation (Krantic et al., 2007). The fragments produced are 50kbp in size, which is consistent with observations of caspase-independent cell death (Cao et al., 2003). Activation of the cell-death pathway ending in AIF is also independent of caspases, since the broad inhibitors zVAD-fmk and zDEVD-fmk do not provide neuroprotection. Inhibition of AIF or knockdown of AIF expression is able to protect against stroke-like conditions (Culmsee et al., 2005). Since AIF relies on passage through the MAC pore to cause cell death, the same regulators of the caspase-dependent mitochondrial pathway are applicable (Tsujimoto, 2003). Bcl-xL prevents AIF translocation to the nucleus (Cao et al., 2003), while tBid and Bax cause AIF efflux from the mitochondria (Cregan et al., 2002; van Loo et al., 2002). Since AIF is attached to the inner mitochondrial membrane, AIF is cleaved from the membrane before it can leave through mitochondrial pores (Donovan & Cotter, 2004). This step is not well-understood, though it is known to be caspase-independent and possibly carried out by tBid and Bax (Donovan & Cotter, 2004; Otera et al., 2005).

Endonuclease G (EndoG) is another well-established mediator of caspase-independent cell death (Li et al., 1997; van Loo et al., 2001). EndoG acts after transient cerebral ischemia (Lee et al., 2005) and oxygen-glucose deprivation (Tanaka et al., 2005), while working independently of caspase-activated DNase (Li et al., 2001; van Loo et al., 2001). Like AIF, EndoG is present in the mitochondrial inter-membrane space, localizes to the nucleus upon release, and causes cell death by cleaving chromatin into fragments. The Bcl-2 family moderates EndoG release from the mitochondria (Donovan & Cotter, 2004); tBid can cause

The mitochondrial permeability transition pore (mPTP) is activated by excess Ca2+ levels, loss of voltage between inner and outer mitochondrial membranes, and high levels of free radicals. It is regulated by Bcl-2 proteins and is capable of releasing cytochrome *c* to bring about caspase-dependent apoptosis. The mPTP is often associated with excitotoxicity, which provides the requisite levels of Ca2+ needed to induce the mPTP to open (Ichas & Mazat, 1998; Martin, 2011). The mPTP is associated with cytochrome *c* release and various reactive oxygen species (ROS). It is involved in oxidative-stress-mediated apoptosis (Baumgartner et

Despite the prevalence of apoptosis in delayed neuronal death, there is another cell-death pathway capable of inducing cell death independently of caspase activation (Kim et al., 2005a; Kroemer & Martin, 2005; Lang-Rollin et al., 2003; Le et al., 2002; Lockshin & Zakeri, 2002). Because it is with features of necrosis, the caspase-independent cell death is also known as necrosis-like cell death (Vande Velde, et al. 2000). Preventing caspase activation by using broad caspase inhibitors such as zVAD-fmk or testing with caspase 3 or 9 knockouts provides only minor protection against cell death after brain ischemia (Himi et al., 1998; Kim et al., 2005b; Le et al., 2002). Dying neurons in the penumbra exhibit 50 kbp DNA fragments, which is atypical of the caspase-dependent chromatin fragmentation that usually results in fragments of 200-1000 bp (MacManus et al., 1997). These findings indicate that caspase-independent, or necrosis-like, cell-death pathways are probably involved in delayed neuronal death. AIF and EndoG may be important players in necrosis-like cell-

AIF is a mitochondrial protein localized in the inner mitochondrial membrane, where it is an oxidoreductase. Upon mitochondrial permeabilization, AIF is released into the cytoplasm and subsequently translocates into the nucleus, where it contributes to chromatin condensation and fragmentation (Krantic et al., 2007). The fragments produced are 50kbp in size, which is consistent with observations of caspase-independent cell death (Cao et al., 2003). Activation of the cell-death pathway ending in AIF is also independent of caspases, since the broad inhibitors zVAD-fmk and zDEVD-fmk do not provide neuroprotection. Inhibition of AIF or knockdown of AIF expression is able to protect against stroke-like conditions (Culmsee et al., 2005). Since AIF relies on passage through the MAC pore to cause cell death, the same regulators of the caspase-dependent mitochondrial pathway are applicable (Tsujimoto, 2003). Bcl-xL prevents AIF translocation to the nucleus (Cao et al., 2003), while tBid and Bax cause AIF efflux from the mitochondria (Cregan et al., 2002; van Loo et al., 2002). Since AIF is attached to the inner mitochondrial membrane, AIF is cleaved from the membrane before it can leave through mitochondrial pores (Donovan & Cotter, 2004). This step is not well-understood, though it is known to be caspase-independent and

possibly carried out by tBid and Bax (Donovan & Cotter, 2004; Otera et al., 2005).

Endonuclease G (EndoG) is another well-established mediator of caspase-independent cell death (Li et al., 1997; van Loo et al., 2001). EndoG acts after transient cerebral ischemia (Lee et al., 2005) and oxygen-glucose deprivation (Tanaka et al., 2005), while working independently of caspase-activated DNase (Li et al., 2001; van Loo et al., 2001). Like AIF, EndoG is present in the mitochondrial inter-membrane space, localizes to the nucleus upon release, and causes cell death by cleaving chromatin into fragments. The Bcl-2 family moderates EndoG release from the mitochondria (Donovan & Cotter, 2004); tBid can cause

al., 2009).

**3.4 Necrosis-like cell death** 

death pathways (Cande et al., 2002; van Loo et al., 2001).

EndoG efflux. The pro-apoptotic protein BNIP3 causes caspase-independent cell death in hypoxia and stroke through the action of EndoG (Zhang et al., 2007b).

### **3.4.1 The BNIP3-activated and EndoG and AIF-mediated neuronal death pathway**

BNIP3 is part of a unique subfamily of death-inducing mitochondrial proteins that includes BNIP3, NIX, BNIP3h and a Caenorhabditis elegans ortholog, ceBNIP3. Expression of BNIP3 can induce death of various cells (Chen et al., 1997), including neurons (Zhang et al., 2007a; Zhang et al., 2007b; Zhang et al., 2007c). Cell death mediated by BNIP3 is characterized through cell transfection studies by early permeabilization of the plasma membrane and damage to the mitochondria without release of cytochrome *c* or activation of caspases (Cizeau et al., 2000; Ray et al., 2000). BNIP3 triggers mPTP opening, decreases mitochondrial membrane potential, and increases generation of ROS once it localizes to the mitochondrial outer membrane (Vande Velde et al., 2000).

The BNIP3 protein has four domains: a PEST domain that targets BNIP3 for degradation, a putative Bcl-2 homology 3 (BH3) domain that is homologous to those on other members of the Bcl-2 family, a CD domain that is conserved from *C. elegans* to humans, and a C-terminal transmembrane domain that is necessary for its mitochondrial localization and for its celldeath-inducing activity (Chen et al., 1999; Farooq et al., 2001; Yasuda et al., 1998). The BH3 domain is often necessary for Bcl-2 proteins to mediate cell death. BNIP3 possesses a BH3 domain that is not necessary for its cell-death-inducing ability in vivo and in vitro (Cizeau et al., 2000; Ray et al., 2000). The mechanism may operate independently of interaction with the Bcl-2 family.

BNIP3 is not detectable in normal neurons but is inducible under hypoxia in a variety of cells and tissues (Bruick, 2000; Guo et al., 2001; Sowter et al., 2001). The BNIP3 promoter contains a functional HIF-1 response element (HRE) that is activated by either hypoxia or forced expression of HIF-1α (Bruick, 2000). HIF-1α accumulation and subsequent activation of BNIP3 is induced by oxidative stress (Zhang et al., 2007a).

HIF-1 is a basic helix-loop-helix PAS domain (BHLH-PAS) transcription factor that normally regulates homeostatic responses to hypoxia in cells (Greijer & van der Wall, 2004). HIF-1 is composed of HIF-1α and HIF-1β; HIF-1 requires heterodimerization of both to function. HIF-1β is constitutively expressed, while HIF-1α expression and stability is dependent on intracellular oxygen levels. Under hypoxia, HIF-1α stabilizes and binds to HIF-1β in order to form HIF-1, which activates genes with HREs in their promoters.

Our work shows that hypoxia increases both BNIP3 and HIF-1α levels in neurons and that knockdown of HIF-1α expression is able to protect cells from hypoxia-induced death (Z. Zhang et al., 2007). Delayed neuronal death is also reduced when cortical neuron cultures are given a dominant-negative form of HIF-1α (HIFdn) via a herpes amplicon (Halterman et al., 1999). Our proposed pathway and other major caspase-independent pathways are shown in figure 1.

### **4. Therapy**

The root of ischemic damage can be traced to a loss of adequate oxygen and glucose due to interrupted blood flow. While this is a singular event responsible for most, if not all, subsequent neuronal death, it is unrealistic to design treatments that are able to restore blood flow in the few seconds to minutes before any damage occurs. Rather, treatment must focus on either prophylactic manipulations of these mechanisms or downstream pathways

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 127

experimental models of ischemia and in various in vivo focal and global ischemia/reperfusion models (Letechipia-Vallejo et al., 2001; Sinha et al., 2001). Melatonin's protective mechanism may lie in its ability to bolster intracellular antioxidative mechanisms. Glutathione peroxidase activity is upregulated by melatonin, as are the gene expressions of Mn-SOD and Cu/Zn-SOD, while preventing the activation of the transcription factor NF-κB. Oxidative stress can activate several cell signalling cascades that may trigger further damage and cell-death programs. Targeting the messengers that mediate this crosstalk may prove as a viable strategy for preventing cell death. The mitogen-activated protein kinases (MAPKs) such as p38, ERK, and JNK/SAPK are important mediators of cell survival and death following ischemic injury; their activation can lead to cell death. Inhibition of their activity reduces cell damage and results in neuroprotection. Other immediate events downstream of oxidative stress, such as degradation of membranes and production of arachidonic acid by

Blocking autophagy more than four hours after cerebral ischemia can be neuroprotective (Puyal & Clarke, 2009), despite controversy about autophagy's role as a protective or damaging mechanism. 3-methyladenine, injected intracerebroventricularly following stroke, reduces the volume of the lesion by almost half, even when given hours after a stroke has occurred. Knockdown of Atg7, the gene coding for beclin 1, also provides protection against hypoxia and ischemia (Koike et al., 2008; Nitatori et al., 1995). Other methods of downregulating beclin 1, and even BNIP3, should yield protection against autophagic cell death. Since there is the possibility that autophagy serves a mainly protective function in some neurons, too broad or unspecific an inhibition may exacerbate injury from stroke. More research needs to be done to determine the exact effects of blocking these autophagy

Hsp70 expression is related to a neuron's ability to survive an ischemic insult. During ischemia, Hsp expression depends on activation of NMDA receptors (Ahn et al., 2008; Lipton, 1999; Saleh et al., 2009). While this receptor is an attractive target for neuroprotection, it must be noted that NMDA receptor overstimulation may play a major role in excitotoxicity, since it mediates calcium influx. If treatment strategies are to be pursued, a balance must be established between the activation of Hsp70 expression and exacerbation of excitotoxic damage. A possible solution is to use melatonin, which is capable of inducing Hsp70 upregulation and has antioxidative effects. Gene therapy to induce the expression of Hsp72 is effective in mice and may also be an option once the technology

Hsp-related therapy primarily relies on preconditioning. Hsps have protective effects only when they exist at sufficient levels in the cytoplasm. That is an unlikely scenario for a patient during a stroke, where no precondition has occurred, ischemic onset is quick and severe, and protein synthesis is halted or slowed. Most of the evidence for Hsp neuroprotection involves pretreatment to induce Hsp expression prior to the ischemic insult. Hsp-based treatment might find utility during reperfusion, if its expression can be induced rapidly and sufficiently and is shown to offer protection against this second wave

phospholipases, may be potential therapeutic targets in stroke.

**4.2 Autophagy-related therapy** 

inducers.

**4.3 Hsp-related therapy** 

becomes more mature.

of injury.

Fig. 1. Caspase-independent cell-death pathways. HIF-1 is induced directly by hypoxia or by oxidative stress and activates the expression of BNIP3 to cause the mitochondrial release of EndoG and AIF. Translocation of EndoG and AIF to the nucleus results in neuronal cell death without cytochrome c release and caspase activation. Bcl-2/Bcl-xL normally binds with beclin 1 to inhibit its activity. Sufficient BNIP3 displacement of Bcl-2/Bcl-xL from beclin 1 can cause runaway autophagy resulting in cell death. Immediate energy failure following stroke or hypoxia results in calcium disregulation and influx, triggering ROS production, phospholipase activity, and the calpain-cathepsin pathway. These processes can effect caspase-independent cell death.

that involve oxidative stress, energy depletion, ion deregulation, loss of protein synthesis, and activation of a host of protective and cell-death-inducing internal cell mechanisms. Due to the complex interactions that lead to delayed neuronal death in stroke, multi-approach strategies must be used. A comprehensive approach targeting as many pathways as possible would theoretically yield the best patient outcomes.

### **4.1 Antioxidants**

Targeting a wide range of proteins and mechanisms involved in oxidative stress may provide beneficial therapeutic interventions for ischemia and reperfusion injury. Application of antioxidant compounds appears to be effective in combating oxidative stress in stroke (Huang et al., 2001). Antioxidant enzymes may protect against apoptosis after cerebral ischemia and reperfusion. Superoxide dismutase (SOD) has a protective role against focal cerebral ischemia. SOD-1 overexpression attenuates apoptotic cell death (Saito et al., 2004).

Melatonin is known for its neuroprotective free radical scavenging and antioxidant properties and may be a candidate for protecting against delayed neuronal death. Melatonin can readily cross the blood–brain barrier and effectively prevents neuronal loss in experimental models of ischemia and in various in vivo focal and global ischemia/reperfusion models (Letechipia-Vallejo et al., 2001; Sinha et al., 2001). Melatonin's protective mechanism may lie in its ability to bolster intracellular antioxidative mechanisms. Glutathione peroxidase activity is upregulated by melatonin, as are the gene expressions of Mn-SOD and Cu/Zn-SOD, while preventing the activation of the transcription factor NF-κB. Oxidative stress can activate several cell signalling cascades that may trigger further damage and cell-death programs. Targeting the messengers that mediate this crosstalk may prove as a viable strategy for preventing cell death. The mitogen-activated protein kinases (MAPKs) such as p38, ERK, and JNK/SAPK are important mediators of cell survival and death following ischemic injury; their activation can lead to cell death. Inhibition of their activity reduces cell damage and results in neuroprotection. Other immediate events downstream of oxidative stress, such as degradation of membranes and production of arachidonic acid by phospholipases, may be potential therapeutic targets in stroke.

### **4.2 Autophagy-related therapy**

126 Advances in the Preclinical Study of Ischemic Stroke

Fig. 1. Caspase-independent cell-death pathways. HIF-1 is induced directly by hypoxia or by oxidative stress and activates the expression of BNIP3 to cause the mitochondrial release of EndoG and AIF. Translocation of EndoG and AIF to the nucleus results in neuronal cell death without cytochrome c release and caspase activation. Bcl-2/Bcl-xL normally binds with beclin 1 to inhibit its activity. Sufficient BNIP3 displacement of Bcl-2/Bcl-xL from beclin 1 can cause runaway autophagy resulting in cell death. Immediate energy failure following stroke or hypoxia results in calcium disregulation and influx, triggering ROS production, phospholipase activity, and the calpain-cathepsin pathway. These processes can

that involve oxidative stress, energy depletion, ion deregulation, loss of protein synthesis, and activation of a host of protective and cell-death-inducing internal cell mechanisms. Due to the complex interactions that lead to delayed neuronal death in stroke, multi-approach strategies must be used. A comprehensive approach targeting as many pathways as possible

Targeting a wide range of proteins and mechanisms involved in oxidative stress may provide beneficial therapeutic interventions for ischemia and reperfusion injury. Application of antioxidant compounds appears to be effective in combating oxidative stress in stroke (Huang et al., 2001). Antioxidant enzymes may protect against apoptosis after cerebral ischemia and reperfusion. Superoxide dismutase (SOD) has a protective role against focal cerebral ischemia. SOD-1 overexpression attenuates apoptotic cell death (Saito et al.,

Melatonin is known for its neuroprotective free radical scavenging and antioxidant properties and may be a candidate for protecting against delayed neuronal death. Melatonin can readily cross the blood–brain barrier and effectively prevents neuronal loss in

effect caspase-independent cell death.

**4.1 Antioxidants** 

2004).

would theoretically yield the best patient outcomes.

Blocking autophagy more than four hours after cerebral ischemia can be neuroprotective (Puyal & Clarke, 2009), despite controversy about autophagy's role as a protective or damaging mechanism. 3-methyladenine, injected intracerebroventricularly following stroke, reduces the volume of the lesion by almost half, even when given hours after a stroke has occurred. Knockdown of Atg7, the gene coding for beclin 1, also provides protection against hypoxia and ischemia (Koike et al., 2008; Nitatori et al., 1995). Other methods of downregulating beclin 1, and even BNIP3, should yield protection against autophagic cell death. Since there is the possibility that autophagy serves a mainly protective function in some neurons, too broad or unspecific an inhibition may exacerbate injury from stroke. More research needs to be done to determine the exact effects of blocking these autophagy inducers.

### **4.3 Hsp-related therapy**

Hsp70 expression is related to a neuron's ability to survive an ischemic insult. During ischemia, Hsp expression depends on activation of NMDA receptors (Ahn et al., 2008; Lipton, 1999; Saleh et al., 2009). While this receptor is an attractive target for neuroprotection, it must be noted that NMDA receptor overstimulation may play a major role in excitotoxicity, since it mediates calcium influx. If treatment strategies are to be pursued, a balance must be established between the activation of Hsp70 expression and exacerbation of excitotoxic damage. A possible solution is to use melatonin, which is capable of inducing Hsp70 upregulation and has antioxidative effects. Gene therapy to induce the expression of Hsp72 is effective in mice and may also be an option once the technology becomes more mature.

Hsp-related therapy primarily relies on preconditioning. Hsps have protective effects only when they exist at sufficient levels in the cytoplasm. That is an unlikely scenario for a patient during a stroke, where no precondition has occurred, ischemic onset is quick and severe, and protein synthesis is halted or slowed. Most of the evidence for Hsp neuroprotection involves pretreatment to induce Hsp expression prior to the ischemic insult. Hsp-based treatment might find utility during reperfusion, if its expression can be induced rapidly and sufficiently and is shown to offer protection against this second wave of injury.

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 129

These caspase inhibitors can be a valuable tool to combat delayed neuronal death, despite many of them being unable to cross the blood brain barrier. Most studies involve the direct injection of the inhibitors into brain tissue or intraventricular space. Seeing as intrinsic caspase-dependent cell death depends on mitochondrial permeability, there is a chance that blocking caspase activation may allow caspase-independent death pathways to occur. As a result, inhibiting only caspases may allow a number of cells to die by alternative means. Caspase inhibitors alone do not help in preserving long-term potentiation and plasticity of neurons after ischemia (Gillardon et al., 1999). Theoretically, blocking as many of the cell death signalling pathways as possible may maximize

A concern with the use of caspase inhibitors in therapy or inhibiting apoptosis in general is that it may increase the probability of developing cancer or autoimmune disorders. This risk must be balanced against the potential neuroprotective effects of directly inhibiting apoptosis. This risk may be minimized if the inhibitors are localized as much as possible to

Recently, more therapeutic strategies have been targeted towards caspase-independent cell death that is mediated by AIF and EndoG. Reducing the levels of AIF in a cell by using neutralizing antibodies (Cregan et al., 2002), RNAi (Strosznajder & Gajkowska, 2006) or gene knockout (Klein et al., 2002) is strongly neuroprotective. Downregulation of EndoG activity has been explored. Our team has found that RNAi inhibition of BNIP3 reduces EndoG translocation and is neuroprotective against hypoxia-induced cell death (Zhang et al., 2007b). Other studies have found that mutant heterozygosity for EndoG in transgenic

AIF and EndoG release can be inhibited by preventing mitochondrial outer membrane permeabilization (MOMP). Blocking MAC activation or preventing mitochondrial rupturing may be neuroprotective. Seeing as most stroke patients are treated for hours after a stroke occurs, when MOMP has already been induced, strategies centred on preventing mitochondrial release of death promoters are limited. Some benefit may still exist for those cases receiving prompt intervention, when treatment can prevent MOMP in affected but not yet compromised mitochondria. Preventing MOMP while simultaneously targeting

Hsp70 is capable of inhibiting AIF release from the mitochondria. This mechanism may be dependent on the C-terminal region of Hsp70 rather than its enzymatic activity (Sun et al., 2006). Hsp70 may be capable of inhibiting the nuclease functions of EndoG in an ATPdependent manner as well (Kalinowska et al., 2005). Hsp70 may offer neuroprotection

MOMP inhibition by targeting upstream factors has achieved significant levels of neuroprotection in vivo and is another therapeutic possibility. For example, it has been found that inhibiting the family of MAPKs can protect against ischemic damage. Treating mice through inhibition of p53 by genetic (Morrison et al., 1996), pharmacological (Culmsee et al., 2001) means, or by using blockers of the JNK signalling pathway (Gao et al., 2005; Guan et al., 2006) has resulted in neuroprotection against ischemia and excitotoxicity,

mice provides resistance to TNF-α-induced cell death (Zhang et al., 2003).

downstream death effectors may prevent cell death (Galluzzi et al., 2009).

presumably in part by reducing mitochondrial permeability.

neuroprotection.

the infarct region.

**4.6 AIF and EndoG** 

through a multitude of pathways.

A recent study has implicated Hsp70 in blocking the release of AIF from the mitochondria. This may be an additional mechanism for preventing delayed neuronal death by inhibiting the activation of caspase-dependent cell-death pathways (Ruchalski et al., 2006).

### **4.4 Protective effects of exogenous growth factors**

Neurotrophins, like Hsps, are exploitable as neuroprotective elements. Exogenous BDNF protects against delayed neuronal death in the rat (Beck et al., 1994; Tsukahara et al., 1994) after ischemia. Administration of VEGF is neuroprotective through inhibition of apoptosis (Hayashi et al., 1998; Jin et al., 2001; Manoonkitiwongsa et al., 2004). Gene therapy strategies for GDNF are also promising (Harvey et al., 2005; Shirakura et al., 2003; Tsai et al., 2000)**.**  Other neurotrophins are similarly able to exert protective actions by inhibiting death or triggering protective mechanisms. Neurotrophins suffer the drawback of being difficult to deliver. Many require administration before or immediately after an ischemic incident to be effective. Various methods have been devised to target neurotrophins to neurons in order to reduce delayed neuronal death.

Neurotrophins are difficult to localize to the neurons in a clinical setting. Most do not cross the blood-brain barrier, and large doses to overcome the minimal localization to brain neurons result in harmful side effects (Ferrer, 2006). The use of viral or ligand vectors to carry neurotrophins have had some success in ischemic models. Murine monoclonal antibody against rat transferrin receptors (OX26-SA) linked to a neurotrophin is capable of neuroprotection when injected into the carotid arteries, though treatment must be promptly administered after ischemia to observe any protective effects (Wu, 2005). Targeting also allows for lower doses to be used, which overcomes the obstacle of otherwise inducing side effects.

At least one study has found an increase in neuronal necrosis following BDNF pre-treatment in cell culture while reducing apoptosis in the same cells (Koh et al., 1995). The mechanism may be via the potentiation of NMDA-mediated Ca2+ influx, which can amplify excitotoxic effects. Another explanation may be that BDNF exacerbates free-radical-induced cell death (Gwag et al., 1995). A patient who has suffered a stroke would virtually never have received pre-treatment with neurotrophins, but the fact that neurotrophins could inadvertently exacerbate damage under certain conditions (Gwag et al., 1995) should be considered when designing neuroprotective strategies.

### **4.5 Caspase inhibitors**

Caspases and their associated players in apoptosis may also be viable targets for preventing delayed neuronal death. Caspase inhibitors, such as the specific caspase-1 inhibitor Ac-WEHD-CHO, and broad capase inhibitors, such as z-VAD-fmk, protect against delayed neuronal death in CA-1 pyramidal cells (Hayashi et al., 2001). Injection of benzyloxycarbonyl-Asp-CH2-dichlorobenzene, a permanent inhibitor of caspases, also offers protection against delayed neuronal death by delaying chromatin condensation and DNA fragmentation (Himi et al., 1998). Administration of the broad inhibitors z-VAD-fmk and z-DEVD-fmk preserves neurological functions in addition to attenuating delayed death (Endres et al., 1998). Upregulation of the activity of intracellular caspase inhibitors is also an option. Induced overexpression of XIAP using viral vectors shows neuroprotective effects (Xu et al., 1999). UCF-101, an HtrA2/Omi inhibitor, prevents apoptosis by regulating Fasmediated proteins in extrinsic apoptosis as well.

These caspase inhibitors can be a valuable tool to combat delayed neuronal death, despite many of them being unable to cross the blood brain barrier. Most studies involve the direct injection of the inhibitors into brain tissue or intraventricular space. Seeing as intrinsic caspase-dependent cell death depends on mitochondrial permeability, there is a chance that blocking caspase activation may allow caspase-independent death pathways to occur. As a result, inhibiting only caspases may allow a number of cells to die by alternative means. Caspase inhibitors alone do not help in preserving long-term potentiation and plasticity of neurons after ischemia (Gillardon et al., 1999). Theoretically, blocking as many of the cell death signalling pathways as possible may maximize neuroprotection.

A concern with the use of caspase inhibitors in therapy or inhibiting apoptosis in general is that it may increase the probability of developing cancer or autoimmune disorders. This risk must be balanced against the potential neuroprotective effects of directly inhibiting apoptosis. This risk may be minimized if the inhibitors are localized as much as possible to the infarct region.

### **4.6 AIF and EndoG**

128 Advances in the Preclinical Study of Ischemic Stroke

A recent study has implicated Hsp70 in blocking the release of AIF from the mitochondria. This may be an additional mechanism for preventing delayed neuronal death by inhibiting

Neurotrophins, like Hsps, are exploitable as neuroprotective elements. Exogenous BDNF protects against delayed neuronal death in the rat (Beck et al., 1994; Tsukahara et al., 1994) after ischemia. Administration of VEGF is neuroprotective through inhibition of apoptosis (Hayashi et al., 1998; Jin et al., 2001; Manoonkitiwongsa et al., 2004). Gene therapy strategies for GDNF are also promising (Harvey et al., 2005; Shirakura et al., 2003; Tsai et al., 2000)**.**  Other neurotrophins are similarly able to exert protective actions by inhibiting death or triggering protective mechanisms. Neurotrophins suffer the drawback of being difficult to deliver. Many require administration before or immediately after an ischemic incident to be effective. Various methods have been devised to target neurotrophins to neurons in order to

Neurotrophins are difficult to localize to the neurons in a clinical setting. Most do not cross the blood-brain barrier, and large doses to overcome the minimal localization to brain neurons result in harmful side effects (Ferrer, 2006). The use of viral or ligand vectors to carry neurotrophins have had some success in ischemic models. Murine monoclonal antibody against rat transferrin receptors (OX26-SA) linked to a neurotrophin is capable of neuroprotection when injected into the carotid arteries, though treatment must be promptly administered after ischemia to observe any protective effects (Wu, 2005). Targeting also allows for lower doses to be used, which overcomes the obstacle of otherwise inducing side

At least one study has found an increase in neuronal necrosis following BDNF pre-treatment in cell culture while reducing apoptosis in the same cells (Koh et al., 1995). The mechanism may be via the potentiation of NMDA-mediated Ca2+ influx, which can amplify excitotoxic effects. Another explanation may be that BDNF exacerbates free-radical-induced cell death (Gwag et al., 1995). A patient who has suffered a stroke would virtually never have received pre-treatment with neurotrophins, but the fact that neurotrophins could inadvertently exacerbate damage under certain conditions (Gwag et al., 1995) should be considered when

Caspases and their associated players in apoptosis may also be viable targets for preventing delayed neuronal death. Caspase inhibitors, such as the specific caspase-1 inhibitor Ac-WEHD-CHO, and broad capase inhibitors, such as z-VAD-fmk, protect against delayed neuronal death in CA-1 pyramidal cells (Hayashi et al., 2001). Injection of benzyloxycarbonyl-Asp-CH2-dichlorobenzene, a permanent inhibitor of caspases, also offers protection against delayed neuronal death by delaying chromatin condensation and DNA fragmentation (Himi et al., 1998). Administration of the broad inhibitors z-VAD-fmk and z-DEVD-fmk preserves neurological functions in addition to attenuating delayed death (Endres et al., 1998). Upregulation of the activity of intracellular caspase inhibitors is also an option. Induced overexpression of XIAP using viral vectors shows neuroprotective effects (Xu et al., 1999). UCF-101, an HtrA2/Omi inhibitor, prevents apoptosis by regulating Fas-

the activation of caspase-dependent cell-death pathways (Ruchalski et al., 2006).

**4.4 Protective effects of exogenous growth factors** 

reduce delayed neuronal death.

designing neuroprotective strategies.

mediated proteins in extrinsic apoptosis as well.

**4.5 Caspase inhibitors** 

effects.

Recently, more therapeutic strategies have been targeted towards caspase-independent cell death that is mediated by AIF and EndoG. Reducing the levels of AIF in a cell by using neutralizing antibodies (Cregan et al., 2002), RNAi (Strosznajder & Gajkowska, 2006) or gene knockout (Klein et al., 2002) is strongly neuroprotective. Downregulation of EndoG activity has been explored. Our team has found that RNAi inhibition of BNIP3 reduces EndoG translocation and is neuroprotective against hypoxia-induced cell death (Zhang et al., 2007b). Other studies have found that mutant heterozygosity for EndoG in transgenic mice provides resistance to TNF-α-induced cell death (Zhang et al., 2003).

AIF and EndoG release can be inhibited by preventing mitochondrial outer membrane permeabilization (MOMP). Blocking MAC activation or preventing mitochondrial rupturing may be neuroprotective. Seeing as most stroke patients are treated for hours after a stroke occurs, when MOMP has already been induced, strategies centred on preventing mitochondrial release of death promoters are limited. Some benefit may still exist for those cases receiving prompt intervention, when treatment can prevent MOMP in affected but not yet compromised mitochondria. Preventing MOMP while simultaneously targeting downstream death effectors may prevent cell death (Galluzzi et al., 2009).

Hsp70 is capable of inhibiting AIF release from the mitochondria. This mechanism may be dependent on the C-terminal region of Hsp70 rather than its enzymatic activity (Sun et al., 2006). Hsp70 may be capable of inhibiting the nuclease functions of EndoG in an ATPdependent manner as well (Kalinowska et al., 2005). Hsp70 may offer neuroprotection through a multitude of pathways.

MOMP inhibition by targeting upstream factors has achieved significant levels of neuroprotection in vivo and is another therapeutic possibility. For example, it has been found that inhibiting the family of MAPKs can protect against ischemic damage. Treating mice through inhibition of p53 by genetic (Morrison et al., 1996), pharmacological (Culmsee et al., 2001) means, or by using blockers of the JNK signalling pathway (Gao et al., 2005; Guan et al., 2006) has resulted in neuroprotection against ischemia and excitotoxicity, presumably in part by reducing mitochondrial permeability.

Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 131

used in studies successfully demonstrating neuroprotection (Babu & Ramanathan, 2011;

Some potential therapeutic targets may alleviate calcium-induced neuronal damage. Calcium/calmodulin-dependent protein kinase kinase (CaM-KK) protects against delayed apoptosis following glutamate by activating Atk and CaM kinase IV (Yano et al., 2005), which both are anti-apoptotic players. Nimodipine (Mossakowski & Gadamski, 1990; Nuglisch et al., 1990), dantrolene (Wei & Perry, 1996), and the tetrapeptide Tyr-Val-Ala-Aspchloromethyl ketone (Ac-YVAD-cmk) (Gray et al., 2001) are all able to block damage due to high cytosolic Ca2+ levels in a variety of stroke models and may be useful in preventing

Energy depletion plays a large role in excitotoxicity. Methods that selectively inhibit poly (ADP-ribose) polymerase-1 (PARP-1) and PARP-2 offer neuroprotection (Chiarugi, 2005) by counteracting energy-consuming activities following ischemia and reducing the drop in high-energy molecules. Additional evidence implicates PARP in a pathway capable of inducing AIF release and activation (Niimura et al., 2006), which indicates that therapies targeting PARP may have a protective effect against AIF-mediated delayed neuronal death. Drugs that inhibit PHRP activation, such as PJ34 (Xu et al., 2010) or hepatocyte growth

factor (Niimura et al., 2006), may be useful as part of multimodal early interventions.

Neuronal cell death following stroke occurs in necrosis, apoptosis and other alternative modes and is mediated through diverse molecular pathways. These pathways provide

The authors would like to thank Ms. Jacqueline Hogue for her assistance in preparing the manuscript. This work was supported by grants from the Canadian Institutes of Health Research, Canadian Stroke Network and Manitoba Health Research Council (to J. Kong) and

Adhami, F., Liao, G., Morozov, Y. M., Schloemer, A., Schmithorst, V. J., Lorenz, J. N., Dunn,

Ahn, J., Piri, N., Caprioli, J., Munemasa, Y., Kim, S. H. & Kwong, J. M. (2008). Expression of

R. S., Vorhees, C. V., Wills-Karp, M., Degen, J. L., Davis, R. J., Mizushima, N., Rakic, P., Dardzinski, B. J., Holland, S. K., Sharp, F. R. & Kuan, C. Y. (2006). Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy. *Am J Pathol*

heat shock transcription factors and heat shock protein 72 in rat retina after intravitreal injection of low dose N-methyl-D-aspartate. *Neurosci Lett* 433, 11-6. Aita, V. M., Liang, X. H., Murty, V. V., Pincus, D. L., Yu, W., Cayanis, E., Kalachikov, S.,

Gilliam, T. C. & Levine, B. (1999). Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. *Genomics* 59, 59-65. Ashkenazi, A. & Dixit, V. M. (1998). Death receptors: signaling and modulation. *Science* 281,

Prass & Dirnagl, 1998).

excitotoxic damage.

**5. Conclusions** 

**6. Acknowledgment** 

**7. References** 

169, 566-83.

1305-8.

therapeutic targets for stroke management.

Shanghai Health Bureau Foundation (2008-2010,2008087 to X. Bi).

### **4.7 MOMP prevention by targeting Bcl-2 proteins**

Regulating Bcl-2 proteins provides protection against delayed neuronal death by preserving mitochondrial integrity. The MAC pore is key in regulating mitochondrial permeability and is under the control of the Bcl-2 family of proteins. Inhibition or upregulation of select members by genetic or pharmacological means can modulate the downstream activation of caspase-dependent apoptosis and AIF- or EndoG-mediated necrosis-like cell death; and they have been investigated for treatment strategies.

Inhibiting the pro-apoptotic BH3-only Bcl-2 proteins prevents MOMP, providing protection against mitochondria-mediated cell death. Pharmacologically blocking Bid with 4-phenylsulfanyl-phenylamine derivatives prevents tBid-induced Smac release, AIF release, caspase-3 activation, and nuclear condensation (Culmsee & Plesnila, 2006; Culmsee et al., 2005). Knockouts of Bid (Plesnila et al., 2002; Plesnila et al., 2001) or Bax genes (Gibson et al., 2001; Tehranian et al., 2008) protect against ischemic cell death in stroke models as well.

Genetic means of boosting the effects of Bcl-2 antiapoptotic proteins provide neuroprotection. Transgenically upregulating the protective Bcl-2 gene provides protection in mice of neurons injured by ischemia (Martinou et al., 1994). A similar effect can be observed when human Bcl-2 is overexpressed with herpes simplex virus vectors (Linnik et al., 1995). Gene therapy using adeno-associated viruses carrying the Bcl-2 gene is also effective (Shimazaki et al., 2000). Human gene therapy, while powerful, is not yet mature, so it will take time for these approaches to be proven effective and integrated into a clinical setting. In the meantime, other methods of upregulating protective Bcl-2 members should be explored.

BDNF is capable of regulating cell-death pathways. BDNF is capable of counter-regulating Bax and Bcl-2 when administered intravenously after ischemia (Schabitz et al., 2000). Neuroprotection is achieved by conjugating the product of the bcl-x gene with the HIV-Tat PTD as a method of delivery (Asoh et al., 2002). The upregulation of Bcl-2 and downregulation of Bax is implicated as part of hypothermia's protective mechanism against ischemic damage. These and other methods for regulating the Bcl-2 family may prove clinically relevant and could be examined for extra neuroprotection when used in combination or with therapies targeting different cell death mechanisms.

### **4.8 Excitotoxicity and calcium-mediated damage prevention**

Excitotoxic damage due to massive Ca2+ influx should be reduced to some extent by either preventing ion disturbance or targeting the resulting structural damage by calpains, free radical production, and caspase activation. Free radical production and caspase activation may demonstrate protective action for mild excitotoxic stress causing delayed neuronal death.

Neuroprotection by blocking glutamate release and reception has been attempted with some success. In experimental stroke models, glutamate blockade provides protection against cell death, but the results do not necessarily translate into human therapy. Blocking the release of glutamate and other excitatory amino acids through the use of various drugs has been unsuccessful in human trials. Too little is known about neuroprotection to rule out glutamate blockers entirely. It is possible that treatment in these trials occurred too late and was unable to block the excitotoxic chain reaction. If the drugs were administered within a couple hours of stroke, then the time conditions would be similar to those found in models used in studies successfully demonstrating neuroprotection (Babu & Ramanathan, 2011; Prass & Dirnagl, 1998).

Some potential therapeutic targets may alleviate calcium-induced neuronal damage. Calcium/calmodulin-dependent protein kinase kinase (CaM-KK) protects against delayed apoptosis following glutamate by activating Atk and CaM kinase IV (Yano et al., 2005), which both are anti-apoptotic players. Nimodipine (Mossakowski & Gadamski, 1990; Nuglisch et al., 1990), dantrolene (Wei & Perry, 1996), and the tetrapeptide Tyr-Val-Ala-Aspchloromethyl ketone (Ac-YVAD-cmk) (Gray et al., 2001) are all able to block damage due to high cytosolic Ca2+ levels in a variety of stroke models and may be useful in preventing excitotoxic damage.

Energy depletion plays a large role in excitotoxicity. Methods that selectively inhibit poly (ADP-ribose) polymerase-1 (PARP-1) and PARP-2 offer neuroprotection (Chiarugi, 2005) by counteracting energy-consuming activities following ischemia and reducing the drop in high-energy molecules. Additional evidence implicates PARP in a pathway capable of inducing AIF release and activation (Niimura et al., 2006), which indicates that therapies targeting PARP may have a protective effect against AIF-mediated delayed neuronal death. Drugs that inhibit PHRP activation, such as PJ34 (Xu et al., 2010) or hepatocyte growth factor (Niimura et al., 2006), may be useful as part of multimodal early interventions.

### **5. Conclusions**

130 Advances in the Preclinical Study of Ischemic Stroke

Regulating Bcl-2 proteins provides protection against delayed neuronal death by preserving mitochondrial integrity. The MAC pore is key in regulating mitochondrial permeability and is under the control of the Bcl-2 family of proteins. Inhibition or upregulation of select members by genetic or pharmacological means can modulate the downstream activation of caspase-dependent apoptosis and AIF- or EndoG-mediated necrosis-like cell death; and they

Inhibiting the pro-apoptotic BH3-only Bcl-2 proteins prevents MOMP, providing protection against mitochondria-mediated cell death. Pharmacologically blocking Bid with 4-phenylsulfanyl-phenylamine derivatives prevents tBid-induced Smac release, AIF release, caspase-3 activation, and nuclear condensation (Culmsee & Plesnila, 2006; Culmsee et al., 2005). Knockouts of Bid (Plesnila et al., 2002; Plesnila et al., 2001) or Bax genes (Gibson et al., 2001; Tehranian et al., 2008) protect against ischemic cell death in

Genetic means of boosting the effects of Bcl-2 antiapoptotic proteins provide neuroprotection. Transgenically upregulating the protective Bcl-2 gene provides protection in mice of neurons injured by ischemia (Martinou et al., 1994). A similar effect can be observed when human Bcl-2 is overexpressed with herpes simplex virus vectors (Linnik et al., 1995). Gene therapy using adeno-associated viruses carrying the Bcl-2 gene is also effective (Shimazaki et al., 2000). Human gene therapy, while powerful, is not yet mature, so it will take time for these approaches to be proven effective and integrated into a clinical setting. In the meantime, other methods of upregulating protective Bcl-2 members should be

BDNF is capable of regulating cell-death pathways. BDNF is capable of counter-regulating Bax and Bcl-2 when administered intravenously after ischemia (Schabitz et al., 2000). Neuroprotection is achieved by conjugating the product of the bcl-x gene with the HIV-Tat PTD as a method of delivery (Asoh et al., 2002). The upregulation of Bcl-2 and downregulation of Bax is implicated as part of hypothermia's protective mechanism against ischemic damage. These and other methods for regulating the Bcl-2 family may prove clinically relevant and could be examined for extra neuroprotection when used in

Excitotoxic damage due to massive Ca2+ influx should be reduced to some extent by either preventing ion disturbance or targeting the resulting structural damage by calpains, free radical production, and caspase activation. Free radical production and caspase activation may demonstrate protective action for mild excitotoxic stress causing delayed neuronal

Neuroprotection by blocking glutamate release and reception has been attempted with some success. In experimental stroke models, glutamate blockade provides protection against cell death, but the results do not necessarily translate into human therapy. Blocking the release of glutamate and other excitatory amino acids through the use of various drugs has been unsuccessful in human trials. Too little is known about neuroprotection to rule out glutamate blockers entirely. It is possible that treatment in these trials occurred too late and was unable to block the excitotoxic chain reaction. If the drugs were administered within a couple hours of stroke, then the time conditions would be similar to those found in models

combination or with therapies targeting different cell death mechanisms.

**4.8 Excitotoxicity and calcium-mediated damage prevention** 

**4.7 MOMP prevention by targeting Bcl-2 proteins** 

have been investigated for treatment strategies.

stroke models as well.

explored.

death.

Neuronal cell death following stroke occurs in necrosis, apoptosis and other alternative modes and is mediated through diverse molecular pathways. These pathways provide therapeutic targets for stroke management.

### **6. Acknowledgment**

The authors would like to thank Ms. Jacqueline Hogue for her assistance in preparing the manuscript. This work was supported by grants from the Canadian Institutes of Health Research, Canadian Stroke Network and Manitoba Health Research Council (to J. Kong) and Shanghai Health Bureau Foundation (2008-2010,2008087 to X. Bi).

### **7. References**


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**7** 

*China* 

**The Matrix Metalloproteinases** 

*Department of Neurology, the First Affiliated Hospital,* 

Matrix metalloproteinases(MMPs) are a family of zinc-containing endopeptidases which can degrade extracellular matrix (ECM) components at physiological PH. Thirty members of MMPs have been found so far [1]. They are widely distributed in plants,vertebrates and invertebrates cells.In human body, MMPs are mainly produced by vascular smooth muscle cells, monocytes, endothelial cells and so on. MMPs are synthesized as latent enzymes (zymogens) that are secreted or membrane-associated and must be proteolytically processed to their active form. Additionally, MMPs can be inhibited by endogenous inhibitors (e.g.

MMP members have similar structures. They are usually composed of six structural

Human MMPs(except MMP14) have a signal peptide sequence. The role of the signal peptide is to guide the post-translation substrate to the cytoplasm endoplasmic reticulum. Propeptide domain contains the conserved sequence of Pro-Arg-Cys-Gly-Val/Asn-Pro-Asp(PRCGV/NPD) which is responsible for maintaining the stability of plasminogen. When the propeptide domain is cut off by the exogenous enzymes, MMPs plasminogen can be

There are two zinc ion binding domains and at least one calcium binding domain in the catalysis domain. Of the 2 zinc ion binding domains, one is in the activation center which is responsible for the catalytic process of MMPs. The other one is the structural zinc ion domain. In the catalysis domain, both gelatinase A and B have a insertion sequence of 175 residues. This insertion sequence is the type-fibronectin binding domain. Studies suggest that this domain may be responsible for the integration between gelatinase and its

**1. Introduction** 

TIMPs, tissue inhibitors of metalloproteinases).

**1.2 The signal peptide and propeptide domain** 

**1.1.1 Construction of MMPs** 

**1.3 The catalysis domain** 

activated.

substrate.

domains with different functions.

**1.1 Construction and function of matrix metalloproteinases** 

**and Cerebral Ischemia** 

Wan Yang and Guangqin Li

*Chongqing Medical University* 


## **The Matrix Metalloproteinases and Cerebral Ischemia**

Wan Yang and Guangqin Li

*Department of Neurology, the First Affiliated Hospital, Chongqing Medical University China* 

### **1. Introduction**

144 Advances in the Preclinical Study of Ischemic Stroke

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Krieglstein, J. (2002). Transforming growth factor-beta 1 increases bad phosphorylation and protects neurons against damage. *J Neurosci* 22, 3898-909. Zhu, Y. Z., Huang, S. H., Tan, B. K., Sun, J., Whiteman, M. & Zhu, Y. C. (2004b).

Antioxidants in Chinese herbal medicines: a biochemical perspective. *Nat Prod Rep*

homologous to C. elegans CED-4, participates in cytochrome c-dependent

Matrix metalloproteinases(MMPs) are a family of zinc-containing endopeptidases which can degrade extracellular matrix (ECM) components at physiological PH. Thirty members of MMPs have been found so far [1]. They are widely distributed in plants,vertebrates and invertebrates cells.In human body, MMPs are mainly produced by vascular smooth muscle cells, monocytes, endothelial cells and so on. MMPs are synthesized as latent enzymes (zymogens) that are secreted or membrane-associated and must be proteolytically processed to their active form. Additionally, MMPs can be inhibited by endogenous inhibitors (e.g. TIMPs, tissue inhibitors of metalloproteinases).

### **1.1 Construction and function of matrix metalloproteinases**

### **1.1.1 Construction of MMPs**

MMP members have similar structures. They are usually composed of six structural domains with different functions.

### **1.2 The signal peptide and propeptide domain**

Human MMPs(except MMP14) have a signal peptide sequence. The role of the signal peptide is to guide the post-translation substrate to the cytoplasm endoplasmic reticulum. Propeptide domain contains the conserved sequence of Pro-Arg-Cys-Gly-Val/Asn-Pro-Asp(PRCGV/NPD) which is responsible for maintaining the stability of plasminogen. When the propeptide domain is cut off by the exogenous enzymes, MMPs plasminogen can be activated.

### **1.3 The catalysis domain**

There are two zinc ion binding domains and at least one calcium binding domain in the catalysis domain. Of the 2 zinc ion binding domains, one is in the activation center which is responsible for the catalytic process of MMPs. The other one is the structural zinc ion domain. In the catalysis domain, both gelatinase A and B have a insertion sequence of 175 residues. This insertion sequence is the type-fibronectin binding domain. Studies suggest that this domain may be responsible for the integration between gelatinase and its substrate.

The Matrix Metalloproteinases and Cerebral Ischemia 147

It is extremely important for pro-MMP-1 to produce MMP-1). MMP-11 is also called stromelysin-3,but it is often classified to other types because of its different structural

The structure of matrilysins lacks hemopexin domain. Matrilysin-1(MMP-7) and matrilysin-2(MMP-26) are included. In addition to degrading ECM, MMP-7 can process cell surface factors, such as pro TNF-and E-cadherin. The matrilysin-2 (MMP-26) can also digest many

There are six types of MT-MMPs, four types of MT-MMPs are type I transmembrane protein(MMP-14,-15, -16,-24), the other two types are GPI-ankyrin(MMP-17,-25). In addition to MT4-MMP, all other MT-MMPs can activate pro MMP-2. These enzymes can also digest some ECM components. MT1-MMP(MMP-14) has the activity to degrade collagen types I, II,

is mainly expressed in the cerebellum. MT6-MMP(MMP-25) is almost exclusively expressed in peripheral blood leucocytes, the original astrocytomas and the glioblastomas. However, it

a. Binding with cell membrane and provide the focus area for the decomposition and

b. In the process of migration to the cell membrane, MT-MMPs are responsible for the cell activation of pro convertase pathway. Therefore, different from other types of MMPs, MT-MMPs have the proteolytic activity once it is inserted into the cell membrane. **c.** MT-MMPs have the substrate recognition sites of other types of MMPs and participate in an important pathway to activate other types of MMPs. It has been proved that MT1-

There are at least seven types of MMPs which are not mentioned in the prevous text. MMP-12 is mainly expressed in macrophages and may take part in the migration of macrophages. MMP-12 can digest elastin and some other proteins. MMP-19 is identified through the liver c-DNA cloning and exists as T-cell antigens in the rheumatoid arthritis patients. Enamelysin (MMP-20) is mainly expressed in newly formed enamel and can digest amelogenin. The function of MMP-22 is not clear. MMP-23 is mainly expressed in the regeneration tissue and

The activation of MMPs is adjusted by three parts: gene transcription, zymogen activation

The genetic study has confirmed that there is promotor polymorphisms in MMP-1, -3, -9, -12 which affects the expression of MMPs gene[8].The expression of MMPs mRNA is affected by

sequence and substrate specificity.

**2.5 Membrane-type MMPs(MT-MMPs)** 

is not expressed in the meningiomas.

MT-MMPs have the following characteristics:

degradation of extracellular matrix protein;

MMP participates in the hydrolysis of MMP-2 and MMP-13.

**3. Influence factors to the activation of matrix metalloproteinases** 

Epilysin (MMP-28) is mainly expressed in keratinocytes[7].

**2.4 Matrilysins** 

ECM components.

III. MT5-MMP(MMP-24)

**2.6 Other types of MMPs** 

and endogenous inhibitors.

**3.1 Gene transcription** 

### **1.4 The hinge domain and hemopexin binding domain**

The hinge domain is located between the catalysis and the hemopexin binding domian and is linked with the terminal amino acid residues of the hemopexin binding domain by the disulfide bond. The hemopexin domain contains four duplicate sequences which has the weak homology with hemopexin and vitronectin. This domain is considered to be relevant to most MMPs' substrate specificity and also plays an important role in the integration between MMPs and tissue inhibitors of metalloproteinases (TIMPs).

### **1.5 The transmembrane domain**

This domain exists in the carboxyl terminal of the Membrane-type MMPs(MT-MMPs) and can fix MT-MMPs to the cell membrane.

MMP members have different characteristics based on the six structural domains.

### **2. Function of matrix metalloproteinases**

MMPs are the essential enzymes that may play a role in the degradation of ECM in the connective tissue. They can degrade almost all components of ECM and play an important role in various physiological and pathological processes in human body. Under physiological conditions, MMPs participate in the process of tissue demodeling such as wound healing, bone resorption, pregnancy, childbirth and breast atrophy. Normal physiological process depends on the control and coordination between MMPs and TIMPs. When infection or other stimulation occurs, the expression and activation of MMP are out of control, which may lead to the excessive degradation of ECM.

MMPs family can be divided into the following six categories based on the strucuture of their substrate, sequence simlilarity and characteristics of structural domain.

### **2.1 Collagenases**

MMP-1, MMP-8,MMP-13 and MMP-18 are included. The main feature of these enzymes is to resolve collagen types I, II, III. Collagenase can also resolve some ECM components and other non-extracellular matrix molecules.

### **2.2 Gelatinases**

Gelatinase A(MMP-2) and gelatinase B(MMP-9) are included. They are secreted or membrane-associated and must be proteolytically processed to their active form. MMP-2 can digest gelatin, collagen types IV, V, VIII, X laminin, elastin and fibronectin. The molecular weight of MMP-9 is 92KD which is the largest one. MMP-9 can be synthesized by various cells, such as astrocytes, vascular endothelial cells, microglias, neutrophils and macrophages. MMP-9 is mainly affected by the regulation of plasminogen activator and its subsrates inclucde gelatinase, collagen types IV, V and elastin[2]. Researches have shown that MMP-2 and MMP-9 may play a role in angiogenesis[3], atherosclerosis[4,5] and ischemic brain injury[6].

### **2.3 Stromelysins**

Stromelysin-1(MMP-3) and stromelysin-2(MMP-10) have the same substrate specificity. However, the proteolytic efficiency of MMP-3 is higher than that of MMP-10. In addition to digesting extracellular matrix components, MMP-3 can also activate some pro-MMPs (e.g. It is extremely important for pro-MMP-1 to produce MMP-1). MMP-11 is also called stromelysin-3,but it is often classified to other types because of its different structural sequence and substrate specificity.

### **2.4 Matrilysins**

146 Advances in the Preclinical Study of Ischemic Stroke

The hinge domain is located between the catalysis and the hemopexin binding domian and is linked with the terminal amino acid residues of the hemopexin binding domain by the disulfide bond. The hemopexin domain contains four duplicate sequences which has the weak homology with hemopexin and vitronectin. This domain is considered to be relevant to most MMPs' substrate specificity and also plays an important role in the integration

This domain exists in the carboxyl terminal of the Membrane-type MMPs(MT-MMPs) and

MMPs are the essential enzymes that may play a role in the degradation of ECM in the connective tissue. They can degrade almost all components of ECM and play an important role in various physiological and pathological processes in human body. Under physiological conditions, MMPs participate in the process of tissue demodeling such as wound healing, bone resorption, pregnancy, childbirth and breast atrophy. Normal physiological process depends on the control and coordination between MMPs and TIMPs. When infection or other stimulation occurs, the expression and activation of MMP are out of

MMPs family can be divided into the following six categories based on the strucuture of

MMP-1, MMP-8,MMP-13 and MMP-18 are included. The main feature of these enzymes is to resolve collagen types I, II, III. Collagenase can also resolve some ECM components and

Gelatinase A(MMP-2) and gelatinase B(MMP-9) are included. They are secreted or membrane-associated and must be proteolytically processed to their active form. MMP-2 can digest gelatin, collagen types IV, V, VIII, X laminin, elastin and fibronectin. The molecular weight of MMP-9 is 92KD which is the largest one. MMP-9 can be synthesized by various cells, such as astrocytes, vascular endothelial cells, microglias, neutrophils and macrophages. MMP-9 is mainly affected by the regulation of plasminogen activator and its subsrates inclucde gelatinase, collagen types IV, V and elastin[2]. Researches have shown that MMP-2 and MMP-9 may play a role in angiogenesis[3], atherosclerosis[4,5] and ischemic

Stromelysin-1(MMP-3) and stromelysin-2(MMP-10) have the same substrate specificity. However, the proteolytic efficiency of MMP-3 is higher than that of MMP-10. In addition to digesting extracellular matrix components, MMP-3 can also activate some pro-MMPs (e.g.

MMP members have different characteristics based on the six structural domains.

**1.4 The hinge domain and hemopexin binding domain** 

**1.5 The transmembrane domain** 

**2.1 Collagenases** 

**2.2 Gelatinases** 

brain injury[6].

**2.3 Stromelysins** 

can fix MT-MMPs to the cell membrane.

other non-extracellular matrix molecules.

**2. Function of matrix metalloproteinases** 

control, which may lead to the excessive degradation of ECM.

their substrate, sequence simlilarity and characteristics of structural domain.

between MMPs and tissue inhibitors of metalloproteinases (TIMPs).

The structure of matrilysins lacks hemopexin domain. Matrilysin-1(MMP-7) and matrilysin-2(MMP-26) are included. In addition to degrading ECM, MMP-7 can process cell surface factors, such as pro TNF-and E-cadherin. The matrilysin-2 (MMP-26) can also digest many ECM components.

### **2.5 Membrane-type MMPs(MT-MMPs)**

There are six types of MT-MMPs, four types of MT-MMPs are type I transmembrane protein(MMP-14,-15, -16,-24), the other two types are GPI-ankyrin(MMP-17,-25). In addition to MT4-MMP, all other MT-MMPs can activate pro MMP-2. These enzymes can also digest some ECM components. MT1-MMP(MMP-14) has the activity to degrade collagen types I, II, III. MT5-MMP(MMP-24)

is mainly expressed in the cerebellum. MT6-MMP(MMP-25) is almost exclusively expressed in peripheral blood leucocytes, the original astrocytomas and the glioblastomas. However, it is not expressed in the meningiomas.

MT-MMPs have the following characteristics:


### **2.6 Other types of MMPs**

There are at least seven types of MMPs which are not mentioned in the prevous text. MMP-12 is mainly expressed in macrophages and may take part in the migration of macrophages. MMP-12 can digest elastin and some other proteins. MMP-19 is identified through the liver c-DNA cloning and exists as T-cell antigens in the rheumatoid arthritis patients. Enamelysin (MMP-20) is mainly expressed in newly formed enamel and can digest amelogenin. The function of MMP-22 is not clear. MMP-23 is mainly expressed in the regeneration tissue and Epilysin (MMP-28) is mainly expressed in keratinocytes[7].

### **3. Influence factors to the activation of matrix metalloproteinases**

The activation of MMPs is adjusted by three parts: gene transcription, zymogen activation and endogenous inhibitors.

### **3.1 Gene transcription**

The genetic study has confirmed that there is promotor polymorphisms in MMP-1, -3, -9, -12 which affects the expression of MMPs gene[8].The expression of MMPs mRNA is affected by

The Matrix Metalloproteinases and Cerebral Ischemia 149

TIMPs inhibit the MMPs activity by two steps. In the stage of zymogen activation, TIMP-2 can form a stable complex with pro MMP-2,as well as TIMP-1 and pro MMP-9. Therefore, they can impede the zymogen self-activation of pro MMP-1. In the stage of activated MMPs, both TIMP-1 and TIMP-2 can directly form a tight 1:1 complex with the activated MMPs and inhibit their activity. Naturally, the TIMP-MMP complexes may also be activated

In recent years, there is growing research interest on MMPs in central nervous system. In the normal central nervous system, MMP-2 and MMP-9 have been found in perivascular cells, brain vascular endothelial cells, astrocytes and microglias. The microglias in cultured rat can secrete MMP-9 once they are activated. Human hippocampal pyramidal cells can also synthesize MMP-9. It is reported that MMP-9 mRNA can be expressed in the developing mouse embryo brain, suggesting that MMP-9 is related to neurodevelopment. Studies also show that MMP-2 is related to the regeneration of axons. A growing number of scholars believe that MMPs plays an important role in the pathologic processes of central nervous

Atherosclerosis is one of the underlying vascular risk factors for developing cerebrovascular disease. It is reported that the gelatinase of MMPs plays a key role in the process of intimal injury and the formation of atherosclerotic lesions. The endothelial cells covered on the plaque have the activity to express MMP-2 and MMP-9. The overexpression of MMPs can dissolve collagens and significantly change the proportion of plaque composition which leads to the relative increase in lipid content and increases the plaque instability. Then the plaque cap thinningzs and splits, eventually leading to the occurrence of cerebral ischemia disease[14,15]. Studies have shown that MMP-8 and MMP-1, MMP-12 might play a decisive role in maintaining the stability of atherosclerotic plaques[16]. The rupture of atherosclerotic plaque partially depends on the activity and content of MMP-9[17]. Another study reported that the focal increase of MMP-9 activity

In the normal state, cerebral vascular endothelial cells can not express or only express a small amount of MMP-9[18]. By the animal experiments, Fukuda[19] confirmed that ischemic brain tissue could produce some active proteases and these proteases led to the rapid and significant degradation of microvessels. Another studies suggested that cerebral ischemia and reperfusion could induce the expression of MMPs. Especially, the activity of MMP-2 and MMP-9 would increase, which was related to cerebral microvascular permeability, blood-brain barrier (BBB) permeability, BBB damage, inflammatory cell invasion and cerebral edema[6,12,13,20]. Gidday et al[21] showed that MMP-9 in ischemic brain tissue played a pro-inflammatory role which helped neutrophil leukocytes migrate from the blood circulation into the tissue. At first, MMP-9 caused BBB damage, then MMP-9 contributed to the proteolysis of microvascular basement membrane and eventually led to neurological

**4. Role of matrix metalloproteinase in cerebral ischemia** 

under certain conditions.

system diseases[5].

**4.1 MMPs and pathogenesis of stroke** 

was an early warning of acute plaque rupture.

**4.2 MMPs and ischemic brain injury** 

a variety of chemical factors, such as ECM components, carcinogens, oncogene, neural hormones, cytokines and corticosteroids. For example, TNF-affects the expression of MMPs gene by affecting the multi-cell system transcription factors (the latter is combined with the specific response elements in the MMPs gene enhancer);Tissue-type plasminogen activator (tPA) and urokinase can induce the expression of MMP-9[9,10]. The signal transduction mechanism of MMPs' activation remains to be elucidated. Previous studies suggest that MAPK pathway may be relevant to MMPs gene expression, transcription factor (AP-1 and NF-KB) is closely relevant to activation of MMPs [10]. The signal transduction mechanisms in different types of cells or for different types of MMPs may be different.

Mengshol et al[11] have showed that p38, JNK and NF-KB are essential for IL-1 to induce cartilage cells to produce MMP-13. For the production of MMP-1, p38 is still essential while JNK and NF-KB are not essential. Studies have also shown that some factors such as glucocorticoid and TGF-, can inhibit MMP gene expression at the genetic level.

### **3.2 Zymogen activation**

After translation and modification, the vast majority of MMPs mRNA is secreted to ECM in the form of zymogen. And it will be activated after hydrolysis of the propeptide domain. The activation mechanism found now includes stepwise activation, activation by MT-MMP, and cell activation. The initial activation of MMP is often associated with plasmin parenzyme, elastase, kallikrein. Among them, plasmin is considered to be the most powerful physiological activator in human body. In addition, SH-reagent (iodoacetic acid, HOCL, oxidized glutathione), denaturant (urea, SDS, NaSCN) and heat treatment have the ability to hydrolyze propeptide domain.

### **3.3 Endogenous inhibitors**

The proteolytic activity of MMP is inhibited by non-specific and specific inhibitors. Nonspecific inhibitors include 2-macroglobulin,1-antiprotease and BB-94(batimistat). Specific inhibitors are the tissue inhibitors of metalloproteinases(TIMPs). TIMPs, which are the coding proteins of multi-gene family, are the natural inhibitors of MMPs. The expression of TIMPs is regulated during development and tissue remodeling. To date, a total of four types of TIMPs have been found in vertebrates. They form the high affinity complexes with activated MMPs at the molar ratio of 1:1 and inhibit the degradation of ECM by blocking the catalysis domain of MMPs. TIMP-1 inhibits the activity of most MMPs, except for MT1- MMP and MMP-2; TIMP-2 inhibits majority of MMPs except MMP-9. In addition, TIMP-2 can form the complexes with MT1-MMP in the cell membrane which may have the regulation to activate the proteolytic activity of MMP-2; TIMP-3 inhibits MMP-1, -2, -3, -9, -13; TIMP-4 inhibits MMP-1, -3, -7, -9 and is highly expressed in human heart. Corresponding to MMPs, TIMPs play a negative role in the regulation of the ECM metabolism. They can prevent the activation of MMPs, inhibit the function and affect the extent of protein breakdown and duration of injury[12]. In some excessive matrix degradation diseases, the imbalance between MMPs and TIMPs leads to a net increase in overall activity of MMPs[13]. Although TIMPs play an important role in preventing excessive matrix degradation caused by MMPs, recent research shows that TIMP-1 and TIMP-2 are the multifunctional proteins with different biological functions. It has been reported that TIMP-1 and TIMP-2 demonstrate growth factor-like activity and inhibit the angiogenesis;while TIMP-3 is associated with apoptosis[7].

a variety of chemical factors, such as ECM components, carcinogens, oncogene, neural hormones, cytokines and corticosteroids. For example, TNF-affects the expression of MMPs gene by affecting the multi-cell system transcription factors (the latter is combined with the specific response elements in the MMPs gene enhancer);Tissue-type plasminogen activator (tPA) and urokinase can induce the expression of MMP-9[9,10]. The signal transduction mechanism of MMPs' activation remains to be elucidated. Previous studies suggest that MAPK pathway may be relevant to MMPs gene expression, transcription factor (AP-1 and NF-KB) is closely relevant to activation of MMPs [10]. The signal transduction mechanisms in different types of cells or for different types of MMPs may be different. Mengshol et al[11] have showed that p38, JNK and NF-KB are essential for IL-1 to induce cartilage cells to produce MMP-13. For the production of MMP-1, p38 is still essential while JNK and NF-KB are not essential. Studies have also shown that some factors such as

glucocorticoid and TGF-, can inhibit MMP gene expression at the genetic level.

After translation and modification, the vast majority of MMPs mRNA is secreted to ECM in the form of zymogen. And it will be activated after hydrolysis of the propeptide domain. The activation mechanism found now includes stepwise activation, activation by MT-MMP, and cell activation. The initial activation of MMP is often associated with plasmin parenzyme, elastase, kallikrein. Among them, plasmin is considered to be the most powerful physiological activator in human body. In addition, SH-reagent (iodoacetic acid, HOCL, oxidized glutathione), denaturant (urea, SDS, NaSCN) and heat treatment have the ability to

The proteolytic activity of MMP is inhibited by non-specific and specific inhibitors. Nonspecific inhibitors include 2-macroglobulin,1-antiprotease and BB-94(batimistat). Specific inhibitors are the tissue inhibitors of metalloproteinases(TIMPs). TIMPs, which are the coding proteins of multi-gene family, are the natural inhibitors of MMPs. The expression of TIMPs is regulated during development and tissue remodeling. To date, a total of four types of TIMPs have been found in vertebrates. They form the high affinity complexes with activated MMPs at the molar ratio of 1:1 and inhibit the degradation of ECM by blocking the catalysis domain of MMPs. TIMP-1 inhibits the activity of most MMPs, except for MT1- MMP and MMP-2; TIMP-2 inhibits majority of MMPs except MMP-9. In addition, TIMP-2 can form the complexes with MT1-MMP in the cell membrane which may have the regulation to activate the proteolytic activity of MMP-2; TIMP-3 inhibits MMP-1, -2, -3, -9, -13; TIMP-4 inhibits MMP-1, -3, -7, -9 and is highly expressed in human heart. Corresponding to MMPs, TIMPs play a negative role in the regulation of the ECM metabolism. They can prevent the activation of MMPs, inhibit the function and affect the extent of protein breakdown and duration of injury[12]. In some excessive matrix degradation diseases, the imbalance between MMPs and TIMPs leads to a net increase in overall activity of MMPs[13]. Although TIMPs play an important role in preventing excessive matrix degradation caused by MMPs, recent research shows that TIMP-1 and TIMP-2 are the multifunctional proteins with different biological functions. It has been reported that TIMP-1 and TIMP-2 demonstrate growth factor-like activity and inhibit the angiogenesis;while TIMP-3 is

**3.2 Zymogen activation** 

hydrolyze propeptide domain.

**3.3 Endogenous inhibitors** 

associated with apoptosis[7].

TIMPs inhibit the MMPs activity by two steps. In the stage of zymogen activation, TIMP-2 can form a stable complex with pro MMP-2,as well as TIMP-1 and pro MMP-9. Therefore, they can impede the zymogen self-activation of pro MMP-1. In the stage of activated MMPs, both TIMP-1 and TIMP-2 can directly form a tight 1:1 complex with the activated MMPs and inhibit their activity. Naturally, the TIMP-MMP complexes may also be activated under certain conditions.

### **4. Role of matrix metalloproteinase in cerebral ischemia**

In recent years, there is growing research interest on MMPs in central nervous system. In the normal central nervous system, MMP-2 and MMP-9 have been found in perivascular cells, brain vascular endothelial cells, astrocytes and microglias. The microglias in cultured rat can secrete MMP-9 once they are activated. Human hippocampal pyramidal cells can also synthesize MMP-9. It is reported that MMP-9 mRNA can be expressed in the developing mouse embryo brain, suggesting that MMP-9 is related to neurodevelopment. Studies also show that MMP-2 is related to the regeneration of axons. A growing number of scholars believe that MMPs plays an important role in the pathologic processes of central nervous system diseases[5].

### **4.1 MMPs and pathogenesis of stroke**

Atherosclerosis is one of the underlying vascular risk factors for developing cerebrovascular disease. It is reported that the gelatinase of MMPs plays a key role in the process of intimal injury and the formation of atherosclerotic lesions. The endothelial cells covered on the plaque have the activity to express MMP-2 and MMP-9. The overexpression of MMPs can dissolve collagens and significantly change the proportion of plaque composition which leads to the relative increase in lipid content and increases the plaque instability. Then the plaque cap thinningzs and splits, eventually leading to the occurrence of cerebral ischemia disease[14,15]. Studies have shown that MMP-8 and MMP-1, MMP-12 might play a decisive role in maintaining the stability of atherosclerotic plaques[16]. The rupture of atherosclerotic plaque partially depends on the activity and content of MMP-9[17]. Another study reported that the focal increase of MMP-9 activity was an early warning of acute plaque rupture.

### **4.2 MMPs and ischemic brain injury**

In the normal state, cerebral vascular endothelial cells can not express or only express a small amount of MMP-9[18]. By the animal experiments, Fukuda[19] confirmed that ischemic brain tissue could produce some active proteases and these proteases led to the rapid and significant degradation of microvessels. Another studies suggested that cerebral ischemia and reperfusion could induce the expression of MMPs. Especially, the activity of MMP-2 and MMP-9 would increase, which was related to cerebral microvascular permeability, blood-brain barrier (BBB) permeability, BBB damage, inflammatory cell invasion and cerebral edema[6,12,13,20]. Gidday et al[21] showed that MMP-9 in ischemic brain tissue played a pro-inflammatory role which helped neutrophil leukocytes migrate from the blood circulation into the tissue. At first, MMP-9 caused BBB damage, then MMP-9 contributed to the proteolysis of microvascular basement membrane and eventually led to neurological

The Matrix Metalloproteinases and Cerebral Ischemia 151

Matrix metalloproteinase inhibitors have been used to treat cancer metastasis. Currently, many MMP inhibitors are also used in experimental models of neurological diseases[6], such as bacterial meningitis, cerebral infarction and experimental allergic meningitis. Studies have shown that the content of MMPs increased after cerebral ischemia and reperfusion, exogenous inhibitors could reduce the ischemic and reperfusion injury[12,13]. Thus, MMPs may become a new potential target for stroke therapy and matrix metalloproteinase

Previous studies have showed that the activated leukocytes could increase the reperfusion injury in the central nervous system. So, the drugs which can inhibit the leukocyte adhesion (including the intracellular adherence factor antibodies) have a neuroprotective effect. Matrix metalloproteinase inhibitors can combine with the divalent cation in vitro, inhibit the

Lee' et al[3] have shown that MMP inhibitors could inhibit the MMP-9 production in brain after stimulated and parenchymal angiogenesis.Horstmann[27] found that MMP-1 not only had the direct proteolytic capacity, but also played a role in the activation cascade of MMPs. It could crack collagen types I, II, III and be involved in the activation of MMP-2, MMP-9. Non-specific matrix metalloproteinase inhibitors are clustered in atherosclerosis tissues and inhibit the activity of MMP-9 in the carotid artery plaques, thereby stabilizing the easily broken atherosclerotic plaques. Matrix metalloproteinase inhibitors can also reduce the incidence of acute plaque rupture by reducing MMP-9 activity. The content of TIMPs is significantly higher in cerebral ischemia-reperfusion. They combine with the corresponding MMPs and prevent the activation of MMPs. They inhibit the function of MMPs, stabilize the ECM, significantly reduce the blood-brain barrier damage and the brain edema after

**5.2 Matrix metalloproteinase inhibitors and therapies for cerebral ischemia** 

Previous studies suggested that MMP-9 monoclonal antibody may significantly reduce the infarction volume in a rat model of ischemia[13]. Clinical studies have also confirmed that MMP-9 is related to the total infarction volume. It is reported that matrix metalloproteinase inhibitors and MMPs neutralizing antibodies can reduce the vasogenic brain edema and infarction volume[26]. In addition, MMPs inhibitors are also effective in preventing

rt-PA and urokinase are effective drugs for acute ischemic stroke. However, the clinical application of thrombolytics is limited by the narrow therapeutic time window and the complication of bleeding after thrombolysis. But if we combine rt-PA or urokinase with the MMP inhibitors (BB-94 or doxycycline)in thrombolysis treatment, the incidence of hemorrhage and the amount of hemorrhage after thrombolysis may be decreased and the thrombolytic time window will be prolonged[28,29]. This is because that if MMPs inhibitors are used before the application of rt-PA or urokinase, MMP-2, MMP-3 and MMP-9 (which have the potential damage for BBB) would be inhibited. BBB would be closed and the integrity of blood vessels would be maintained, thus might increase the safety of the

**5. Effect of inhibiting matrix metalloproteinase on cerebral ischemia** 

inhibitors can be used for treatment of cerebrovascular diseases[2].

**5.1 Matrix metalloproteinase inhibitors and cerebral ischemia** 

leukocytes function, and reduce the reperfusion injury.

atherosclerosis and the ischemic brain damage.

thrombolytic therapy[6,30].

ischemia[12].

damage. The possible mechanism may be the following: In the ischemic brain tissue, MMP-9 is mainly expressed in vascular endothelial cells, the increased MMP-9 may act on the tight junctions and basement membrane among the BBB endothelial cells which leads to the BBB damage,increase of permeability and vasogenic brain edema. The worst is the occurrence of herniation[22]. In addition, the degradation of vascular basement membrane makes neutrophil leukocytes exudated to the brain tissue. MMP-2 and MMP-9 expressed by macrophages may contribute to their entry into the ischemic lesions and promote the wound healing after focal stroke [13]. However, MMP-9 expressed by neutrophil leukocytes may also contribute to the ischemic damage of brain. Zalewska et al[23] proposed that activated MMP-9 may act on a certain link of the cell apoptosis cascade in the hippocampus CAI area after transient ischemic brain. Previous experimental studies also showed that MMP-9 could degrade the myelin basic protein of brain white matter and lead to the damage after ischemic brain[24]. Early studies have showed that injecting MMP-2 to the rat brain can contribute to the opening of BBB. MMP-2 and MMP-9 destroy the capillary tight junctions and basement membrane by protein hydrolysis after brain ischemia, thus leading to vasogenic brain edema. In 1996, Rosenberg et al[25] monitored of the expression of MMP-9 in rats with first onset cerebral infarction. They found the upregulated expression of MMP-9 4 hours after the occlusion of middle cerebral artery,. Within 12 hours and 24 hours, the expression of MMP-9 in the infarction site significantly increased which was consistent with the peak of the vasogenic brain edema, suggesting that MMP-9 played an important role in secondary brain damage and vasogenic edema. Montaner' et al[26] also showed that the activity of MMP-9 abnormally increased in the early stage of stroke and pro-inflammatory response, while the activity of MMP-2 appeared in the repair phase of vascular regeneration. Autopsy results showed that one week after infarction, MMP-9 was expressed in neutrophils, and one week later the macrophages expressing matrilysin and MMP-2 were observed. Montaner et al also found that the level of plasma MMP-2 was higher in the patients with previous history of stroke. The above results suggested that MMP-9 plays an important role in secondary brain damage and vasogenic brain edema,while MMP-2 is involved in tissue repair and nerve regeneration.

Previous studies also suggested that MMP-9 may also related to the hemorrhage translation after tPA or urokinase thrombolysis[24].

### **4.3 MMPs and cerebral ischemia reperfusion**

Animal model studies showed that 3 hours and 48 hours of reperfusion occurred after cerebral ischemia, BBB was opened and the opening reached to the peak in the 48th hour; The first opening was related to the increased level of MMP-2 while the second opening occurred in the stage when the level of MMP-9 was significantly higher; the content of MMP-2 reached to the peak 5 days after reperfusion and the repair process began at the same time; the content of TIMP-1 significantly increased in the 48th hour while that of TIMP-2 increased to the maximum on day 5[12]. These results suggested that reperfusion may affect MMPs and TIMPs, while MMPs and TIMPs promoted the reperfusion injury by complex ways. When the synthetic inhibitors of MMPs (BB-1101 ) was applicated to inhibit MMPs, both the BBB's first opening and cerebral edema after reperfusion were prevented, suggesting that BBB's opening and brain edema after reperfusion were related to MMPs.

damage. The possible mechanism may be the following: In the ischemic brain tissue, MMP-9 is mainly expressed in vascular endothelial cells, the increased MMP-9 may act on the tight junctions and basement membrane among the BBB endothelial cells which leads to the BBB damage,increase of permeability and vasogenic brain edema. The worst is the occurrence of herniation[22]. In addition, the degradation of vascular basement membrane makes neutrophil leukocytes exudated to the brain tissue. MMP-2 and MMP-9 expressed by macrophages may contribute to their entry into the ischemic lesions and promote the wound healing after focal stroke [13]. However, MMP-9 expressed by neutrophil leukocytes may also contribute to the ischemic damage of brain. Zalewska et al[23] proposed that activated MMP-9 may act on a certain link of the cell apoptosis cascade in the hippocampus CAI area after transient ischemic brain. Previous experimental studies also showed that MMP-9 could degrade the myelin basic protein of brain white matter and lead to the damage after ischemic brain[24]. Early studies have showed that injecting MMP-2 to the rat brain can contribute to the opening of BBB. MMP-2 and MMP-9 destroy the capillary tight junctions and basement membrane by protein hydrolysis after brain ischemia, thus leading to vasogenic brain edema. In 1996, Rosenberg et al[25] monitored of the expression of MMP-9 in rats with first onset cerebral infarction. They found the upregulated expression of MMP-9 4 hours after the occlusion of middle cerebral artery,. Within 12 hours and 24 hours, the expression of MMP-9 in the infarction site significantly increased which was consistent with the peak of the vasogenic brain edema, suggesting that MMP-9 played an important role in secondary brain damage and vasogenic edema. Montaner' et al[26] also showed that the activity of MMP-9 abnormally increased in the early stage of stroke and pro-inflammatory response, while the activity of MMP-2 appeared in the repair phase of vascular regeneration. Autopsy results showed that one week after infarction, MMP-9 was expressed in neutrophils, and one week later the macrophages expressing matrilysin and MMP-2 were observed. Montaner et al also found that the level of plasma MMP-2 was higher in the patients with previous history of stroke. The above results suggested that MMP-9 plays an important role in secondary brain damage and vasogenic brain edema,while MMP-2 is

Previous studies also suggested that MMP-9 may also related to the hemorrhage translation

Animal model studies showed that 3 hours and 48 hours of reperfusion occurred after cerebral ischemia, BBB was opened and the opening reached to the peak in the 48th hour; The first opening was related to the increased level of MMP-2 while the second opening occurred in the stage when the level of MMP-9 was significantly higher; the content of MMP-2 reached to the peak 5 days after reperfusion and the repair process began at the same time; the content of TIMP-1 significantly increased in the 48th hour while that of TIMP-2 increased to the maximum on day 5[12]. These results suggested that reperfusion may affect MMPs and TIMPs, while MMPs and TIMPs promoted the reperfusion injury by complex ways. When the synthetic inhibitors of MMPs (BB-1101 ) was applicated to inhibit MMPs, both the BBB's first opening and cerebral edema after reperfusion were prevented, suggesting that BBB's opening and brain edema after reperfusion were related

involved in tissue repair and nerve regeneration.

**4.3 MMPs and cerebral ischemia reperfusion** 

after tPA or urokinase thrombolysis[24].

to MMPs.

### **5. Effect of inhibiting matrix metalloproteinase on cerebral ischemia**

Matrix metalloproteinase inhibitors have been used to treat cancer metastasis. Currently, many MMP inhibitors are also used in experimental models of neurological diseases[6], such as bacterial meningitis, cerebral infarction and experimental allergic meningitis. Studies have shown that the content of MMPs increased after cerebral ischemia and reperfusion, exogenous inhibitors could reduce the ischemic and reperfusion injury[12,13]. Thus, MMPs may become a new potential target for stroke therapy and matrix metalloproteinase inhibitors can be used for treatment of cerebrovascular diseases[2].

### **5.1 Matrix metalloproteinase inhibitors and cerebral ischemia**

Previous studies have showed that the activated leukocytes could increase the reperfusion injury in the central nervous system. So, the drugs which can inhibit the leukocyte adhesion (including the intracellular adherence factor antibodies) have a neuroprotective effect. Matrix metalloproteinase inhibitors can combine with the divalent cation in vitro, inhibit the leukocytes function, and reduce the reperfusion injury.

Lee' et al[3] have shown that MMP inhibitors could inhibit the MMP-9 production in brain after stimulated and parenchymal angiogenesis.Horstmann[27] found that MMP-1 not only had the direct proteolytic capacity, but also played a role in the activation cascade of MMPs. It could crack collagen types I, II, III and be involved in the activation of MMP-2, MMP-9. Non-specific matrix metalloproteinase inhibitors are clustered in atherosclerosis tissues and inhibit the activity of MMP-9 in the carotid artery plaques, thereby stabilizing the easily broken atherosclerotic plaques. Matrix metalloproteinase inhibitors can also reduce the incidence of acute plaque rupture by reducing MMP-9 activity. The content of TIMPs is significantly higher in cerebral ischemia-reperfusion. They combine with the corresponding MMPs and prevent the activation of MMPs. They inhibit the function of MMPs, stabilize the ECM, significantly reduce the blood-brain barrier damage and the brain edema after ischemia[12].

### **5.2 Matrix metalloproteinase inhibitors and therapies for cerebral ischemia**

Previous studies suggested that MMP-9 monoclonal antibody may significantly reduce the infarction volume in a rat model of ischemia[13]. Clinical studies have also confirmed that MMP-9 is related to the total infarction volume. It is reported that matrix metalloproteinase inhibitors and MMPs neutralizing antibodies can reduce the vasogenic brain edema and infarction volume[26]. In addition, MMPs inhibitors are also effective in preventing atherosclerosis and the ischemic brain damage.

rt-PA and urokinase are effective drugs for acute ischemic stroke. However, the clinical application of thrombolytics is limited by the narrow therapeutic time window and the complication of bleeding after thrombolysis. But if we combine rt-PA or urokinase with the MMP inhibitors (BB-94 or doxycycline)in thrombolysis treatment, the incidence of hemorrhage and the amount of hemorrhage after thrombolysis may be decreased and the thrombolytic time window will be prolonged[28,29]. This is because that if MMPs inhibitors are used before the application of rt-PA or urokinase, MMP-2, MMP-3 and MMP-9 (which have the potential damage for BBB) would be inhibited. BBB would be closed and the integrity of blood vessels would be maintained, thus might increase the safety of the thrombolytic therapy[6,30].

The Matrix Metalloproteinases and Cerebral Ischemia 153

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### **6. Conclusion**

MMPs may play an important role in the brain ischemia and reperfusion by degrading the ECM and destroying the blood -brain barrier which can lead to the vasogenic brain edema and secondary brain injury. The matrix metalloproteinase inhibitors and MMPs neutralizing antibodies can reduce the vasogenic brain edema and infarction volume. In addition, MMPs inhibitors are also effective in preventing atherosclerosis and the ischemic brain damage. Thus, MMPs may become a new potential target for stroke therapy and matrix metalloproteinase inhibitors can be used for the treatment of cerebrovascular diseases.

### **7. References**


MMPs may play an important role in the brain ischemia and reperfusion by degrading the ECM and destroying the blood -brain barrier which can lead to the vasogenic brain edema and secondary brain injury. The matrix metalloproteinase inhibitors and MMPs neutralizing antibodies can reduce the vasogenic brain edema and infarction volume. In addition, MMPs inhibitors are also effective in preventing atherosclerosis and the ischemic brain damage. Thus, MMPs may become a new potential target for stroke therapy and matrix metalloproteinase inhibitors can be used for the treatment of cerebrovascular diseases.

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**6. Conclusion** 

**7. References** 


**8** 

Jun Hyun Yoo

*Republic of Korea* 

**Folate Deficiency Enhances Delayed Neuronal Death in the Hippocampus** 

*Department of Family Medicine, Samsung Medical Center, Samsung* 

*Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul,* 

Transient cerebral ischemia, in which the brain is temporarily deprived of nutrients and oxygen, results in delayed degeneration of vulnerable neurons within the CA1 region of the hippocampus. The pathophysiology of cerebral ischemic disease is a complex series of cellular biochemical process that involves intracellular ATP depletion, excitotoxicity, oxidative stress, microvascular injury, hypercoagulable hemostatic activation, post-ischemic inflammation and final cell death of neuronal, glial, and endothelial cells (Brouns et al, 2009;

Folate is an essential micronutrient as a methyl donor for the DNA nucleotides synthesis and cytosine methylation for the control of gene expression. Clinically, folate deficiency is linked to megaloblastic anemia and atherothrobotic vascular disease. On the biochemical basis, folate deficiency increases nuclear DNA damage via uracil misincorporation and which induces chromosome breaks (Blount et al, 1997; Fenech, 2010). A metabolic consequence of folate deficiency is the accumulation of intermediate metabolite, homocysteine. Dietary folate deficiency has been shown to decrease mitochondrial folate concentration and mitochondrial DNA content and increase mitochondrial DNA deletion in brain, leading to leakage of ROS and increase of oxidative stress (Chang et al, 2007; Ho et al, 2003; Crott et al, 2005). Electron microscopic finding showed mitochondrial degeneration in the endothelium and perivascular fibrosis in microvascular wall in the rat brain (Kim et al,

Homocysteine is a toxic amino acid to neuronal and vascular endothelial cells. Numerous epidemiological studies have recognized the association of folate deficiency and hyperhomocysteinemia with increased risk of vascular disease and ischemic stroke (Yoo et al, 1998, 2000, Kang et al, 1992). Hyperhomocysteinemia produces complex alterations in the blood vessels including oxidative stress, endothelial dysfunction and inflammatory response via the activation of transcription factor such as nuclear factor-kB (NF-kB) or activiator protein-1(AP-1). Homocysteiene upregulate E-selectin, P-selectin, ICAM-1, V-CAM-1, MCP-1 via activation of NF-kB, and AP-1 (Hwang et al, 2008; Woo et al, 2008). No study has yet evaluated the morphological characteristics of the folate-deficient hippocampus after transient forebrain ischemia. This study examined the delayed neuronal

**1. Introduction** 

Jin et al, 2010).

2002).

**After Transient Cerebral Ischemia** 


### **Folate Deficiency Enhances Delayed Neuronal Death in the Hippocampus After Transient Cerebral Ischemia**

Jun Hyun Yoo

*Department of Family Medicine, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea* 

### **1. Introduction**

154 Advances in the Preclinical Study of Ischemic Stroke

[29] JIANG Guo-hui,LI Guang-qin,LI Jie.Inhibtion of matrix metalloproteinase-9 can

[30] Montaner J, Molina CA, Monasterio J, et al. Matrix metalloproteinase-9 pretreatment

rats.Journal of Brain and Nervous Diseases,2009,17:354-358

stroke. Circulation,2003,107:598-603.

prolonging urokinase thrombolytic time window after embolic stroke in

level predicts intracranial hemorrhagic complications after thrombolysis in human

Transient cerebral ischemia, in which the brain is temporarily deprived of nutrients and oxygen, results in delayed degeneration of vulnerable neurons within the CA1 region of the hippocampus. The pathophysiology of cerebral ischemic disease is a complex series of cellular biochemical process that involves intracellular ATP depletion, excitotoxicity, oxidative stress, microvascular injury, hypercoagulable hemostatic activation, post-ischemic inflammation and final cell death of neuronal, glial, and endothelial cells (Brouns et al, 2009; Jin et al, 2010).

Folate is an essential micronutrient as a methyl donor for the DNA nucleotides synthesis and cytosine methylation for the control of gene expression. Clinically, folate deficiency is linked to megaloblastic anemia and atherothrobotic vascular disease. On the biochemical basis, folate deficiency increases nuclear DNA damage via uracil misincorporation and which induces chromosome breaks (Blount et al, 1997; Fenech, 2010). A metabolic consequence of folate deficiency is the accumulation of intermediate metabolite, homocysteine. Dietary folate deficiency has been shown to decrease mitochondrial folate concentration and mitochondrial DNA content and increase mitochondrial DNA deletion in brain, leading to leakage of ROS and increase of oxidative stress (Chang et al, 2007; Ho et al, 2003; Crott et al, 2005). Electron microscopic finding showed mitochondrial degeneration in the endothelium and perivascular fibrosis in microvascular wall in the rat brain (Kim et al, 2002).

Homocysteine is a toxic amino acid to neuronal and vascular endothelial cells. Numerous epidemiological studies have recognized the association of folate deficiency and hyperhomocysteinemia with increased risk of vascular disease and ischemic stroke (Yoo et al, 1998, 2000, Kang et al, 1992). Hyperhomocysteinemia produces complex alterations in the blood vessels including oxidative stress, endothelial dysfunction and inflammatory response via the activation of transcription factor such as nuclear factor-kB (NF-kB) or activiator protein-1(AP-1). Homocysteiene upregulate E-selectin, P-selectin, ICAM-1, V-CAM-1, MCP-1 via activation of NF-kB, and AP-1 (Hwang et al, 2008; Woo et al, 2008).

No study has yet evaluated the morphological characteristics of the folate-deficient hippocampus after transient forebrain ischemia. This study examined the delayed neuronal

Folate Deficiency Enhances Delayed Neuronal Death

**2.5 Examination of neuronal apoptosis: TUNEL staining** 

and coverslipped with Canada Balsam (Kanto Chemical).

excitation light and a barrier filter (Schmued and Hopkins, 2000).

buffer and mounted on gelatin-coated slides.

**2.7 Immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG)** 

**2.8 Platelet endothelial cell adhesion molecule-1 (PECAM-1) staining** 

Canada Balsam (Kanto Chemical).

**staining** 

in the Hippocampus After Transient Cerebral Ischemia 157

mounted on the gelatin-coated slides. After dehydration the sections were mounted in

The sections in the hippocampal CA1 region were stained using terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) staining. The sections were washed in 0.1 M PBS (pH 7.4) for 30 min before being incubated in blocking solution (3% H2O2 in 0.01 M PBS) at room temperature for 20 min, and were then washed in PBS for 5 min and treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) at 4°C for 2 min. Next, the sections were washed 3 times, and then incubated in TUNEL reaction mixture according to kit instructions (Roche Molecular Biochemicals, Mannheim, Germany). The TUNEL reaction mixture was prepared with a 1:2 dilution of the enzyme solution. The sections were washed 3 times with PBS (10 min per wash) before being incubated in converter-POD (Roche Molecular Biochemicals) at 37°C for 30 min and treated with DAB-substrate solution for 1.5-2 min. After washing the sections 3 times, the sections were counterstained with methyl green, dehydrated

**2.6 Examination of neuronal damage: Fluoro-Jade B (F-J B) histofluorescence** 

**A**ccording to the experiment of Candelario-Jalil et al (2003), the sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% F-J B (Histochem, Jefferson, AR) staining solution. After washing, the sections were placed on a slide warmer (approximately 50°C), and then examined using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450-490 nm)

At designated times (30 min, 3 h, 6 h, 12 h, 24 h, 2 days, 3 days and 4 days) after the surgery, sham- and ischemia-operated animals (*n* = 7 at each time point) were used for this experiment. To obtain the exact data in this study, tissues of sham-operated and operated animals were processed under the same conditions. The sections were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min, 150 μM/ml RNase A for 1 h at 37ºC, 50 mM sodium hydroxide in 40% ethanol for 10 min. The sections were incubated with mouse anti-8-OHdG antiserum (1:100) (Bail et al., 1996; Won et al., 1999, 2001) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After washing 3 times for 10 min with PBS, the sections were incubated sequentially, in goat anti-mouse IgG and Vectastain (Vector), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS 3 times for 10 min each. The sections were visualized using 3,3'-diaminobenzidine tetrachloride (Sigma) in 0.1 M Tris-

Immunohistochemistry for PECAM-1 (final mediator of neutrophil transendothelial migration) was conducted according to the method by Hwang et al (2005b). In brief, the sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS and 10%

death and morphologic changes in the hippocampal CA1 region after transient forebrain ischemia in a gerbil model.

### **2. Materials and methods**

### **2.1 Experimental animals, diets, measurements of body weight and serum homocysteine level**

For a detailed description of the present experimental method is referred to the published article (Hwang IK et al, 2008). The animals were fed with the respective diet *ad libitum* for 3 months. After 3 months on the folate deficient-diets (FAD), blood was taken for analysis of homocysteine levels. Homocysteine levels in serum samples were quantified with the use of an high performance liquid chromatography(Yoo et al, 1998).

### **2.2 Induction of transient forebrain ischemia and tissue processing for histology**

After 3 months of folate deficient-diet, animals were anesthetized with isoflurane in 33% oxygen and 67% nitrous oxide. Bilateral common carotid arteries were isolated and occluded using non-traumatic aneurysm clips. After 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. The body temperature under freeregulating or normothermic (37 ± 0.5ºC) conditions was monitored with a rectal temperature probe and maintained during and after the surgery until the complete recovery from anesthesia. Thereafter, animals were kept on the thermal incubator to maintain the body temperature of animals until the euthanasia. Sham-operated animals served as controls: these sham-operated animals were subjected to the same surgical procedures except no occlusion of common carotid artery.

For the tissue preparation, sham- and ischemia-operated animals were anesthetized and perfused transcardially with 0.1 M phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brains were removed and postfixed in the same fixative for 6 hours. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight.

### **2.3 Examination of neuronal damage: Cresyl violet staining**

The sections in the hippocampal CA1 region were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma) was dissolved at 1.0% (*w*/*v*) in distilled water, and glacial acetic acid was added to this solution. The sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kanto Chemical, Tokyo, Japan). All animals (n=7 at each time) were sampled according to the time lines to evaluate the evolving histopathologic changes (3 hour, 12 hour, 1 day, 2day, 3day, 4day after reperfusion).

### **2.4 Examination of neuronal damage: NeuN immunohistochemistry**

The sections in the hippocampal CA1 region were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min and 10% goat serum in 0.05 M PBS for 30 min. The sections were next incubated with diluted mouse anti-NeuN (diluted 1:1,000, Chemicon International, Temecula, CA) overnight at room temperature. Thereafter the tissues were exposed to biotinylated goat anti-mouse IgG and streptavidin peroxidase complex (Vector, Burlingame, CA). And they were visualized with 3,3'-diaminobenzidine in 0.1 M Tris-HCl buffer and

death and morphologic changes in the hippocampal CA1 region after transient forebrain

For a detailed description of the present experimental method is referred to the published article (Hwang IK et al, 2008). The animals were fed with the respective diet *ad libitum* for 3 months. After 3 months on the folate deficient-diets (FAD), blood was taken for analysis of homocysteine levels. Homocysteine levels in serum samples were quantified with the use of

**2.2 Induction of transient forebrain ischemia and tissue processing for histology**  After 3 months of folate deficient-diet, animals were anesthetized with isoflurane in 33% oxygen and 67% nitrous oxide. Bilateral common carotid arteries were isolated and occluded using non-traumatic aneurysm clips. After 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. The body temperature under freeregulating or normothermic (37 ± 0.5ºC) conditions was monitored with a rectal temperature probe and maintained during and after the surgery until the complete recovery from anesthesia. Thereafter, animals were kept on the thermal incubator to maintain the body temperature of animals until the euthanasia. Sham-operated animals served as controls: these sham-operated animals were subjected to the same surgical procedures except no

For the tissue preparation, sham- and ischemia-operated animals were anesthetized and perfused transcardially with 0.1 M phosphate-buffered saline (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (pH 7.4). The brains were removed and postfixed in the same fixative for 6 hours. The brain tissues were cryoprotected by infiltration

The sections in the hippocampal CA1 region were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma) was dissolved at 1.0% (*w*/*v*) in distilled water, and glacial acetic acid was added to this solution. The sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kanto Chemical, Tokyo, Japan). All animals (n=7 at each time) were sampled according to the time lines to evaluate the evolving histopathologic changes (3 hour, 12 hour, 1 day, 2day, 3day,

The sections in the hippocampal CA1 region were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min and 10% goat serum in 0.05 M PBS for 30 min. The sections were next incubated with diluted mouse anti-NeuN (diluted 1:1,000, Chemicon International, Temecula, CA) overnight at room temperature. Thereafter the tissues were exposed to biotinylated goat anti-mouse IgG and streptavidin peroxidase complex (Vector, Burlingame, CA). And they were visualized with 3,3'-diaminobenzidine in 0.1 M Tris-HCl buffer and

**2.1 Experimental animals, diets, measurements of body weight and serum** 

an high performance liquid chromatography(Yoo et al, 1998).

**2.3 Examination of neuronal damage: Cresyl violet staining** 

**2.4 Examination of neuronal damage: NeuN immunohistochemistry** 

ischemia in a gerbil model.

**homocysteine level** 

**2. Materials and methods** 

occlusion of common carotid artery.

with 30% sucrose overnight.

4day after reperfusion).

mounted on the gelatin-coated slides. After dehydration the sections were mounted in Canada Balsam (Kanto Chemical).

### **2.5 Examination of neuronal apoptosis: TUNEL staining**

The sections in the hippocampal CA1 region were stained using terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) staining. The sections were washed in 0.1 M PBS (pH 7.4) for 30 min before being incubated in blocking solution (3% H2O2 in 0.01 M PBS) at room temperature for 20 min, and were then washed in PBS for 5 min and treated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) at 4°C for 2 min. Next, the sections were washed 3 times, and then incubated in TUNEL reaction mixture according to kit instructions (Roche Molecular Biochemicals, Mannheim, Germany). The TUNEL reaction mixture was prepared with a 1:2 dilution of the enzyme solution. The sections were washed 3 times with PBS (10 min per wash) before being incubated in converter-POD (Roche Molecular Biochemicals) at 37°C for 30 min and treated with DAB-substrate solution for 1.5-2 min. After washing the sections 3 times, the sections were counterstained with methyl green, dehydrated and coverslipped with Canada Balsam (Kanto Chemical).

### **2.6 Examination of neuronal damage: Fluoro-Jade B (F-J B) histofluorescence staining**

**A**ccording to the experiment of Candelario-Jalil et al (2003), the sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% F-J B (Histochem, Jefferson, AR) staining solution. After washing, the sections were placed on a slide warmer (approximately 50°C), and then examined using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450-490 nm) excitation light and a barrier filter (Schmued and Hopkins, 2000).

### **2.7 Immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG)**

At designated times (30 min, 3 h, 6 h, 12 h, 24 h, 2 days, 3 days and 4 days) after the surgery, sham- and ischemia-operated animals (*n* = 7 at each time point) were used for this experiment. To obtain the exact data in this study, tissues of sham-operated and operated animals were processed under the same conditions. The sections were sequentially treated with 0.3% hydrogen peroxide in PBS for 30 min, 150 μM/ml RNase A for 1 h at 37ºC, 50 mM sodium hydroxide in 40% ethanol for 10 min. The sections were incubated with mouse anti-8-OHdG antiserum (1:100) (Bail et al., 1996; Won et al., 1999, 2001) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After washing 3 times for 10 min with PBS, the sections were incubated sequentially, in goat anti-mouse IgG and Vectastain (Vector), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed with PBS 3 times for 10 min each. The sections were visualized using 3,3'-diaminobenzidine tetrachloride (Sigma) in 0.1 M Trisbuffer and mounted on gelatin-coated slides.

### **2.8 Platelet endothelial cell adhesion molecule-1 (PECAM-1) staining**

Immunohistochemistry for PECAM-1 (final mediator of neutrophil transendothelial migration) was conducted according to the method by Hwang et al (2005b). In brief, the sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS and 10%

Folate Deficiency Enhances Delayed Neuronal Death

throughtout the 3 months of observation.

in CD-group (Figs. 1D, 1G, 1K and 1O).

after transient cerebral ischemia

sham-operated groups (Fig. 4).

**3.4 Change in PECAM-1 immunoreactivity** 

group was much higher than that in CD-group (Fig. 6).

**3.3 Change in 8-hydroxy-deoxyguanosine immunoreactivity** 

**3.2 Neuronal damage** 

in the Hippocampus After Transient Cerebral Ischemia 159

homocysteine were determined and found to be 5- to 8-fold higher in gerbils subjected to FAD compared to CD-(control diet) group. The body weight gain during the first diet month was significantly lower in the FAD group than in the CD group, this was consistent

Cresyl violet and NeuN stainings show the positive pyramidal neurons on the first day. The cell densities decreased over the time after ischemia/reperfusion. Two days after ischemia/reperfusion, neurodegeneration were found in the neurons in the FAD-group (Figs. 1F, 1N), when no significant changes were seen in the CD-group (Figs. 1B, 1J). In FADgroup, CA1 pyramidal neurons showed cytoplasmic shrinkage and chromatic condensation. Starting from day three after ischemia/reperfusion, pyramidal neurons in FAD-group showed delayed neuronal death, which became morphologically similar to that of day four

Delayed neuronal death in the CA1 region was identified using TUNEL or F-J B staining. CA1 pyramidal neurons in the CD- and FAD-groups 1 day after ischemia/reperfusion did not show TUNEL or F-J B staining (Figs. 2B, 2F, 2J, 2N). Two days after ischemia/reperfusion, pyramidal neurons in the FAD-group showed TUNEL or F-J B staining representing neurodegeneration (Figs. 2G, 2O). Four days after ischemia/ reperfusion, pyramidal neurons in the CD-group showed TUNEL or F-J B staining (Figs. 2D, 2L), but TUNEL or F-J B stained pyramidal neurons decreased in FAD-group (Figs. 2H, 2P). These shows that folate deficiency enhances delayed neuronal death in the hippocampus

In this study, we found a significant difference in 8- hydroxy-deoxyguanosine immunoreactivity between the CD- and FAD-groups after ischemia/reperfusion (Figs. 3, 4). In both the sham-operated groups, very weak 8- hydroxy-deoxyguanosine immunoreactivity was detected in the CA1 region (Figs. 3A, 3E). The oxidative change in both groups began to increase at 30 min after ischemia/reperfusion, which the peak changes were noted at 12 hour after ischemia/reperfusion (Figs. 3C, 3G, Fig 4). At 12 hour after ischemia, 8- hydroxy-deoxyguanosine immunoreactivity in FAD-group was much higher than that in CD-group (Fig. 4). Thereafter, it decreased with time (Figs. 3D and 4). Four days after ischemic insult, 8-OHdG immunoreactivity in both groups was lower than that in the

PECAM-1 immunoreactivity in microvessels in the hippocampal CA1 region changed after ischemia/reperfusion (Figs. 5, 6). In the CD- and FAD-fed-sham-operated groups, microvessels showed weak PECAM-1 immunoreactivity (Fig. 5A, 5B), and this immunoreactivity increased with time after ischemic insult in both of these groups (Figs. 5C-5H, Fig 6). PECAM-1 immunoreactivity in CA1 in both groups increased significantly 3 days after ischemia/reperfusion (Figs. 5G, 5H, Fig 6) and PECAM-1 immunoreactivity in FAD-

normal horse serum in 0.05 M PBS. The sections were next incubated with diluted mouse anti-PECAM-1 antibody (diluted 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) overnight. Thereafter, the tissues were exposed to biotinylated horse anti-mouse IgG and streptavidin–peroxidase complex (Vector). The sections were visualized with DAB in 0.1 M Tris-HCl buffer and mounted on the gelatin-coated slides.

### **2.9 Immunohistochemistry for glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1)**

In order to examine the degree of reactive gliosis in the CA1 region in the CD- and FADtreated groups after ischemia/reperfusion, we carried out immunohistochemical staining with rabbit anti-GFAP (diluted 1:1,000, Chemicon) for astrocytes and rabbit anti-Iba-1 (diluted 1:500, Wako, Osaka, Japan) for microglia according to the above mentioned-method (see the NeuN immunohistochemistry). The tissues were exposed to biotinylated goat antirabbit IgG (diluted 1:200; Vector) and streptavidin peroxidase complex (diluted 1:200; Vector). And they were visualized with DAB in 0.1 M Tris-HCl buffer and mounted on the gelatin-coated slides. After dehydration the sections were mounted in Canada Balsam (Kanto Chemical).

### **2.10 Quantification of data**

All measurements were performed in order to ensure objectivity in blind conditions, by two observers for each experiment, carrying out the measures of control and experimental samples under the same conditions.

The number of survived pyramidal cells in the stratum pyramidale within the CA1 region was counted using an AxioM1 light microscope (Carl Zeiss) photomicroscope at a magnification of 400×. Histologic analysis was performed by a blinded observer and the average of the right and left survived cell numbers (neurons per 1 mm liner length) in a single section of the dorsal hippocampal CA1 region was calculated as reported by Kirino group (1986). Five sections of cresyl violet/NeuN and TUNEL/F-J B staining from each animal were used for counting.

Fifteen sections from a animal were randomly selected from the corresponding areas of the hippocampus in order to quantitatively analyze 8-OHdG, GFAP, Iba-1 and PECAM-1 immunoreactivity in the hippocampal CA1 region. The mid-point areas of the hippocampal CA1 region were measured on the monitor at a magnification of 25-50×. Images of all 8-OHdG, GFAP, Iba-1 and PECAM-1 immunoreactive structures taken from 3 layers (strata oriens, pyramidale and radiatum in the hippocampus proper, and molecular, granule cell and polymorphic layers in the dentate gyrus) were obtained through light microscope (Carl Zeiss, Germany). The staining intensity of all 8-OHdG, GFAP, Iba-1 and PECAM-1 immunoreactive structures was evaluated on the basis of a optical density (OD).

### **3. Results**

**3.1 Folate deficient change of body weight and serum concentration of homocysteine**  Folate deficiency rendered the FAD-(folate deficient-diet) group susceptible to ischemia/reperfusion. After 3 months on the folate deficient-diets, serum levels of homocysteine were determined and found to be 5- to 8-fold higher in gerbils subjected to

FAD compared to CD-(control diet) group. The body weight gain during the first diet month was significantly lower in the FAD group than in the CD group, this was consistent throughtout the 3 months of observation.

### **3.2 Neuronal damage**

158 Advances in the Preclinical Study of Ischemic Stroke

normal horse serum in 0.05 M PBS. The sections were next incubated with diluted mouse anti-PECAM-1 antibody (diluted 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) overnight. Thereafter, the tissues were exposed to biotinylated horse anti-mouse IgG and streptavidin–peroxidase complex (Vector). The sections were visualized with DAB in 0.1 M

In order to examine the degree of reactive gliosis in the CA1 region in the CD- and FADtreated groups after ischemia/reperfusion, we carried out immunohistochemical staining with rabbit anti-GFAP (diluted 1:1,000, Chemicon) for astrocytes and rabbit anti-Iba-1 (diluted 1:500, Wako, Osaka, Japan) for microglia according to the above mentioned-method (see the NeuN immunohistochemistry). The tissues were exposed to biotinylated goat antirabbit IgG (diluted 1:200; Vector) and streptavidin peroxidase complex (diluted 1:200; Vector). And they were visualized with DAB in 0.1 M Tris-HCl buffer and mounted on the gelatin-coated slides. After dehydration the sections were mounted in Canada Balsam

All measurements were performed in order to ensure objectivity in blind conditions, by two observers for each experiment, carrying out the measures of control and experimental

The number of survived pyramidal cells in the stratum pyramidale within the CA1 region was counted using an AxioM1 light microscope (Carl Zeiss) photomicroscope at a magnification of 400×. Histologic analysis was performed by a blinded observer and the average of the right and left survived cell numbers (neurons per 1 mm liner length) in a single section of the dorsal hippocampal CA1 region was calculated as reported by Kirino group (1986). Five sections of cresyl violet/NeuN and TUNEL/F-J B staining from each

Fifteen sections from a animal were randomly selected from the corresponding areas of the hippocampus in order to quantitatively analyze 8-OHdG, GFAP, Iba-1 and PECAM-1 immunoreactivity in the hippocampal CA1 region. The mid-point areas of the hippocampal CA1 region were measured on the monitor at a magnification of 25-50×. Images of all 8-OHdG, GFAP, Iba-1 and PECAM-1 immunoreactive structures taken from 3 layers (strata oriens, pyramidale and radiatum in the hippocampus proper, and molecular, granule cell and polymorphic layers in the dentate gyrus) were obtained through light microscope (Carl Zeiss, Germany). The staining intensity of all 8-OHdG, GFAP, Iba-1 and PECAM-1 immunoreactive structures was evaluated on the basis of a

**3.1 Folate deficient change of body weight and serum concentration of homocysteine**  Folate deficiency rendered the FAD-(folate deficient-diet) group susceptible to ischemia/reperfusion. After 3 months on the folate deficient-diets, serum levels of

**2.9 Immunohistochemistry for glial fibrillary acidic protein (GFAP) and ionized** 

Tris-HCl buffer and mounted on the gelatin-coated slides.

**calcium-binding adapter molecule 1 (Iba-1)** 

(Kanto Chemical).

**2.10 Quantification of data** 

samples under the same conditions.

animal were used for counting.

optical density (OD).

**3. Results** 

Cresyl violet and NeuN stainings show the positive pyramidal neurons on the first day. The cell densities decreased over the time after ischemia/reperfusion. Two days after ischemia/reperfusion, neurodegeneration were found in the neurons in the FAD-group (Figs. 1F, 1N), when no significant changes were seen in the CD-group (Figs. 1B, 1J). In FADgroup, CA1 pyramidal neurons showed cytoplasmic shrinkage and chromatic condensation. Starting from day three after ischemia/reperfusion, pyramidal neurons in FAD-group showed delayed neuronal death, which became morphologically similar to that of day four in CD-group (Figs. 1D, 1G, 1K and 1O).

Delayed neuronal death in the CA1 region was identified using TUNEL or F-J B staining. CA1 pyramidal neurons in the CD- and FAD-groups 1 day after ischemia/reperfusion did not show TUNEL or F-J B staining (Figs. 2B, 2F, 2J, 2N). Two days after ischemia/reperfusion, pyramidal neurons in the FAD-group showed TUNEL or F-J B staining representing neurodegeneration (Figs. 2G, 2O). Four days after ischemia/ reperfusion, pyramidal neurons in the CD-group showed TUNEL or F-J B staining (Figs. 2D, 2L), but TUNEL or F-J B stained pyramidal neurons decreased in FAD-group (Figs. 2H, 2P). These shows that folate deficiency enhances delayed neuronal death in the hippocampus after transient cerebral ischemia

### **3.3 Change in 8-hydroxy-deoxyguanosine immunoreactivity**

In this study, we found a significant difference in 8- hydroxy-deoxyguanosine immunoreactivity between the CD- and FAD-groups after ischemia/reperfusion (Figs. 3, 4). In both the sham-operated groups, very weak 8- hydroxy-deoxyguanosine immunoreactivity was detected in the CA1 region (Figs. 3A, 3E). The oxidative change in both groups began to increase at 30 min after ischemia/reperfusion, which the peak changes were noted at 12 hour after ischemia/reperfusion (Figs. 3C, 3G, Fig 4). At 12 hour after ischemia, 8- hydroxy-deoxyguanosine immunoreactivity in FAD-group was much higher than that in CD-group (Fig. 4). Thereafter, it decreased with time (Figs. 3D and 4). Four days after ischemic insult, 8-OHdG immunoreactivity in both groups was lower than that in the sham-operated groups (Fig. 4).

### **3.4 Change in PECAM-1 immunoreactivity**

PECAM-1 immunoreactivity in microvessels in the hippocampal CA1 region changed after ischemia/reperfusion (Figs. 5, 6). In the CD- and FAD-fed-sham-operated groups, microvessels showed weak PECAM-1 immunoreactivity (Fig. 5A, 5B), and this immunoreactivity increased with time after ischemic insult in both of these groups (Figs. 5C-5H, Fig 6). PECAM-1 immunoreactivity in CA1 in both groups increased significantly 3 days after ischemia/reperfusion (Figs. 5G, 5H, Fig 6) and PECAM-1 immunoreactivity in FADgroup was much higher than that in CD-group (Fig. 6).

Folate Deficiency Enhances Delayed Neuronal Death

in the Hippocampus After Transient Cerebral Ischemia 161

Fig. 2. TUNEL and Fluoro-Jade B (F-JB) staining of the CA1 region in the sham-operated (A,E,I,M) and ischemia-operated groups 1 days (B,F,J,N), 2 days (C,G,K,O), and 4 days (D,H,L,P) after ischemia/reperfusion and feeding with a folic acid-deficient or control diet. Two days after ischemia/reperfusion, TUNEL- or F-JBpositive pyramidal neurons are observed in stratum pyramidale (SP) of the folate-deficient diet-treated group. Four days after ischemia-reperfusion, TUNEL or F-JB reaction decreases in pyramidal neurons in the SP of the folate-deficient diet-treated group. SO, stratum oriens; SR, stratum radiatum.

Fig. 1. Cresyl violet (CV) and NeuN staining of the CA1 region in sham-operated (A,E,I,M) and ischemia-operated groups 2 days (B,F,J,N), 3 days (C,G,K,O) and 4 days (D,H,L,P) after ischemia/reperfusion and feeding with a folic acid-deficient or control diet. Two days after ischemia/reperfusion, CV- or NeuN-positive pyramidal neurons in the folate-deficient diettreated group show cytoplasmic shrinkage and chromatic condensation. Three days after ischemia/reperfusion, CV- or NeuN-positive pyramidal neurons in the folate-deficient diettreated group show ''delayed neuronal death'' like that in the control diet-treated group 4 days after ischemia/reperfusion. SO, stratum oriens; SP, stratum pyramidale; SR,stratum radiatum.

Fig. 1. Cresyl violet (CV) and NeuN staining of the CA1 region in sham-operated (A,E,I,M) and ischemia-operated groups 2 days (B,F,J,N), 3 days (C,G,K,O) and 4 days (D,H,L,P) after ischemia/reperfusion and feeding with a folic acid-deficient or control diet. Two days after ischemia/reperfusion, CV- or NeuN-positive pyramidal neurons in the folate-deficient diettreated group show cytoplasmic shrinkage and chromatic condensation. Three days after ischemia/reperfusion, CV- or NeuN-positive pyramidal neurons in the folate-deficient diettreated group show ''delayed neuronal death'' like that in the control diet-treated group 4 days after ischemia/reperfusion. SO, stratum oriens; SP, stratum pyramidale; SR,stratum

radiatum.

Fig. 2. TUNEL and Fluoro-Jade B (F-JB) staining of the CA1 region in the sham-operated (A,E,I,M) and ischemia-operated groups 1 days (B,F,J,N), 2 days (C,G,K,O), and 4 days (D,H,L,P) after ischemia/reperfusion and feeding with a folic acid-deficient or control diet. Two days after ischemia/reperfusion, TUNEL- or F-JBpositive pyramidal neurons are observed in stratum pyramidale (SP) of the folate-deficient diet-treated group. Four days after ischemia-reperfusion, TUNEL or F-JB reaction decreases in pyramidal neurons in the SP of the folate-deficient diet-treated group. SO, stratum oriens; SR, stratum radiatum.

Folate Deficiency Enhances Delayed Neuronal Death

Bars indicate means ± SEM.

in the Hippocampus After Transient Cerebral Ischemia 163

Fig. 4. Relative optical density (ROD) as percentage of 8-hydroxy-deoxyguanosine immunoreactivity in the CA1 region after transient ischemia (n= 5-7 per group; aP < 0.05 significantly different from the control diet- or folate-deficient diet-fed sham-operated group, bP < 0.05 significantly different from the control diet- or folate-deficient diet-treated preadjacent group, cP < 0.05 between the control diet- and the folate deficient diet-groups).

Fig. 5. Immunohistochemistry for platelet endothelial cell adhesion molecule-1 (PECAM-1) in the CA1 region in sham-operated (A,E) and in ischemia-operated groups at 3 hr (B,F), 1 day (C,G), and 3 days (D,H) after ischemia/reperfusion in control diet- and folate-deficient diet-groups. In control diet-(A) and folate-deficient diet -sham-operated (E) groups, weak PECAM-1 immunoreactivity is detected in microvessels. Three days after ischemia /reperfusion, PECAM-1 immunoreactivity in both groups increased significantly (G,H); the immunoreactivity in folate deficient diet-group is higher than that in control diet-group.

SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum.

Fig. 3. Immunohistochemistry for 8-hydroxy-deoxyguanosine in the CA1 region in the control diet- and folate deficient diet-sham(A,E) and ischemia-operated groups at 3 hr (B,F), 12 hr (C,G), and 2 days (D,H) after ischemia/reperfusion. At 12 hr after ischemic insult, 8- hydroxy-deoxyguanosine immunoreactivity in both groups is highest in CA1 (C,G), showing more dense immunoreactivity in folate-deficient diet- than in the control dietgroup. Two days after ischemia/reperfusion, 8- hydroxy-deoxyguanosine immunoreactivity in folate-deficient diet-group is much lower than that in control diet-group (D,H). SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum.

Fig. 3. Immunohistochemistry for 8-hydroxy-deoxyguanosine in the CA1 region in the control diet- and folate deficient diet-sham(A,E) and ischemia-operated groups at 3 hr (B,F), 12 hr (C,G), and 2 days (D,H) after ischemia/reperfusion. At 12 hr after ischemic insult, 8- hydroxy-deoxyguanosine immunoreactivity in both groups is highest in CA1 (C,G), showing more dense immunoreactivity in folate-deficient diet- than in the control dietgroup. Two days after ischemia/reperfusion, 8- hydroxy-deoxyguanosine immunoreactivity

in folate-deficient diet-group is much lower than that in control diet-group (D,H).

SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum.

Fig. 4. Relative optical density (ROD) as percentage of 8-hydroxy-deoxyguanosine immunoreactivity in the CA1 region after transient ischemia (n= 5-7 per group; aP < 0.05 significantly different from the control diet- or folate-deficient diet-fed sham-operated group, bP < 0.05 significantly different from the control diet- or folate-deficient diet-treated preadjacent group, cP < 0.05 between the control diet- and the folate deficient diet-groups). Bars indicate means ± SEM.

Fig. 5. Immunohistochemistry for platelet endothelial cell adhesion molecule-1 (PECAM-1) in the CA1 region in sham-operated (A,E) and in ischemia-operated groups at 3 hr (B,F), 1 day (C,G), and 3 days (D,H) after ischemia/reperfusion in control diet- and folate-deficient diet-groups. In control diet-(A) and folate-deficient diet -sham-operated (E) groups, weak PECAM-1 immunoreactivity is detected in microvessels. Three days after ischemia /reperfusion, PECAM-1 immunoreactivity in both groups increased significantly (G,H); the immunoreactivity in folate deficient diet-group is higher than that in control diet-group. SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum.

Folate Deficiency Enhances Delayed Neuronal Death

in the Hippocampus After Transient Cerebral Ischemia 165

Fig. 7. Immunohistochemistry for GFAP representing astrocytes and Iba-1 representing microglia in the CA1 region in sham-operated (A,E,I,M) and ischemia-operated groups 2 days (B,F,J,N), 3 days (C,G,K,O), and 4 days (D,H,L,P) after ischemia/reperfusion and feeding with folate-deficient diet or control diet. GFAP immunoreactivity-punctuated astrocytes in folate-deficient diet-group are detected 3 days after ischemia/reperfusion, whereas, in control diet-group, they are detected 4 days after ischemia/reperfusion. An increase of Iba-1-immunoreactive microglia is noted 3 days after ischemia/reperfusion in the stratum pyramidale of folate-deficient diet -group, whereas, in control diet-group, Iba-1-immunoreactive microglia is markedly increased 4 days after ischemia/reperfusion.

Fig. 6. Relative optical density (ROD) as percentage of PECAM-1 immunoreactivity in the CA1 region after transient ischemia (n 5 7 per group; aP < 0.05 significantly different from the control diet- or folate deficient diet treated sham-operated group, bP < 0.05 significantly different from the CD- or folate-deficient diet-treated preadjacent group, cP < 0.05 between the control diet- and the folate-deficient diet-treated groups). Bars indicate means ± SEM.

### **3.5 Reactive gliosis**

Significant morphological changes were observed in glial cells in the CA1 region in the CDand FAD-groups after ischemia/reperfusion. This change began 2 days after ischemia/reperfusion (Figs. 7).

**Astrocytes.** In the FAD-sham-operated group, weak GFAP immunoreactivity was detected in the CA1 region as in the CD-group (Fig. 7A, 7E). GFAP immunoreactive astrocytes had thin processes. Two days after ischemia/reperfusion, many astrocytes showed morphological changes in both groups (Fig. 7B, 7F), although at this time many more astrocytes in the FAD-treated group showed hypertrophied processes. Three days after ischemia/reperfusion, GFAP immunoreactive astrocytes in the FAD-treated group showed punctuated cytoplasm, whereas in the CD-group the cytoplasm of GFAP immunoreactive astrocytes was hypertrophied (Fig. 7C, 7G). Four days after ischemia/reperfusion, the processes of GFAP-immunoreactive astrocytes became hypertrophied, and the number and immunoreactivity in GFAP-immunoreactive astrocytes in the FAD-group were higher in the CD-group (Figs. 7D, 7H).

**Microglia.** Iba-1 immunoreactivity in the FAD-sham-operated group was similar to that in the CD-sham-operated group (Figs. 7I, 7M). Microglia in the CD-group were activated 2 days after ischemia/reperfusion, and many activated microglia in the FAD-group had aggregated to the stratum pyramidale, in which pyramidal neurons showed delayed neuronal death (Fig. 7J, 7N). Three days after ischemia/reperfusion, Iba-1 immunoreactive microglia in the FAD-group were concentrated in the stratum pyramidale of the CA1 region (Fig. 7O), whereas in the CD-group Iba-1 immunoreactive microglia were dispersed in CA1 (Fig. 7K). Four days after ischemia/reperfusion, microgliosis in the FAD-group was severer than in the CD-group (Figs. 7L, 7P).

Fig. 6. Relative optical density (ROD) as percentage of PECAM-1 immunoreactivity in the CA1 region after transient ischemia (n 5 7 per group; aP < 0.05 significantly different from the control diet- or folate deficient diet treated sham-operated group, bP < 0.05 significantly different from the CD- or folate-deficient diet-treated preadjacent group, cP < 0.05 between the control diet- and the folate-deficient diet-treated groups). Bars indicate means ± SEM.

Significant morphological changes were observed in glial cells in the CA1 region in the CDand FAD-groups after ischemia/reperfusion. This change began 2 days after

**Astrocytes.** In the FAD-sham-operated group, weak GFAP immunoreactivity was detected in the CA1 region as in the CD-group (Fig. 7A, 7E). GFAP immunoreactive astrocytes had thin processes. Two days after ischemia/reperfusion, many astrocytes showed morphological changes in both groups (Fig. 7B, 7F), although at this time many more astrocytes in the FAD-treated group showed hypertrophied processes. Three days after ischemia/reperfusion, GFAP immunoreactive astrocytes in the FAD-treated group showed punctuated cytoplasm, whereas in the CD-group the cytoplasm of GFAP immunoreactive astrocytes was hypertrophied (Fig. 7C, 7G). Four days after ischemia/reperfusion, the processes of GFAP-immunoreactive astrocytes became hypertrophied, and the number and immunoreactivity in GFAP-immunoreactive astrocytes in the FAD-group were higher in the

**Microglia.** Iba-1 immunoreactivity in the FAD-sham-operated group was similar to that in the CD-sham-operated group (Figs. 7I, 7M). Microglia in the CD-group were activated 2 days after ischemia/reperfusion, and many activated microglia in the FAD-group had aggregated to the stratum pyramidale, in which pyramidal neurons showed delayed neuronal death (Fig. 7J, 7N). Three days after ischemia/reperfusion, Iba-1 immunoreactive microglia in the FAD-group were concentrated in the stratum pyramidale of the CA1 region (Fig. 7O), whereas in the CD-group Iba-1 immunoreactive microglia were dispersed in CA1 (Fig. 7K). Four days after ischemia/reperfusion, microgliosis in the FAD-group was severer

**3.5 Reactive gliosis** 

ischemia/reperfusion (Figs. 7).

CD-group (Figs. 7D, 7H).

than in the CD-group (Figs. 7L, 7P).

Fig. 7. Immunohistochemistry for GFAP representing astrocytes and Iba-1 representing microglia in the CA1 region in sham-operated (A,E,I,M) and ischemia-operated groups 2 days (B,F,J,N), 3 days (C,G,K,O), and 4 days (D,H,L,P) after ischemia/reperfusion and feeding with folate-deficient diet or control diet. GFAP immunoreactivity-punctuated astrocytes in folate-deficient diet-group are detected 3 days after ischemia/reperfusion, whereas, in control diet-group, they are detected 4 days after ischemia/reperfusion. An increase of Iba-1-immunoreactive microglia is noted 3 days after ischemia/reperfusion in the stratum pyramidale of folate-deficient diet -group, whereas, in control diet-group, Iba-1-immunoreactive microglia is markedly increased 4 days after ischemia/reperfusion.

Folate Deficiency Enhances Delayed Neuronal Death

endoplasmic reticulum (Dalton et al, 1997).

**4.2 Homocysteine and apoptosis pathway** 

ischemia (Allan et al., 2005).

**4.3 Hyperhomocysteinemia and hypercoagulable state of blood** 

in the Hippocampus After Transient Cerebral Ischemia 167

metabotropic glutamate receptors (mGluRl), leading to activation of protein kinase C and increased intracellular IP3 formation, increasing the intracellular calcium ion, especially in

Homocysteine-induced ROS generation enhances the activation of NF-kB (Chern et al., 2001). NF-kB is one of the transcriptional factors that can be controlled by the cellular redox status. NF-kB plays a role in the control of oxidative stress-mediated apoptosis. In the oxidative conditions, neuronal cell death derives from excessive calcium influx and ROS leading to excitotoxicity. In a transient middle cerebral artery occlusion experiment, increased DNA binding was detected after reperfusion following 2 hour ischemia (Schneider et al., 1999), suggesting the activation of NF-kB. Increased transcriptional activity of NF-kB has been identified in mouse models of both permanent and transient cerebral ischemia using kB-dependent β-globin reporter gene assay (Schneider et al., 1999). NF-kB target genes include proinflammatory cytokines shown to be expressed in cerebral ischemia. TNF, IL-6, inducible nitric oxide synthase, intercellular adhesion molecule 1 (ICAM-1), and matrix metallopeptidase (MMP) 9 are major players in the post-ischemic inflammation of brain (Wang et al., 2007; Gilmore, 2008). IL-1 is another possible inducer of NF-kB activity in the ischemic brain (Kunz et al., 2008). Both IL-1α and IL-1β are rapidly induced in cerebral

The mechanism by which hyperhomocysteinemia can cause the hypercoagulable state of blood and an increased risk of thrombosis has poorly established. There have been growing evidences from the various aspects. In vitro study of cultured cells showed a toxic effect of homocysteine on endothelial cell viability ( Wall, 1980). Cultured endothelial cells under high concentration of homocysteine were not viable with copper that led to the oxidation of homocysteine, concomitant with hydrogen peroxide generation (Starkebaum and Harlan, 1986). Homocysteine inhibited the synthesis of prostacyclin, a potent inhibitor of platelets in cultured cells (Wang,1993). In vitro studies have shown that high concentration of homocysteine promote blood clotting cascade. Homocysteine activated factor V on cultured endothelial cells (Rodgers and Kane, 1986) and inhibited protein C activation in cultured endothelial cells (Rodgers, 1990). At concentrations greater than 5mmol/L, homocysteine inhibited thrombomodulin surface expression (thrombomodulin promote activation of the anticoagulant protein C and inhibit procoagulant activity of thrombin) (Lentz , 1991). Homocysteine blocked tissue-type plasminogen activiator in endothelial cells (Hajjar, 1993). Homocysteine increased platelet adhesion (Blann, 1992), and induced tissue factor (Fryer, 1993), and suppressed anticoagulant, heparan sulfate expression (Nishinaga, 1993). It has been documented that homocysteine level as low as 8 micromol/L increased affinity of lipoprotein(a) for plasmin modified fibrin surfaces, inhibiting plasminogen activation (Harpel, 1992). In vivo studies showed an abnormally increased biosynthesis of thromboxane A2 in patients with CBS deficiency (Di Minno, 1993), and endothelial dysfunction (Lentz, 1996). It have been reported that impaired regulation of endotheliumde-rived relaxing factor & nitrogen oxides (Stamler, 1993) and oxidation of low-density lipoprotein in vitro (Pathasarathy, 1987). Folate deficiency may contribute the development of atherothrombogenic condition. In the rat model, dietary folate deficiency, a major cause

### **4. Discussion**

Folate deficiency is common condition, especially in geriatric population which is caused by environmental and genetic factor. The genetic variant of methylenetetrahydrofolate reductase is very common (10-15%). Moderate hyperhomocysteinemia (15-30 μmol/L) is very common condition which is linked to increased risk of artherothrombotic vascular disease (Yoo et al). Low dietary intake of folic acid is associated with increased homocysteine levels and an increased risk of heart disease and stroke (Giles et al., 1995; hankey GJ et al, 2001). Homocysteine has direct effects on the endothelium (Wall et al,1980; Kamath et al., 2006; Lominadze et al., 2006) and astrocytes (Kranich et al., 1998), which are mediate signaling between endothelium and neurons (Nedergaard et al., 2003). In addition, the treatment of folic acid with vitamin B12 and B6 improves the blood-brain barrier function in human (Lehmann et al., 2003). Among the hypoxic brain damage, most sensitive are the pyramidal neurons in the CA1 region of the hippocampus. In experimental animal, transient forebrain ischemia, which temporarily deprives the brain of glucose as well as oxygen, results in the insidious delayed degeneration of specific vulnerable neurons within the CA1 region of the hippocampus (Kirino, 1982; Pulsinelli et al., 1982).

In FAD-group, cresyl violet or NeuN positive neurons began to decrease day 2 after ischemia/reperfusion, while in CD-group, cresyl violet or NeuN positive neurons began to decrease day 3 after ischemia/reperfusion. Delayed neuronal death of CA1 pyramidal neurons in the CD-groups occurred day 4 after ischemia/reperfusion, whereas in the FADgroup, delayed neuronal death in CA1 pyramidal neurons occurred day 3 after ischemia/reperfusion. In addition, CA1 pyramidal neurons in the FAD-group showed TUNEL or F-J B staining representing neurodegeneration day 2 after ischemia/reperfusion. This is the first report that neuronal damage in the ischemic CA1 region is accelerated by folate deficiency.

### **4.1 Excitotoxicity of homocysteine**

Homocysteine is easily carried into neuronal cells via a specific membrane transporter, leading to high intracellular homocysteine concentrations (Grieve et al., 1992). It has been shown that homocysteine and its metabolic derivatives activate both group I metabotropic glutamate receptors (mGluR) (Dalton et al, 1997) and NMDA receptors (Pullan et al., 1987), suggesting the role of homocysteine-induced excitotoxicity. Homocysteine can play as an endogenous glutamate receptor agonist (Lipton et al, 1997; Do et al, 1986; Ito et al, 1991) by activating on N-methyl-D-aspartate(NMDA) receptor subtype. The oxidative product of homocysteine, homocysteic acid, can functions as an excitatory neurotransmitter by activating NMDA receptor (Olney et al, 1987) The neurotoxicity of homocysteic acid in the brain can be partially abrogated by using a NMDA antagonist, suggesting a role for agonistic function (Olney et al, 1987; Kim et al, 1987).

Depending on glycine concentration, homocysteine showed dual response. In the condition of low glycine, homocysteine acts as a antagonist of the glycine site of the NMDA receptor, resulting in neuroprotective function. However, in the situation of high glycine levels after brain ischemia, homocysteine can bind and activate NMDA receptor, leading to excitotoxic damage (Lipton et al, 1997). These actions suggest that folate deficiency accompanied by hyperhomocysteinemia may contribute to the early brain damage after ischemia.

In addition, homocysteine has been reported to induce an extra-cellular signal regulated kinase in the hippocampus(Robert et al, 2005). Homocysteine also activates group I

Folate deficiency is common condition, especially in geriatric population which is caused by environmental and genetic factor. The genetic variant of methylenetetrahydrofolate reductase is very common (10-15%). Moderate hyperhomocysteinemia (15-30 μmol/L) is very common condition which is linked to increased risk of artherothrombotic vascular disease (Yoo et al). Low dietary intake of folic acid is associated with increased homocysteine levels and an increased risk of heart disease and stroke (Giles et al., 1995; hankey GJ et al, 2001). Homocysteine has direct effects on the endothelium (Wall et al,1980; Kamath et al., 2006; Lominadze et al., 2006) and astrocytes (Kranich et al., 1998), which are mediate signaling between endothelium and neurons (Nedergaard et al., 2003). In addition, the treatment of folic acid with vitamin B12 and B6 improves the blood-brain barrier function in human (Lehmann et al., 2003). Among the hypoxic brain damage, most sensitive are the pyramidal neurons in the CA1 region of the hippocampus. In experimental animal, transient forebrain ischemia, which temporarily deprives the brain of glucose as well as oxygen, results in the insidious delayed degeneration of specific vulnerable neurons within the CA1

In FAD-group, cresyl violet or NeuN positive neurons began to decrease day 2 after ischemia/reperfusion, while in CD-group, cresyl violet or NeuN positive neurons began to decrease day 3 after ischemia/reperfusion. Delayed neuronal death of CA1 pyramidal neurons in the CD-groups occurred day 4 after ischemia/reperfusion, whereas in the FADgroup, delayed neuronal death in CA1 pyramidal neurons occurred day 3 after ischemia/reperfusion. In addition, CA1 pyramidal neurons in the FAD-group showed TUNEL or F-J B staining representing neurodegeneration day 2 after ischemia/reperfusion. This is the first report that neuronal damage in the ischemic CA1 region is accelerated by

Homocysteine is easily carried into neuronal cells via a specific membrane transporter, leading to high intracellular homocysteine concentrations (Grieve et al., 1992). It has been shown that homocysteine and its metabolic derivatives activate both group I metabotropic glutamate receptors (mGluR) (Dalton et al, 1997) and NMDA receptors (Pullan et al., 1987), suggesting the role of homocysteine-induced excitotoxicity. Homocysteine can play as an endogenous glutamate receptor agonist (Lipton et al, 1997; Do et al, 1986; Ito et al, 1991) by activating on N-methyl-D-aspartate(NMDA) receptor subtype. The oxidative product of homocysteine, homocysteic acid, can functions as an excitatory neurotransmitter by activating NMDA receptor (Olney et al, 1987) The neurotoxicity of homocysteic acid in the brain can be partially abrogated by using a NMDA antagonist, suggesting a role for

Depending on glycine concentration, homocysteine showed dual response. In the condition of low glycine, homocysteine acts as a antagonist of the glycine site of the NMDA receptor, resulting in neuroprotective function. However, in the situation of high glycine levels after brain ischemia, homocysteine can bind and activate NMDA receptor, leading to excitotoxic damage (Lipton et al, 1997). These actions suggest that folate deficiency accompanied by

In addition, homocysteine has been reported to induce an extra-cellular signal regulated kinase in the hippocampus(Robert et al, 2005). Homocysteine also activates group I

hyperhomocysteinemia may contribute to the early brain damage after ischemia.

region of the hippocampus (Kirino, 1982; Pulsinelli et al., 1982).

**4. Discussion** 

folate deficiency.

**4.1 Excitotoxicity of homocysteine** 

agonistic function (Olney et al, 1987; Kim et al, 1987).

metabotropic glutamate receptors (mGluRl), leading to activation of protein kinase C and increased intracellular IP3 formation, increasing the intracellular calcium ion, especially in endoplasmic reticulum (Dalton et al, 1997).

### **4.2 Homocysteine and apoptosis pathway**

Homocysteine-induced ROS generation enhances the activation of NF-kB (Chern et al., 2001). NF-kB is one of the transcriptional factors that can be controlled by the cellular redox status. NF-kB plays a role in the control of oxidative stress-mediated apoptosis. In the oxidative conditions, neuronal cell death derives from excessive calcium influx and ROS leading to excitotoxicity. In a transient middle cerebral artery occlusion experiment, increased DNA binding was detected after reperfusion following 2 hour ischemia (Schneider et al., 1999), suggesting the activation of NF-kB. Increased transcriptional activity of NF-kB has been identified in mouse models of both permanent and transient cerebral ischemia using kB-dependent β-globin reporter gene assay (Schneider et al., 1999). NF-kB target genes include proinflammatory cytokines shown to be expressed in cerebral ischemia. TNF, IL-6, inducible nitric oxide synthase, intercellular adhesion molecule 1 (ICAM-1), and matrix metallopeptidase (MMP) 9 are major players in the post-ischemic inflammation of brain (Wang et al., 2007; Gilmore, 2008). IL-1 is another possible inducer of NF-kB activity in the ischemic brain (Kunz et al., 2008). Both IL-1α and IL-1β are rapidly induced in cerebral ischemia (Allan et al., 2005).

### **4.3 Hyperhomocysteinemia and hypercoagulable state of blood**

The mechanism by which hyperhomocysteinemia can cause the hypercoagulable state of blood and an increased risk of thrombosis has poorly established. There have been growing evidences from the various aspects. In vitro study of cultured cells showed a toxic effect of homocysteine on endothelial cell viability ( Wall, 1980). Cultured endothelial cells under high concentration of homocysteine were not viable with copper that led to the oxidation of homocysteine, concomitant with hydrogen peroxide generation (Starkebaum and Harlan, 1986). Homocysteine inhibited the synthesis of prostacyclin, a potent inhibitor of platelets in cultured cells (Wang,1993). In vitro studies have shown that high concentration of homocysteine promote blood clotting cascade. Homocysteine activated factor V on cultured endothelial cells (Rodgers and Kane, 1986) and inhibited protein C activation in cultured endothelial cells (Rodgers, 1990). At concentrations greater than 5mmol/L, homocysteine inhibited thrombomodulin surface expression (thrombomodulin promote activation of the anticoagulant protein C and inhibit procoagulant activity of thrombin) (Lentz , 1991). Homocysteine blocked tissue-type plasminogen activiator in endothelial cells (Hajjar, 1993). Homocysteine increased platelet adhesion (Blann, 1992), and induced tissue factor (Fryer, 1993), and suppressed anticoagulant, heparan sulfate expression (Nishinaga, 1993). It has been documented that homocysteine level as low as 8 micromol/L increased affinity of lipoprotein(a) for plasmin modified fibrin surfaces, inhibiting plasminogen activation (Harpel, 1992). In vivo studies showed an abnormally increased biosynthesis of thromboxane A2 in patients with CBS deficiency (Di Minno, 1993), and endothelial dysfunction (Lentz, 1996). It have been reported that impaired regulation of endotheliumde-rived relaxing factor & nitrogen oxides (Stamler, 1993) and oxidation of low-density lipoprotein in vitro (Pathasarathy, 1987). Folate deficiency may contribute the development of atherothrombogenic condition. In the rat model, dietary folate deficiency, a major cause

Folate Deficiency Enhances Delayed Neuronal Death

condition are more vulnerable to ischemic DNA damage.

accumulation in neurons by impairing DNA repair (Kruman et al., 2002).

**4.5 Folate deficiency and platelet endothelial cell adhesion molecule-1** 

express it.( Newman, 1997; Wang, 2003).

Adhesion and trans-endothelial migration of leucocytes play a significant roles in the pathophysiologic events in brain inflammation after stroke. Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa protein member of the immunoglobulin gene superfamily, which is expressed on the surface of platelets, monocytes, neutrophils, selected T cell subsets and on endothelial cell intercellular junctions (Newman, 1997). Expression levels of PECAM-1 differ in the type of organ tissues. It is highly expressed in kidney, lung, and trachea, while its level is at lower in brain, heart and liver. But, fibroblasts, epithelial cells, muscle, nonvascular cells or red blood cells do not

Muller et al. (1993) showed for the first time that monocytes or neutrophils treated with the specific antibodies for PECAM-1 blocked transmigration across the endothelial monolayer in vitro assay. Blocking endothelial cell junctional PECAM-1 also inhibited leukocyte transmigration, indicating that PECAM-1 molecules on both the endothelial cell as well as the leukocyte side contributed to the transmigration process. Most of PECAM in endothelium is distributed in the intercellular junctions, and 15% is on the exposed apical surface. Qing et al(2001) found that anti-PECAM-1 antibody or PECAM-Ig chimeric molecule injection blocked the T cell trafficking into the CNS in TCR transgenic mice during inflammation. Rosenblum et al.(1994) demonstrated that anti-CD31 mAb injection before the damage of endothelium in pial arteriole of mouse doubled the platelet aggregation time. Vaporciyan et al. (1993) also showed that antibody to human PECAM-1 could block in vivo accumulation of rat neutrophils into the peritoneal cavity and the alveolar compartment of the lung. These results suggest that PECAM-1 plays a key role in the transendothelial migration of leukocytes in the process of inflammation. Gumina et al.(1996) showed that

in the Hippocampus After Transient Cerebral Ischemia 169

pronounced in FAD-group. This result indicates that CA1 neurons in folate deficient

Endres et al. (2005) reported that cerebral lesion volumes after ischemia and 72-hour reperfusion were significantly increased by 2.1-fold in folic acid-deficient 129/SV wild-type mice versus controls on a normal diet, and this could not be explained by obvious differences in physiological parameters. They also reported that abasic sites, a marker of oxidative DNA damage, are significantly increased in DNA from the ischemic brains of folate-deficient 129/SV wild-type mice at early time points after MCA occlusion than control mice (Endres et al., 2005). These are supported by those of previous studies which found that folate deficiency in humans induces extensive chromosome damage, fragile site expression, micronucleus formation, and increased uracil levels in bone marrow cell DNA (Blount et al., 1997; Crott et al., 2001). The misincorporation of uracil appears to be a key event in the neurotoxicity associated with folate deficiency, because the pretreatment of culture medium with thymidine and hypoxanthine (precursors of purines) reduces neuronal cell death induced by methyl donor deficiency (Blount et al., 1997). Folate deficiency could cause the misincorporation of uracil into the DNA of proliferating cells caused by the impairment of deoxynucleoside triphosphate pools (Pogribny et al., 1997; Mol et al., 1999). In addition, homocysteine is rapidly taken up by neurons via a specific membrane transporter. Increased levels of homocysteine in cell nuclei may induce DNA strand breaks by disturbing the DNA methylation cycle (Blount et al., 1997) or may promote DNA damage

of hyperhomocysteinemia, was associated with 20-fold enhanced macrophage-derived tissue factor activity and increased ADP- and thrombin-induced platelet aggregation (Durand et al, 1996). In vitro endothelial cell study, cell treated with homocysteine showed a significant decrease in glutathione peroxidase transcription and activity, suggesting the impairment of endothelial ability to detoxify oxidative stress and leading to attenuation of bioavailable nitric oxide, a potent anti-thrombotic factor (Upchurch, 1997)

Recent oligo-array technology data validated by real time reverse transcriptase-polymerase chain reaction showed the changed gene expression in animal fed folate deficient diet, suffering from hyperhomocystinemia. Folate deficiency upregulate integrin beta-3, Rap1b, glycoprotein V, platelet-endothelial cell adhesion molecule-1(PECAM-1) and von Willebrand factor, leading to platelet activation and aggregation. In addition, upregulation of coagulation factor XIIIa, plasminogen activator inhibitor-1, and down regulation of tissuetype plasminogen activator were observed (Ebbesen LS et al, 2006).

### **4.4 Oxidative stress and neurotoxicity in hyperhomocysteinemia and folate deficiency**

The highly reactive sulfhydryl group in the homocysteine is readily oxidized to generate reactive oxygen species (Starkebaum and Harlan, 1986), suggesting that homocysteine can cause cell injury through a mechanism involving oxidative damage. The oxidative stress has been noted that hyperhomocysteinemia and folate deficiency induces or potentiates the toxic effects on the neuronal cells in vivo or in vitro. In early study, Wall et al.(1980) showed homocysteine oxidation is related to hydrogen peroxide generation. In human neuroblastoma cells cultured in folate-deprived media, oxidative stress played a role for homocysteine toxicity in neuronal cells (Ho et al, 2003). The cytotoxicity of homocysteine was compromised by antioxidants including N-acetyl cysteine, vitamin E or C (Reis et al, 2002; Wyse et al, 2002). Antoxidants vitamin including vitamin E or C prevented memory dysfunction induced by homocysteine administration in the rats (Reis et al, 2002) and the reduction of Na-K ATPase activity caused by hyperhomocyeteinemia in rats(Wyse et al, 2002). Folate deficiency decreased the proliferating cells in the dentate gyrus of adult mice hippocampus (Kruman et al, 2005). Folate deprivation led to pronounced hyperhomocysteinemia and reactive oxygen species. Folate deficient condition increased amyloid-beta-induced apoptosis, while high level of folate supplementation abrogated the reactive oxygen species generation by amyloid-beta(Ho et al, 2003). Folate deprivation in neuroblastoma cells showed an increased immunoreactivity of phospho-tau (Ho et al, 2003). In apolipoprotein E-deficient mice, iron challenge increased oxidative stress in folate deprived animals, but not in vitamin E. Oxidative damage can be mitigated by folate supplementation by reducing intracellular superoxide generation or scavenging hydrogen peroxide. (Shea and Rogers, 2002). In primary culture of rat cerebellar granular cells, homocysteine neurotoxicity was partially prevented by NMDA receptor antagonist. Homocysteine-induced neuronal death was effectively blocked by the combination of catalase and superoxide dismutase or

on the oxidative stress and excitotoxicity(Kim and Pae, 1996). A number of evidence supports the roles of DNA damage and apoptosis in the pathogenesis of several neurodegenerative disorders, including cerebral ischemia (Liu et al., 1996; Won et al., 1999, 2001; Bazan, 2005). In the present study, 8-hydroxy-deoxyguanosine immunoreactivity in the CA1 region in FAD-group increased in advance of that in CDgroup, and its peak level was noted at 12 hour after ischemia/reperfusion, which was more

catalase alone. These findings support that the homocysteine-induced neurotoxicity is based

of hyperhomocysteinemia, was associated with 20-fold enhanced macrophage-derived tissue factor activity and increased ADP- and thrombin-induced platelet aggregation (Durand et al, 1996). In vitro endothelial cell study, cell treated with homocysteine showed a significant decrease in glutathione peroxidase transcription and activity, suggesting the impairment of endothelial ability to detoxify oxidative stress and leading to attenuation of

Recent oligo-array technology data validated by real time reverse transcriptase-polymerase chain reaction showed the changed gene expression in animal fed folate deficient diet, suffering from hyperhomocystinemia. Folate deficiency upregulate integrin beta-3, Rap1b, glycoprotein V, platelet-endothelial cell adhesion molecule-1(PECAM-1) and von Willebrand factor, leading to platelet activation and aggregation. In addition, upregulation of coagulation factor XIIIa, plasminogen activator inhibitor-1, and down regulation of tissue-

**4.4 Oxidative stress and neurotoxicity in hyperhomocysteinemia and folate deficiency**  The highly reactive sulfhydryl group in the homocysteine is readily oxidized to generate reactive oxygen species (Starkebaum and Harlan, 1986), suggesting that homocysteine can cause cell injury through a mechanism involving oxidative damage. The oxidative stress has been noted that hyperhomocysteinemia and folate deficiency induces or potentiates the toxic effects on the neuronal cells in vivo or in vitro. In early study, Wall et al.(1980) showed homocysteine oxidation is related to hydrogen peroxide generation. In human neuroblastoma cells cultured in folate-deprived media, oxidative stress played a role for homocysteine toxicity in neuronal cells (Ho et al, 2003). The cytotoxicity of homocysteine was compromised by antioxidants including N-acetyl cysteine, vitamin E or C (Reis et al, 2002; Wyse et al, 2002). Antoxidants vitamin including vitamin E or C prevented memory dysfunction induced by homocysteine administration in the rats (Reis et al, 2002) and the reduction of Na-K ATPase activity caused by hyperhomocyeteinemia in rats(Wyse et al, 2002). Folate deficiency decreased the proliferating cells in the dentate gyrus of adult mice hippocampus (Kruman et al, 2005). Folate deprivation led to pronounced hyperhomocysteinemia and reactive oxygen species. Folate deficient condition increased amyloid-beta-induced apoptosis, while high level of folate supplementation abrogated the reactive oxygen species generation by amyloid-beta(Ho et al, 2003). Folate deprivation in neuroblastoma cells showed an increased immunoreactivity of phospho-tau (Ho et al, 2003). In apolipoprotein E-deficient mice, iron challenge increased oxidative stress in folate deprived animals, but not in vitamin E. Oxidative damage can be mitigated by folate supplementation by reducing intracellular superoxide generation or scavenging hydrogen peroxide. (Shea and Rogers, 2002). In primary culture of rat cerebellar granular cells, homocysteine neurotoxicity was partially prevented by NMDA receptor antagonist. Homocysteine-induced neuronal death was effectively blocked by the combination of catalase and superoxide dismutase or catalase alone. These findings support that the homocysteine-induced neurotoxicity is based

A number of evidence supports the roles of DNA damage and apoptosis in the pathogenesis of several neurodegenerative disorders, including cerebral ischemia (Liu et al., 1996; Won et al., 1999, 2001; Bazan, 2005). In the present study, 8-hydroxy-deoxyguanosine immunoreactivity in the CA1 region in FAD-group increased in advance of that in CDgroup, and its peak level was noted at 12 hour after ischemia/reperfusion, which was more

bioavailable nitric oxide, a potent anti-thrombotic factor (Upchurch, 1997)

type plasminogen activator were observed (Ebbesen LS et al, 2006).

on the oxidative stress and excitotoxicity(Kim and Pae, 1996).

pronounced in FAD-group. This result indicates that CA1 neurons in folate deficient condition are more vulnerable to ischemic DNA damage.

Endres et al. (2005) reported that cerebral lesion volumes after ischemia and 72-hour reperfusion were significantly increased by 2.1-fold in folic acid-deficient 129/SV wild-type mice versus controls on a normal diet, and this could not be explained by obvious differences in physiological parameters. They also reported that abasic sites, a marker of oxidative DNA damage, are significantly increased in DNA from the ischemic brains of folate-deficient 129/SV wild-type mice at early time points after MCA occlusion than control mice (Endres et al., 2005). These are supported by those of previous studies which found that folate deficiency in humans induces extensive chromosome damage, fragile site expression, micronucleus formation, and increased uracil levels in bone marrow cell DNA (Blount et al., 1997; Crott et al., 2001). The misincorporation of uracil appears to be a key event in the neurotoxicity associated with folate deficiency, because the pretreatment of culture medium with thymidine and hypoxanthine (precursors of purines) reduces neuronal cell death induced by methyl donor deficiency (Blount et al., 1997). Folate deficiency could cause the misincorporation of uracil into the DNA of proliferating cells caused by the impairment of deoxynucleoside triphosphate pools (Pogribny et al., 1997; Mol et al., 1999). In addition, homocysteine is rapidly taken up by neurons via a specific membrane transporter. Increased levels of homocysteine in cell nuclei may induce DNA strand breaks by disturbing the DNA methylation cycle (Blount et al., 1997) or may promote DNA damage accumulation in neurons by impairing DNA repair (Kruman et al., 2002).

### **4.5 Folate deficiency and platelet endothelial cell adhesion molecule-1**

Adhesion and trans-endothelial migration of leucocytes play a significant roles in the pathophysiologic events in brain inflammation after stroke. Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa protein member of the immunoglobulin gene superfamily, which is expressed on the surface of platelets, monocytes, neutrophils, selected T cell subsets and on endothelial cell intercellular junctions (Newman, 1997). Expression levels of PECAM-1 differ in the type of organ tissues. It is highly expressed in kidney, lung, and trachea, while its level is at lower in brain, heart and liver. But, fibroblasts, epithelial cells, muscle, nonvascular cells or red blood cells do not express it.( Newman, 1997; Wang, 2003).

Muller et al. (1993) showed for the first time that monocytes or neutrophils treated with the specific antibodies for PECAM-1 blocked transmigration across the endothelial monolayer in vitro assay. Blocking endothelial cell junctional PECAM-1 also inhibited leukocyte transmigration, indicating that PECAM-1 molecules on both the endothelial cell as well as the leukocyte side contributed to the transmigration process. Most of PECAM in endothelium is distributed in the intercellular junctions, and 15% is on the exposed apical surface. Qing et al(2001) found that anti-PECAM-1 antibody or PECAM-Ig chimeric molecule injection blocked the T cell trafficking into the CNS in TCR transgenic mice during inflammation. Rosenblum et al.(1994) demonstrated that anti-CD31 mAb injection before the damage of endothelium in pial arteriole of mouse doubled the platelet aggregation time. Vaporciyan et al. (1993) also showed that antibody to human PECAM-1 could block in vivo accumulation of rat neutrophils into the peritoneal cavity and the alveolar compartment of the lung. These results suggest that PECAM-1 plays a key role in the transendothelial migration of leukocytes in the process of inflammation. Gumina et al.(1996) showed that

Folate Deficiency Enhances Delayed Neuronal Death

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Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN,

Brouns R, De Deyn PP. (2009). The complexity of neurobiological processes in acute

Candelario-Jalil E, Alvarez D, Merino N, León OS. (2003). Delayed treatment with

Chang CM, Yu CC, Lu HT, Chou YF, Huang RF. (2007). Folate deprivation promotes

Chern, C.L., Huang, R.F., Chen, Y.H., Cheng, J.T., Liu, T.Z., (2001). Folate deficiency-induced

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Dalton, M.L., Gadson Jr., P.F., Wrenn, R.W., Rosenquist, T.H., (1997). Homocysteine signal

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nimesulide reduces measures of oxidative stress following global ischemic brain

mitochondrial oxidative decay: DNA large deletions, cytochrome c oxidase dysfunction, membrane depolarization and superoxide overproduction in rat liver.

oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhanced activation of NF-kappa B in

reductase C677T polymorphism does not alter folic acid deficiency-induced uracil incorporation into primary human lymphocyte DNA in vitro. Carcinogenesis 22:

cascade: production of phospholipids, activation of protein kinase C, and the

homophilic interaction up-regulates α6β1 on transmigrated neutrophils in vivo and plays a functional role in the ability of α6 integrins to mediate leukocyte migration

Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery

Strisciuglio P, Andria G, Patrono C, Mancini M. (1993). Abnormally high thromboxane biosynthesis in homozygous homocystinuria. Evidence for platelet

antibodies to PECAM-1 reduce myocardial infarct size in both rat. and Murohara et al(1996) showed blockade of platelet endothelial cell adhesion molecule-1 protects against myocardial ischemia and reperfusion injury in cats.

Brain ischaemia eventually enhances local inflammatory reaction. Accumulated leucocytes adhere to endothelium, probably leading to the microvasculature occlusion (Schmid-Schonbein, 1987; del Zoppo et al, 1991). Hwang et al. (2005b) demonstrated that transient ischaemia in gerbils results in a significant increase of PECAM-1 immunoreactivity in the hippocampus. PECAM-1 expression was particularly prominent in the vulnerable neurons of the hippoccampal CA1 region. PECAM-1 immunoreactivity was significantly increased by 4 days after ischaema. In addition, serum sPECAM-1 levels in ischemic group were higher than those of sham group. Zaremba and Losy (2002). reported that sPECAM-1 increases significantly in serum and in CSF in patients within 24 h after ischaemic stroke, compared with control group. In addition, serum and CSF sPECAM-1 levels within 24 h after ischaemic stroke correlated to volume of early brain CT hypodense areas, indicating the cerebral hypoperfusion. This suggests that PECAM-1 may be involved in inflammatory response mediated extent of early ischaemic brain damage. Also, sPECAM-1 levels within 24 h and at second week after ischaemic stroke correlated positively with neurological stroke severity, and with the degree of functional disability within 24 h of stroke and at second week after the incident. Therefore, initial sPECAM-1 might be of predictive value for the short-term outcome of stroke ( Zaremba and Losy, 2002b).

O'Brien et al(2003) demonstrated that PECAM-1 mediates neutrophil migration through IL-1 beta stimulated endothelial cells. It has shown that hyperhomocysteinemia at moderate level activates human monocyte and induces cytokine expression including tumor necrosis alpha, IL-1 beta, IL-6, IL-8, and IL-12 (Su et al, 2005). In this experiment, PECAM-1 immunoreactivity in the CA1 region was higher in folate deficient group than in the controls. This result suggests that folate deficiency and elevated homocysteine can enhance inflammatory response in post ischemic condition through NF-kB activation. Increased gliosis in folate deficient group may be due to elevations of PECAM-1 immunoreactivity and of its protein level in vessels, inducing the transmigration of lymphocytes and neutrophils (Michiels et al., 1998; Dangerfield et al., 2002; Hwang et al., 2005b).

In summary, folate deficiency was found to induce early and significant neuronal death and gliosis in CA1 with concomitant oxidative DNA damage. These findings suggest that folate deficiency accelerate the pathological neuronal loss and inflammation that are activated after the onset of transient cerebral mild ischemia.

### **5. Acknowledgements**

The author wish to thank Hwang IK, Yoo KY, Suh HW, Kim YS, Kwon DY, Kwon YG, and Won MH for excellent technical work. This work was supported by BioFoods Project in Korea Biotechnology Research Plan under the Ministry of Science and Technology of the Korea Government; Contract grant number: M10510120004-05N1012-00411; MRC program of MOST/KOSEF; R13-2005-022-01002-0; Samsung Biomedical Research Institute; CA5-220-1.

### **6. References**

Allan SM, Tyrrell PJ, Rothwell NJ. (2005). Interleukin-1 and neuronal injury. Nat Rev Immunol 5:629-640.

antibodies to PECAM-1 reduce myocardial infarct size in both rat. and Murohara et al(1996) showed blockade of platelet endothelial cell adhesion molecule-1 protects against

Brain ischaemia eventually enhances local inflammatory reaction. Accumulated leucocytes adhere to endothelium, probably leading to the microvasculature occlusion (Schmid-Schonbein, 1987; del Zoppo et al, 1991). Hwang et al. (2005b) demonstrated that transient ischaemia in gerbils results in a significant increase of PECAM-1 immunoreactivity in the hippocampus. PECAM-1 expression was particularly prominent in the vulnerable neurons of the hippoccampal CA1 region. PECAM-1 immunoreactivity was significantly increased by 4 days after ischaema. In addition, serum sPECAM-1 levels in ischemic group were higher than those of sham group. Zaremba and Losy (2002). reported that sPECAM-1 increases significantly in serum and in CSF in patients within 24 h after ischaemic stroke, compared with control group. In addition, serum and CSF sPECAM-1 levels within 24 h after ischaemic stroke correlated to volume of early brain CT hypodense areas, indicating the cerebral hypoperfusion. This suggests that PECAM-1 may be involved in inflammatory response mediated extent of early ischaemic brain damage. Also, sPECAM-1 levels within 24 h and at second week after ischaemic stroke correlated positively with neurological stroke severity, and with the degree of functional disability within 24 h of stroke and at second week after the incident. Therefore, initial sPECAM-1 might be of predictive value for

O'Brien et al(2003) demonstrated that PECAM-1 mediates neutrophil migration through IL-1 beta stimulated endothelial cells. It has shown that hyperhomocysteinemia at moderate level activates human monocyte and induces cytokine expression including tumor necrosis alpha, IL-1 beta, IL-6, IL-8, and IL-12 (Su et al, 2005). In this experiment, PECAM-1 immunoreactivity in the CA1 region was higher in folate deficient group than in the controls. This result suggests that folate deficiency and elevated homocysteine can enhance inflammatory response in post ischemic condition through NF-kB activation. Increased gliosis in folate deficient group may be due to elevations of PECAM-1 immunoreactivity and of its protein level in vessels, inducing the transmigration of lymphocytes and

In summary, folate deficiency was found to induce early and significant neuronal death and gliosis in CA1 with concomitant oxidative DNA damage. These findings suggest that folate deficiency accelerate the pathological neuronal loss and inflammation that are activated

The author wish to thank Hwang IK, Yoo KY, Suh HW, Kim YS, Kwon DY, Kwon YG, and Won MH for excellent technical work. This work was supported by BioFoods Project in Korea Biotechnology Research Plan under the Ministry of Science and Technology of the Korea Government; Contract grant number: M10510120004-05N1012-00411; MRC program of MOST/KOSEF; R13-2005-022-01002-0; Samsung Biomedical Research Institute; CA5-220-1.

Allan SM, Tyrrell PJ, Rothwell NJ. (2005). Interleukin-1 and neuronal injury. Nat Rev

neutrophils (Michiels et al., 1998; Dangerfield et al., 2002; Hwang et al., 2005b).

myocardial ischemia and reperfusion injury in cats.

the short-term outcome of stroke ( Zaremba and Losy, 2002b).

after the onset of transient cerebral mild ischemia.

**5. Acknowledgements** 

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methyl-*D*-aspartate receptor. Proc Natl Acad Sci U S A 94: 5923-5928. Liu PK, Hsu CY, Dizdaroglu M, Floyd RA, Kow YW, Karakaya A, Rabow LE, Cui JK. (1996).

hypoxic endothelial cells. Cell Adhes Commun 5: 367-374.

reperfusion injury in cats. *J. Immunol.* 156:3550–3557

hyperhomocyst(e)inemia. J Clin Invest 98:24

reperfusion. J Neurosci 16: 6795-6806.

endothelial cells. J Clin Invest 92:1381

NMDA receptor. Brain Res. Bull. 19, 597-602.

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transmigration is independent of monolayer PECAM-1 signaling or localization.

And Samson, L.(1987). L-homocysteic acid: an endogenous excitotoxic ligand of the

uracil in preneoplastic DNA from folate/methyl-deficient rats. Carcinogenesis 18:

(1987). Excitatory amino acid receptor potency and subclass specificity of sulfur-


**9** 

*USA* 

**Glial Cells, Inflammation and Heat Shock** 

*Department of Anesthesia, Stanford University School of Medicine, Stanford, CA* 

Each year approximately 795,000 people in the United States suffer a new or recurrent stroke and it is the third leading cause of death after heart disease and cancer (Lloyd-Jones et al., 2010). The estimated cost of stroke was \$73.7 billion nationwide in 2010, the majority of which was related to payments for inpatient care and rehabilitation for significant morbidity (hemiparesis, aphasia and loss of independence). Stroke is broadly divided into two categories: ischemic and hemorrhagic. The former is related to too little blood supplied to the brain, secondary to thrombus or embolus, and the latter results from excess blood escaping into the cranial cavity. Ischemic brain injury represents conditions including focal ischemia, with subsequent loss of blood flow and nutrients to one area of the brain, and global ischemia, as seen in cardiopulmonary arrest and resuscitation which, when brief, results specifically in neuronal death in the CA1 region of the hippocampus (Pulsinelli, 1985). In either case, decreased cerebral blood flow initiates a cascade of ATP depletion, ion gradient disruption, excessive glutamate release, formation of reactive oxygen species and increased lactic acidosis that leads to neuronal death (Doyle et al., 2008). To date, the only FDA-approved treatment for focal ischemic stroke is recombinant tissue plasminogen activator which aims to restore blood flow by recanalization of the occluded vessel (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). In global ischemia, multiple clinical trials have demonstrated that therapeutic hypothermia increases survival and improves neurologic outcome (Bernard et al., 2002; Sahota and Savitz, 2011; The Hypothermia after Cardiac Arrest Study Group, 2002). Though the exact mechanisms remain unclear, effects on several different pathways have been observed. In spite of this one treatment modality, survival to hospital discharge after cardiac arrest and attempted resuscitation remains a dismal 5-18% depending on the cause and rapidity of the response (de Vreede-

To date, more than one hundred potential pharmacological strategies for stroke have failed to show improved outcome in phase III trials. As such, the role of central nervous system glial cells has recently come under scrutiny as work focused on neurons alone has failed to reverse neuronal death in ischemic areas of the brain. Glial cells (microglia, astrocytes and oligodendrocytes) constitute over 70% of the total cell population in the CNS and are active contributors to neuromodulatory, neurotrophic and neuroimmune events in the brain and

**1. Introduction** 

Swagemakers et al., 1997; Eckstein et al., 2005).

**Proteins in Cerebral Ischemia** 

Vivianne L. Tawfik, Robin E. White and Rona Giffard


### **Glial Cells, Inflammation and Heat Shock Proteins in Cerebral Ischemia**

Vivianne L. Tawfik, Robin E. White and Rona Giffard *Department of Anesthesia, Stanford University School of Medicine, Stanford, CA USA* 

### **1. Introduction**

176 Advances in the Preclinical Study of Ischemic Stroke

Wall RT, Harlan JM, Harker LA, Striker GE. (1980). Homocysteine-induced endothelial cell injury in vitro: A model for the study of vascular injury. Thromb Res 18:113 Wang J, Dudman NPB, Wilcken DE. (1993). Effects of homocys-teine and related

Wang Q, Tang XN, Yenari MA. (2007). The inflammatory response in stroke. J

Wang Q, Sun AY, Simonyi A, Jensen MD, Shelat PB, Rottinghaus GE, MacDonald RS, Miller

Wang Y, Su X, Sorenson CM, Sheibani N. (2003). Tissue-specific distributions of

Won MH, Kang TC, Jeon GS, Lee JC, Kim DY, Choi EM, Lee KH, Choi CD, Chung MH, Cho

Woo CW, Siow YL, O K. (2008). Homocysteine induces monocyte chemoattractant protein-1

Wyse, A.T., Zugno, A.I., Streck, E. L., Matte, C., Calcagnotto, T., Wannmacher, C.M. and

Yoo JH, Chung CS, Kang SS. (1998). Relation of plasma homocyst(e)ine to cerebral infarction

Yoo JH, Park JE, Hong KP, Lee SH, Kim DK, Lee WR, Park SC. (1999). Moderate

Yoo JH, Choi GD, Kang SS. (2000). Pathogenicity of thermolabile methylenetetrahydrofolate reductase for vascular dementia. Arterioscler Thromb Vasc Biol 20:1921-1925. Yoo JH, Lee SC. (2001). Elevated levels of plasma homocyst(e)ine and asymmetric dimethylarginine in elderly patients with stroke. Atherosclerosis 158:425-430. Zaremba J, Losy J. (2002). sPECAM-1 in serum and CSF of acute ischaemic stroke patients.

Zaremba J, Losy J. (2002). Soluble platelet endothelial cell adhesion molecule-1 in ischaemic

stroke patients is related to the extent of early brain damage. Central-European

risk of coronary artery disease in Koreans. Thromb Res 97: 77-84.

cells. Thromb Haemost 6:1047

deficits. J Neurosci Res 82:138-148.

Heart and Circulatory Physiology 284: H1008-H1017.

and C treatment. Neurochem. Res 27, 1685-1689.

and cerebral atherosclerosis. Stroke 29: 2478-2483.

Acta Neurologica Scandinavica 106: 292–298.

Journal of Immunology 27: 90–96.

Neuroimmunol 184:53-68.

Neurosci Lett 301: 139-142.

283(3):1282-92

compounds on prostacyclin production by cultured human vascular endothelial

DK, Lubahn DE, Weisman GA, Sun GY. (2005). Neuroprotective mechanisms of curcumin against cerebral ischemia-induced neuronal apoptosis and behavioral

alternatively spliced human PECAM-1 isoforms. American Journal of Physiology.

SS. (1999). Immunohistochemical detection of oxidative DNA damage induced by ischemia-reperfusion insults in gerbil hippocampus in vivo. Brain Res 836: 70-78. Won MH, Kang TC, Park SK, Jeon GS, Kim Y, Seo JH, Choi E, Chung MH, Cho SS. (2001).

The alterations of *N*-methyl-*D*-aspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia-reperfusion insult.

expression in hepatocytes mediated via activator protein-1 activation. J Biol Chem

Wajner, M. (2002). Inhibition of Na (+), K(+)-ATPase activity in hippocampus of rats subjected to acute administration of homocysteine is prevented by vitamins E

hyperhomocyst(e)inemia is associated with the presence of coronary artery disease and the severity of coronary atherosclerosis in Koreans. Thromb Res 94: 45-52. Yoo JH, Park SC. (2000). Low plasma folate in combination with the 677 C to T

methylenetetrahydrofolate reductase polymorphism is associated with increased

Each year approximately 795,000 people in the United States suffer a new or recurrent stroke and it is the third leading cause of death after heart disease and cancer (Lloyd-Jones et al., 2010). The estimated cost of stroke was \$73.7 billion nationwide in 2010, the majority of which was related to payments for inpatient care and rehabilitation for significant morbidity (hemiparesis, aphasia and loss of independence). Stroke is broadly divided into two categories: ischemic and hemorrhagic. The former is related to too little blood supplied to the brain, secondary to thrombus or embolus, and the latter results from excess blood escaping into the cranial cavity. Ischemic brain injury represents conditions including focal ischemia, with subsequent loss of blood flow and nutrients to one area of the brain, and global ischemia, as seen in cardiopulmonary arrest and resuscitation which, when brief, results specifically in neuronal death in the CA1 region of the hippocampus (Pulsinelli, 1985). In either case, decreased cerebral blood flow initiates a cascade of ATP depletion, ion gradient disruption, excessive glutamate release, formation of reactive oxygen species and increased lactic acidosis that leads to neuronal death (Doyle et al., 2008). To date, the only FDA-approved treatment for focal ischemic stroke is recombinant tissue plasminogen activator which aims to restore blood flow by recanalization of the occluded vessel (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). In global ischemia, multiple clinical trials have demonstrated that therapeutic hypothermia increases survival and improves neurologic outcome (Bernard et al., 2002; Sahota and Savitz, 2011; The Hypothermia after Cardiac Arrest Study Group, 2002). Though the exact mechanisms remain unclear, effects on several different pathways have been observed. In spite of this one treatment modality, survival to hospital discharge after cardiac arrest and attempted resuscitation remains a dismal 5-18% depending on the cause and rapidity of the response (de Vreede-Swagemakers et al., 1997; Eckstein et al., 2005).

To date, more than one hundred potential pharmacological strategies for stroke have failed to show improved outcome in phase III trials. As such, the role of central nervous system glial cells has recently come under scrutiny as work focused on neurons alone has failed to reverse neuronal death in ischemic areas of the brain. Glial cells (microglia, astrocytes and oligodendrocytes) constitute over 70% of the total cell population in the CNS and are active contributors to neuromodulatory, neurotrophic and neuroimmune events in the brain and

GGlial Cells, Inflammation and Heat Shock Proteins in Cerebral Ischemia 179

Overall, this chapter seeks to review the importance of astrocytes and microglia in the postischemic inflammatory response and the role of heat shock proteins in modulating

Microglia are monocyte-derived CNS tissue macrophages that are phenotypically adapted to the neural environment. As such, they are characterized by minimal phagocytic activity and low expression of the macrophage-specific antigen, CD45 (Kreutzberg, 1996) which may be used to differentiate resident microglia from infiltrating macrophages (Babcock et al., 2003). Microglia constitutively and inducibly express a variety of immune-related receptors on their surface including cytokine, chemokine, prostaglandin, pattern recognition and complement receptors (Aloisi, 2001). In addition, microglia can function as antigen presenting cells, and through CD40 on their membranes can form an "immunological synapse" with CD40L-expressing T-cells recruited centrally (Gerritse et al., 1996). As a result, microglia can respond to diverse stresses by performing innate immune functions such as phagocytosis, and can release potentially beneficial factors such as glial-derived

Microglia can release a host of factors to protect the CNS; however, when activated after ischemia by necrotic cell debris and other substances, they can produce free radicals, proinflammatory cytokines (IL-1β, TNFα, IL-6 and interferon-γ), reactive oxygen species, matrix metalloproteinases and glutamate in an aberrant fashion (Lucin and Wyss-Coray, 2009; Yenari et al., 2010). Such compounds are instrumental in the subsequent activation of astrocytes (see below), induction of cell adhesion molecules and T-lymphocyte recruitment into the CNS following various injuries (Liu and Hong, 2003; Sweitzer et al., 2002). Several studies have been performed testing inhibition of microglial activation as a strategy to protect neurons following ischemic injury. Transgenic mice lacking the pattern recognition receptor TLR4 (Hyakkoku et al., 2010), mice overexpressing the anti-inflammatory cytokine IL-10 (De Bilbao et al., 2009) and treatment with a neutralizing antibody against TNF-alpha (Barone et al., 1997), all resulted in suppressed microglial activation and significantly decreased infarct size following focal cerebral ischemia. Taken together, these studies suggest that inhibition of the post-ischemia inflammatory response is a viable option for stroke treatment. Although microglia quickly respond to ischemia by producing proinflammatory cytokines (Yenari et al., 2010), proliferating (Denes et al., 2007) and exhibiting an altered cell morphology (Tanaka et al., 2003), only a small percentage (1-8%) of the microglia in the corpus callosum and lesion penumbra, and no microglia in the lesion core, express Hsp72 early after focal ischemia (Soriano et al., 1994). This indicates that a robust increase in Hsp72 protein expression is not a normal aspect of the post-ischemic

Interestingly, the majority of inflammatory mediators produced by microglia after stroke are produced by NFB pathway activation (Yenari et al., 2010). Hsp72 has been implicated in modulation of inflammation by suppressing NFB through multiple interactions. Previous work has shown that Hsp72 directly binds to the NFB:IB complex, thus preventing IB phosphorylation and subsequent NFB activation (Feinstein et al., 1996; Zheng et al., 2008). Ran et al. (2004) further demonstrated that Hsp72 binds directly to IB kinase- (IKK-), an essential regulatory component of the IKK complex, blocking activation of IB and release of

inflammation and outcome after cerebral ischemia.

**2. Microglia: Immune cells of the CNS** 

neurotrophic factor (GDNF).

microglial phenotype.

spinal cord (Pellerin, 2005; Reichenbach and Wolburg, 2005). Once thought of merely as neuronal support cells, astrocytes and microglia, in their physiologic role, dynamically control synaptic function and neuronal activity by performing a variety of crucial functions. Microglia, the intrinsic macrophages of the CNS, provide immune surveillance against invading pathogens or nervous system insults (Aloisi, 2001) while astrocytes regulate synaptic glutamate levels, contribute to the blood-brain-barrier and supply neuronal growth factors (Liberto et al., 2004). Given their complex involvement in normal CNS function, glial cells must be considered in any strategy focused on neuronal preservation after ischemic injury.

Heat shock proteins (HSP) are a phylogenetically conserved group of chaperones that assist in ATP-dependent protein folding, translocation across membranes, suppression of protein aggregation, presentation of substrates for degradation and modulate a host of other intracellular processes (Hartl, 1996). The 70 kDa heat shock protein family (HSP70) is the most extensively studied group of chaperones and consists of at least twelve constitutive and inducible proteins which aid in a coordinated response to cellular stressors. The most important members include: the constitutively expressed primarily cytosolic Hsc70/Hsp73, the heat inducible cytosolic form Hsp70/72, the glucose regulated mitochondrial protein Grp75/mortalin/mtHsp70 and the endoplasmic reticulum glucose regulated protein Grp78/BiP. The HSP70s are structurally comprised of a 44 kDa amino-terminal ATPase domain, 18 kDa carboxyl-terminal substrate-binding domain and a more variable 10 kDa segment that terminates in the highly conserved EEVD sequence that regulates intramolecular interactions and ATPase activity (Freeman et al., 1995). Our laboratory has demonstrated that the carboxyl-terminal domain of Hsp72 is sufficient to protect astrocytes from oxygen-glucose deprivation and decrease infarct volume after transient middle cerebral artery occlusion (MCAO) (Sun et al., 2006b).

Extensive work using overexpression and knockout of HSP70 family members has highlighted integral cytoprotective, anti-apoptotic and immune regulatory roles for these proteins. Induction of Hsp70/72 by heat stress or targeted overexpression in multiple experimental disease models including stroke (Rajdev et al., 2000), sepsis (Ryan et al., 1992), renal injury (Jo et al., 2006) and acute lung injury (Villar et al., 1994) demonstrated decreased organ injury and enhanced survival. Lee et al. (2001) showed that while *hsp70.1* knockout mice have a normal baseline phenotype; cerebral infarct volume was 30% larger and mortality was higher than in wild type littermates after focal ischemia. In addition, using a combined *hsp70.1/3* knockout (the two genes are separated by only 8kb on chromosome 17 and show 99% homology), Lee et al. (2004) later determined that cytochrome *c* release into the cytosol and levels of activated caspase-3 were increased after MCAO suggesting that Hsp 70/72 plays a role in preventing initiation of apoptosis after injury. The *hsp70.1/3* knockout also exhibited an enhanced inflammatory response to cecal perforation and ligation (an animal model of acute respiratory distress syndrome/sepsis) as evidenced by increased NF-B activation, TNF- and IL-6 expression and lung injury highlighting a role in controlling immune function in injury states (Singleton and Wischmeyer, 2006). In this same model, Weiss et al. (2002) demonstrated that targeted overexpression of Hsp70 in rat lung significantly attenuated interstitial and alveolar edema, protein exudation and dramatically decreased neutrophil accumulation leading to improvement of acute respiratory distress syndrome.

Overall, this chapter seeks to review the importance of astrocytes and microglia in the postischemic inflammatory response and the role of heat shock proteins in modulating inflammation and outcome after cerebral ischemia.

### **2. Microglia: Immune cells of the CNS**

178 Advances in the Preclinical Study of Ischemic Stroke

spinal cord (Pellerin, 2005; Reichenbach and Wolburg, 2005). Once thought of merely as neuronal support cells, astrocytes and microglia, in their physiologic role, dynamically control synaptic function and neuronal activity by performing a variety of crucial functions. Microglia, the intrinsic macrophages of the CNS, provide immune surveillance against invading pathogens or nervous system insults (Aloisi, 2001) while astrocytes regulate synaptic glutamate levels, contribute to the blood-brain-barrier and supply neuronal growth factors (Liberto et al., 2004). Given their complex involvement in normal CNS function, glial cells must be considered in any strategy focused on neuronal preservation after ischemic

Heat shock proteins (HSP) are a phylogenetically conserved group of chaperones that assist in ATP-dependent protein folding, translocation across membranes, suppression of protein aggregation, presentation of substrates for degradation and modulate a host of other intracellular processes (Hartl, 1996). The 70 kDa heat shock protein family (HSP70) is the most extensively studied group of chaperones and consists of at least twelve constitutive and inducible proteins which aid in a coordinated response to cellular stressors. The most important members include: the constitutively expressed primarily cytosolic Hsc70/Hsp73, the heat inducible cytosolic form Hsp70/72, the glucose regulated mitochondrial protein Grp75/mortalin/mtHsp70 and the endoplasmic reticulum glucose regulated protein Grp78/BiP. The HSP70s are structurally comprised of a 44 kDa amino-terminal ATPase domain, 18 kDa carboxyl-terminal substrate-binding domain and a more variable 10 kDa segment that terminates in the highly conserved EEVD sequence that regulates intramolecular interactions and ATPase activity (Freeman et al., 1995). Our laboratory has demonstrated that the carboxyl-terminal domain of Hsp72 is sufficient to protect astrocytes from oxygen-glucose deprivation and decrease infarct volume after transient middle

Extensive work using overexpression and knockout of HSP70 family members has highlighted integral cytoprotective, anti-apoptotic and immune regulatory roles for these proteins. Induction of Hsp70/72 by heat stress or targeted overexpression in multiple experimental disease models including stroke (Rajdev et al., 2000), sepsis (Ryan et al., 1992), renal injury (Jo et al., 2006) and acute lung injury (Villar et al., 1994) demonstrated decreased organ injury and enhanced survival. Lee et al. (2001) showed that while *hsp70.1* knockout mice have a normal baseline phenotype; cerebral infarct volume was 30% larger and mortality was higher than in wild type littermates after focal ischemia. In addition, using a combined *hsp70.1/3* knockout (the two genes are separated by only 8kb on chromosome 17 and show 99% homology), Lee et al. (2004) later determined that cytochrome *c* release into the cytosol and levels of activated caspase-3 were increased after MCAO suggesting that Hsp 70/72 plays a role in preventing initiation of apoptosis after injury. The *hsp70.1/3* knockout also exhibited an enhanced inflammatory response to cecal perforation and ligation (an animal model of acute respiratory distress syndrome/sepsis) as evidenced by increased NF-B activation, TNF- and IL-6 expression and lung injury highlighting a role in controlling immune function in injury states (Singleton and Wischmeyer, 2006). In this same model, Weiss et al. (2002) demonstrated that targeted overexpression of Hsp70 in rat lung significantly attenuated interstitial and alveolar edema, protein exudation and dramatically decreased neutrophil accumulation leading to improvement of acute

cerebral artery occlusion (MCAO) (Sun et al., 2006b).

respiratory distress syndrome.

injury.

Microglia are monocyte-derived CNS tissue macrophages that are phenotypically adapted to the neural environment. As such, they are characterized by minimal phagocytic activity and low expression of the macrophage-specific antigen, CD45 (Kreutzberg, 1996) which may be used to differentiate resident microglia from infiltrating macrophages (Babcock et al., 2003). Microglia constitutively and inducibly express a variety of immune-related receptors on their surface including cytokine, chemokine, prostaglandin, pattern recognition and complement receptors (Aloisi, 2001). In addition, microglia can function as antigen presenting cells, and through CD40 on their membranes can form an "immunological synapse" with CD40L-expressing T-cells recruited centrally (Gerritse et al., 1996). As a result, microglia can respond to diverse stresses by performing innate immune functions such as phagocytosis, and can release potentially beneficial factors such as glial-derived neurotrophic factor (GDNF).

Microglia can release a host of factors to protect the CNS; however, when activated after ischemia by necrotic cell debris and other substances, they can produce free radicals, proinflammatory cytokines (IL-1β, TNFα, IL-6 and interferon-γ), reactive oxygen species, matrix metalloproteinases and glutamate in an aberrant fashion (Lucin and Wyss-Coray, 2009; Yenari et al., 2010). Such compounds are instrumental in the subsequent activation of astrocytes (see below), induction of cell adhesion molecules and T-lymphocyte recruitment into the CNS following various injuries (Liu and Hong, 2003; Sweitzer et al., 2002). Several studies have been performed testing inhibition of microglial activation as a strategy to protect neurons following ischemic injury. Transgenic mice lacking the pattern recognition receptor TLR4 (Hyakkoku et al., 2010), mice overexpressing the anti-inflammatory cytokine IL-10 (De Bilbao et al., 2009) and treatment with a neutralizing antibody against TNF-alpha (Barone et al., 1997), all resulted in suppressed microglial activation and significantly decreased infarct size following focal cerebral ischemia. Taken together, these studies suggest that inhibition of the post-ischemia inflammatory response is a viable option for stroke treatment. Although microglia quickly respond to ischemia by producing proinflammatory cytokines (Yenari et al., 2010), proliferating (Denes et al., 2007) and exhibiting an altered cell morphology (Tanaka et al., 2003), only a small percentage (1-8%) of the microglia in the corpus callosum and lesion penumbra, and no microglia in the lesion core, express Hsp72 early after focal ischemia (Soriano et al., 1994). This indicates that a robust increase in Hsp72 protein expression is not a normal aspect of the post-ischemic microglial phenotype.

Interestingly, the majority of inflammatory mediators produced by microglia after stroke are produced by NFB pathway activation (Yenari et al., 2010). Hsp72 has been implicated in modulation of inflammation by suppressing NFB through multiple interactions. Previous work has shown that Hsp72 directly binds to the NFB:IB complex, thus preventing IB phosphorylation and subsequent NFB activation (Feinstein et al., 1996; Zheng et al., 2008). Ran et al. (2004) further demonstrated that Hsp72 binds directly to IB kinase- (IKK-), an essential regulatory component of the IKK complex, blocking activation of IB and release of

GGlial Cells, Inflammation and Heat Shock Proteins in Cerebral Ischemia 181

development (Daneman et al., 2010), ablation of astrocytes in the process of CNS restoration leads to failure of blood-brain barrier repair, an enhanced infiltration of leukocytes and subsequent excitotoxic neuronal death (Bush et al., 1999). In addition, astrocytes have been postulated to mediate functional hyperemia, the coupling of neuronal activity with increased cerebral blood flow, via changes in intracellular calcium in astrocytic endfeet leading to release of vasoactive substances (cyclooxygenase, adenosine) and modulation of adjacent arterioles (Iadecola and Nedergaard, 2007; Takano et al., 2006). It is estimated that 56% of rat cortical synapses are ensheathed by astrocyte domains (Chao et al., 2002) and an individual astrocyte occupies an exclusive, non-overlapping territory; each interfacing with the vasculature and thousands of synapses suggesting a complex process of synaptic integration (Bushong et al., 2004). Indeed, mice lacking GFAP exhibit increased infarct following ischemia, possibly due to blood-brain barrier and cerebral blood flow dysfunction (Nawashiro et al., 2000). The implication is that following ischemia, astrocytes are poised to influence penumbral blood flow and provision of neuronal nutrients in a coordinated

While it was established over a hundred years ago that neurons of the CA1 region of the hippocampus are selectively vulnerable to forebrain ischemia (Pulsinelli, 1985); evidence for injury to astrocytes has been more recent (Petito et al., 1998). The two main types of astrocytes found in the CNS are protoplasmic astrocytes found in the gray matter and fibrous astrocytes found in the white matter. This is important because astrocytes isolated from different brain regions exhibit varying sensitivity to oxygen-glucose deprivation (OGD) with striatal cells most vulnerable followed by hippocampal and cortical astrocytes (Xu et al., 2001). *In vivo*, using the middle cerebral artery occlusion (MCAO) model of focal ischemia, Lukaszevicz et al. (2002) demonstrated selective degeneration of protoplasmic cortical astrocytes with associated breakdown of the blood brain barrier. Yu et al. (1989) first demonstrated that cultured astrocytes are sensitive to hypoxia, exhibiting swelling and 80% suppression of glutamate uptake after 12-24 hours of oxygen deprivation. Using in situ hybridization and immunohistochemistry, Liu et al. (1999) demonstrated an early decline in mRNA and protein for GFAP in the ischemic core after middle cerebral artery occlusion (MCAO) with a corresponding increase in astrocyte markers in the penumbra, both of which temporally preceded neuronal death. In agreement, Zhao et al. (2003) showed early loss of GFAP after traumatic brain injury. Early *in vitro* work determined that co-culture of neurons with astrocytes protected them from OGD; specifically, when cultured alone and exposed to 4 hour of OGD only 5% of neurons survived compared to 75% survival in mixed cultures (Vibulsreth et al., 1987). Neurons co-cultured with astrocytes have also been shown to survive exposure to 100-fold higher concentrations of glutamate (Rosenberg and Aizenman, 1989). Taken together, these studies suggest that therapeutics aimed at maintaining

astrocyte viability and function may protect neurons from ischemic injury.

Another key function of astrocytes is the control of extracellular glutamate homeostasis through sodium-dependent uptake via the excitatory amino acid transporters (EAATs) (Danbolt, 2001). The glutamate-aspartate transporter (GLAST/EAAT1) and the glutamate transporter-1 (GLT-1/EAAT2) are primarily localized in astrocytes. GLT-1 is the most

**3.3 Astrocytes as regulators of synaptic glutamate** 

fashion.

**3.2 Astrocytes and ischemic injury** 

NFB. Mice overexpressing Hsp72 have decreased infarcts compared to their wild-type counterparts, along with attenuated microglial activation and TNF- production (Rajdev et al., 2000; Zheng et al., 2008). Microglia isolated from these mice showed decreased toxicity towards cultured astrocytes, accompanied by decreased NFB signaling. Thus, the use of heat shock proteins to inhibit NFB signaling in microglia may be an effective treatment for stroke by inhibiting a plethora of downstream factors that ultimately lead to further glial cell activation and neuronal cell death.

### **3. Astrocytes: Multiple roles in physiology & pathophysiology**

Historically, astrocytes were considered to be passive elements in the CNS and neurotransmitter receptor expression was believed to be solely a characteristic of neurons. To the contrary, astrocytes express a variety of receptors on their surface, including metabotropic glutamate receptors, GABA receptors, adenosine receptors and the mu, delta and kappa opioid receptors, among others (Kettenmann and Steinhauser, 2005). In addition, these cells express a variety of ion channels on their surface, including Ca2+ channels. These are important because astrocytes are thought to function as part of a syncytia linked by connexins in order to transfer information in the form of ATP and Ca2+ (Kielian, 2008). In a model of ischemia, Cotrina et al. (1998) demonstrated that gap junctions remain open after oxygen-glucose deprivation (OGD) and may contribute to infarct evolution through direct astrocytic intercellular communication.

Astrocytes derive from the neuroectoderm and express a series of "marker antigens" during development such as the cytoskeletal protein vimentin and nestin (Eliasson et al., 1999) and the fatty acid binding protein brain lipid binding protein (Schmid et al., 2006). Once they reach their adult phenotype, other proteins are expressed including Aldh1L1 and glial fibrillary acidic protein (GFAP) (Cahoy et al., 2008). This intermediate filament is commonly considered to be astrocyte-specific, though it may also be found on reactive choroid plexus epithelium cells and neuronal precursor cells (Reichenbach and Wolburg, 2005) and there are also astrocyte populations that are GFAP negative (Kimelberg, 2004). GFAP functions as a structural protein and enhancement of GFAP remains the mainstay for demonstrating astrocytic reactivity in the CNS (Eng et al., 2000), however, it is important to note that only 15% of the total astrocyte cell volume is labeled with GFAP (Bushong et al., 2002). Remarkably, GFAP knockout mice do not exhibit an altered phenotype at baseline; however, after trauma, astrocyte hypertrophy is suppressed, scar formation is less organized and healing is slowed (Pekny et al., 1995; Pekny and Pekna, 2004). In further work using a lesion model of the entorhinal cortex, Wilhelmsson et al. (2004) demonstrated that double GFAP-/- Vim-/- mice display increased neuronal loss in the dentate gyrus at day 4 postinjury but enhanced synaptic regeneration at day 10. These data suggest a two-part response of reactive astrocytes to CNS injury: beneficial for neuronal survival in the initial post-injury period and detrimental to CNS regeneration in the recovery phase (Pekny and Nilsson, 2005).

### **3.1 Role in the blood-brain barrier**

Astrocytes play an integral role in the structure of the blood-brain barrier (BBB), which limits the entry of circulating elements into the nervous system. Though recent data suggests that pericytes, not astrocytes, are required for the formation of the BBB during development (Daneman et al., 2010), ablation of astrocytes in the process of CNS restoration leads to failure of blood-brain barrier repair, an enhanced infiltration of leukocytes and subsequent excitotoxic neuronal death (Bush et al., 1999). In addition, astrocytes have been postulated to mediate functional hyperemia, the coupling of neuronal activity with increased cerebral blood flow, via changes in intracellular calcium in astrocytic endfeet leading to release of vasoactive substances (cyclooxygenase, adenosine) and modulation of adjacent arterioles (Iadecola and Nedergaard, 2007; Takano et al., 2006). It is estimated that 56% of rat cortical synapses are ensheathed by astrocyte domains (Chao et al., 2002) and an individual astrocyte occupies an exclusive, non-overlapping territory; each interfacing with the vasculature and thousands of synapses suggesting a complex process of synaptic integration (Bushong et al., 2004). Indeed, mice lacking GFAP exhibit increased infarct following ischemia, possibly due to blood-brain barrier and cerebral blood flow dysfunction (Nawashiro et al., 2000). The implication is that following ischemia, astrocytes are poised to influence penumbral blood flow and provision of neuronal nutrients in a coordinated fashion.

### **3.2 Astrocytes and ischemic injury**

180 Advances in the Preclinical Study of Ischemic Stroke

NFB. Mice overexpressing Hsp72 have decreased infarcts compared to their wild-type counterparts, along with attenuated microglial activation and TNF- production (Rajdev et al., 2000; Zheng et al., 2008). Microglia isolated from these mice showed decreased toxicity towards cultured astrocytes, accompanied by decreased NFB signaling. Thus, the use of heat shock proteins to inhibit NFB signaling in microglia may be an effective treatment for stroke by inhibiting a plethora of downstream factors that ultimately lead to further glial cell

Historically, astrocytes were considered to be passive elements in the CNS and neurotransmitter receptor expression was believed to be solely a characteristic of neurons. To the contrary, astrocytes express a variety of receptors on their surface, including metabotropic glutamate receptors, GABA receptors, adenosine receptors and the mu, delta and kappa opioid receptors, among others (Kettenmann and Steinhauser, 2005). In addition, these cells express a variety of ion channels on their surface, including Ca2+ channels. These are important because astrocytes are thought to function as part of a syncytia linked by connexins in order to transfer information in the form of ATP and Ca2+ (Kielian, 2008). In a model of ischemia, Cotrina et al. (1998) demonstrated that gap junctions remain open after oxygen-glucose deprivation (OGD) and may contribute to infarct evolution through direct

Astrocytes derive from the neuroectoderm and express a series of "marker antigens" during development such as the cytoskeletal protein vimentin and nestin (Eliasson et al., 1999) and the fatty acid binding protein brain lipid binding protein (Schmid et al., 2006). Once they reach their adult phenotype, other proteins are expressed including Aldh1L1 and glial fibrillary acidic protein (GFAP) (Cahoy et al., 2008). This intermediate filament is commonly considered to be astrocyte-specific, though it may also be found on reactive choroid plexus epithelium cells and neuronal precursor cells (Reichenbach and Wolburg, 2005) and there are also astrocyte populations that are GFAP negative (Kimelberg, 2004). GFAP functions as a structural protein and enhancement of GFAP remains the mainstay for demonstrating astrocytic reactivity in the CNS (Eng et al., 2000), however, it is important to note that only 15% of the total astrocyte cell volume is labeled with GFAP (Bushong et al., 2002). Remarkably, GFAP knockout mice do not exhibit an altered phenotype at baseline; however, after trauma, astrocyte hypertrophy is suppressed, scar formation is less organized and healing is slowed (Pekny et al., 1995; Pekny and Pekna, 2004). In further work using a lesion model of the entorhinal cortex, Wilhelmsson et al. (2004) demonstrated that double GFAP-/- Vim-/- mice display increased neuronal loss in the dentate gyrus at day 4 postinjury but enhanced synaptic regeneration at day 10. These data suggest a two-part response of reactive astrocytes to CNS injury: beneficial for neuronal survival in the initial post-injury period and detrimental to CNS regeneration in the recovery phase (Pekny and Nilsson,

Astrocytes play an integral role in the structure of the blood-brain barrier (BBB), which limits the entry of circulating elements into the nervous system. Though recent data suggests that pericytes, not astrocytes, are required for the formation of the BBB during

**3. Astrocytes: Multiple roles in physiology & pathophysiology** 

activation and neuronal cell death.

astrocytic intercellular communication.

**3.1 Role in the blood-brain barrier** 

2005).

While it was established over a hundred years ago that neurons of the CA1 region of the hippocampus are selectively vulnerable to forebrain ischemia (Pulsinelli, 1985); evidence for injury to astrocytes has been more recent (Petito et al., 1998). The two main types of astrocytes found in the CNS are protoplasmic astrocytes found in the gray matter and fibrous astrocytes found in the white matter. This is important because astrocytes isolated from different brain regions exhibit varying sensitivity to oxygen-glucose deprivation (OGD) with striatal cells most vulnerable followed by hippocampal and cortical astrocytes (Xu et al., 2001). *In vivo*, using the middle cerebral artery occlusion (MCAO) model of focal ischemia, Lukaszevicz et al. (2002) demonstrated selective degeneration of protoplasmic cortical astrocytes with associated breakdown of the blood brain barrier. Yu et al. (1989) first demonstrated that cultured astrocytes are sensitive to hypoxia, exhibiting swelling and 80% suppression of glutamate uptake after 12-24 hours of oxygen deprivation. Using in situ hybridization and immunohistochemistry, Liu et al. (1999) demonstrated an early decline in mRNA and protein for GFAP in the ischemic core after middle cerebral artery occlusion (MCAO) with a corresponding increase in astrocyte markers in the penumbra, both of which temporally preceded neuronal death. In agreement, Zhao et al. (2003) showed early loss of GFAP after traumatic brain injury. Early *in vitro* work determined that co-culture of neurons with astrocytes protected them from OGD; specifically, when cultured alone and exposed to 4 hour of OGD only 5% of neurons survived compared to 75% survival in mixed cultures (Vibulsreth et al., 1987). Neurons co-cultured with astrocytes have also been shown to survive exposure to 100-fold higher concentrations of glutamate (Rosenberg and Aizenman, 1989). Taken together, these studies suggest that therapeutics aimed at maintaining astrocyte viability and function may protect neurons from ischemic injury.

#### **3.3 Astrocytes as regulators of synaptic glutamate**

Another key function of astrocytes is the control of extracellular glutamate homeostasis through sodium-dependent uptake via the excitatory amino acid transporters (EAATs) (Danbolt, 2001). The glutamate-aspartate transporter (GLAST/EAAT1) and the glutamate transporter-1 (GLT-1/EAAT2) are primarily localized in astrocytes. GLT-1 is the most

GGlial Cells, Inflammation and Heat Shock Proteins in Cerebral Ischemia 183

has now emerged that indicates a direct role for Hsp72 in regulation of cell death by apoptosis and potentially even necrosis (Giffard and Yenari, 2004). Mitochondria are central to both cell death pathways; severe ischemia renders mitochondria unable to produce ATP and in less extreme stress conditions, mitochondria may increase production of reactive oxygen species (ROS), lose membrane potential and undergo changes in respiratory function (Dugan and Kim-Han, 2004). Ischemia can activate mitochondrial cytochrome *c* which translocates to the cytosol where it interacts with Apaf1 to form the apoptosome and activate caspase 9, initiating a cascade leading to DNA fragmentation (Chan, 2004; Leist and Jaattela, 2001). We have shown that overexpression of Hsp72 in cultured astrocytes subjected to glucose deprivation leads to decreased formation of ROS, stabilization of the mitochondrial membrane potential and prevention of increases in state IV respiration suggesting decreased cytochrome *c* release and activation of apoptosis (Ouyang et al., 2006). Furthermore, in the MCAO model of ischemia, we have shown that transfection of Hsp72 leads to inhibition of apoptosis-inducing factor (AIF) translocation to the nucleus thereby blocking caspase-independent apoptosis (Sun et al., 2006b). This is supported by previous work by Ravagnan et al. (2001) demonstrating that Hsp72 protects Apaf -/- cells against death via an interaction with AIF. For a comprehensive review of the role of Hsp72 in cell

Mitochondrial dysfunction leading to a loss of ATP production impairs many of the energydemanding neuroprotective functions of astrocytes after ischemic injury including ion homeostasis and neurotransmitter turnover (Bambrick et al., 2004). Mortalin forms part of the mitochondrial protein import machinery by binding a translocase in the inner membrane to form an ATP-dependent motor (Voos et al., 1999) and while it is not heat inducible it has been shown to increase after a variety of other stressors including glucose deprivation, oxidative stress and focal cerebral ischemia (Hadari et al., 1997; Lee, 2001;

Using LXSN-mortalin-transduced astrocytes, our laboratory has shown that overexpression of mortalin produces mitochondrial protection after glucose deprivation (Voloboueva et al., 2008). Specifically, we found decreased hydroethidine fluorescence (an indicator of the accumulation of reactive oxygen species (ROS)) and preserved mitochondrial membrane potential as measured by tetramethyl rhodamine staining (TMRE), a dye whose sequestration by mitochondria depends on the mitochondrial membrane potential, in astrocytes expressing increased levels of mortalin. In addition, mortalin overexpression preserved ATP levels in astrocytes subjected to oxygen-glucose deprivation and enhanced cell survival. In a more clinically relevant model of stroke, middle cerebral artery occlusion (MCAO), we further investigated the role of mortalin in mitochondrial protection. Rats overexpressing mortalin in astrocytes and neurons by direct intraventricular injection of a DNA plasmid encoding mortalin were subjected to MCAO and found to have a reduction in infarct area, decreased ROS and lipid oxidation compared to vector-transfected controls. Similar to our *in vitro* data we showed that mortalin overexpression reduced the ischemiainduced depletion of ATP and maintained electron transport chain complex IV activity (Xu

To investigate the specific role of astrocytic mitochondrial inhibition in ischemia we treated astrocyte cultures with the Krebs cycle inhibitor, fluorocitrate (Voloboueva et al., 2007).

death please see Giffard et al. (2008).

Massa et al., 1995).

et al., 2009).

**4.3 Mitochondrial protection and mortalin/mitochondrial Hsp70** 

studied astrocyte transporter and is suggested to be responsible for over 90% of synaptic glutamate clearance (Tanaka et al., 1997). Dysregulation of synaptic glutamate clearance by these transporters has been implicated in many disease processes (Gegelashvili and Schousboe, 1997; Maragakis and Rothstein, 2001; Rothstein et al., 1996). For example, glutamate levels have been shown to increase 50 times from baseline after ischemia and glutamate efflux from astrocytes has been suggested to occur by reversal of glutamate transport (Mitani et al., 1994; Seki et al., 1999). Transient MCAO leads to downregulation of GLT-1 which precedes neuronal death and antisense knockdown of GLT-1 exacerbates neuronal death in the same model (Rao et al., 2001a; Rao et al., 2001b; Rao et al., 2000). Furthermore, using pre-treatment with ceftriaxone, a known inducer of GLT-1, Chu et al. (2007) demonstrated a dose-dependent decrease in infarct volume and levels of the proinflammatory cytokine TNF after MCAO. In addition, work from our laboratory showed that upregulation of GLT-1 in astrocytes using ceftriaxone decreases CA1 neuronal cell death in a global ischemia model (Ouyang et al., 2007). Complete knock-out of GLT-1 results in spontaneous seizures, selective death of CA1 neurons and 20% survival of animals at 12 weeks (Tanaka et al., 1997) and mice lacking GLT-1 display enhanced neuronal death after brief ischemia compared to wild type controls (Mitani and Tanaka, 2003). These findings underline the importance of exquisite regulation of synaptic glutamate by astrocytes in maintaining neuronal integrity.

### **4. Heat shock proteins affect astrocyte regulation of ischemia**

### **4.1 A role for Hsp72 in ischemia**

Our laboratory has been particularly interested in the role of astrocytic heat shock proteins as regulators of ischemic injury. Initial studies demonstrated induction of Hsp72 in cultured astrocytes exposed to heat shock or OGD (Bergeron et al., 1996) and further work confirmed that Hsp72 overexpression in astrocytes exposed to glucose deprivation (Xu and Giffard, 1997) or oxygen-glucose deprivation (Papadopoulos et al., 1996) was cytoprotective. Interestingly, overexpression of Hsp72 in astrocytes was shown to protect co-cultured neurons from ischemic injury (Xu et al., 1999); highlighting the integral role of astrocytes in neuronal homeostasis and survival. As discussed above, we have also demonstrated that the carboxyl-terminal domain of Hsp72 is sufficient to protect astrocytes from oxygen-glucose deprivation by suppressing protein aggregation and further decreases infarct volume after transient middle cerebral artery occlusion (MCAO) (Sun et al., 2006b). Astrocytes in the CA1 region of the brain, which is particularly sensitive to forebrain ischemia, lose glutamate transporter expression and activity prior to the death of CA1 neurons (Chen et al., 2005; Ouyang et al., 2007; Yeh et al., 2005). We have shown that astrocyte-targeted overexpression of Hsp72 not only protects CA1 neurons from transient forebrain ischemia, but also preserves GLT-1 immunoreactivity in the region (Xu et al., 2010) suggesting a possible mechanism for the observed protection.

### **4.2 Hsp72 as a regulator of apoptosis**

Multiple studies have highlighted a neuroprotective role of Hsp72 overexpression in models of ischemia (Hoehn et al., 2001; Rajdev et al., 2000; van der Weerd et al., 2005). The mechanism was initially attributed to the known chaperone functions of Hsp72 including maintaining correct protein folding and inhibiting aggregation, however, a body of work

studied astrocyte transporter and is suggested to be responsible for over 90% of synaptic glutamate clearance (Tanaka et al., 1997). Dysregulation of synaptic glutamate clearance by these transporters has been implicated in many disease processes (Gegelashvili and Schousboe, 1997; Maragakis and Rothstein, 2001; Rothstein et al., 1996). For example, glutamate levels have been shown to increase 50 times from baseline after ischemia and glutamate efflux from astrocytes has been suggested to occur by reversal of glutamate transport (Mitani et al., 1994; Seki et al., 1999). Transient MCAO leads to downregulation of GLT-1 which precedes neuronal death and antisense knockdown of GLT-1 exacerbates neuronal death in the same model (Rao et al., 2001a; Rao et al., 2001b; Rao et al., 2000). Furthermore, using pre-treatment with ceftriaxone, a known inducer of GLT-1, Chu et al. (2007) demonstrated a dose-dependent decrease in infarct volume and levels of the proinflammatory cytokine TNF after MCAO. In addition, work from our laboratory showed that upregulation of GLT-1 in astrocytes using ceftriaxone decreases CA1 neuronal cell death in a global ischemia model (Ouyang et al., 2007). Complete knock-out of GLT-1 results in spontaneous seizures, selective death of CA1 neurons and 20% survival of animals at 12 weeks (Tanaka et al., 1997) and mice lacking GLT-1 display enhanced neuronal death after brief ischemia compared to wild type controls (Mitani and Tanaka, 2003). These findings underline the importance of exquisite regulation of synaptic glutamate by

astrocytes in maintaining neuronal integrity.

**4.1 A role for Hsp72 in ischemia** 

mechanism for the observed protection.

**4.2 Hsp72 as a regulator of apoptosis** 

**4. Heat shock proteins affect astrocyte regulation of ischemia** 

Our laboratory has been particularly interested in the role of astrocytic heat shock proteins as regulators of ischemic injury. Initial studies demonstrated induction of Hsp72 in cultured astrocytes exposed to heat shock or OGD (Bergeron et al., 1996) and further work confirmed that Hsp72 overexpression in astrocytes exposed to glucose deprivation (Xu and Giffard, 1997) or oxygen-glucose deprivation (Papadopoulos et al., 1996) was cytoprotective. Interestingly, overexpression of Hsp72 in astrocytes was shown to protect co-cultured neurons from ischemic injury (Xu et al., 1999); highlighting the integral role of astrocytes in neuronal homeostasis and survival. As discussed above, we have also demonstrated that the carboxyl-terminal domain of Hsp72 is sufficient to protect astrocytes from oxygen-glucose deprivation by suppressing protein aggregation and further decreases infarct volume after transient middle cerebral artery occlusion (MCAO) (Sun et al., 2006b). Astrocytes in the CA1 region of the brain, which is particularly sensitive to forebrain ischemia, lose glutamate transporter expression and activity prior to the death of CA1 neurons (Chen et al., 2005; Ouyang et al., 2007; Yeh et al., 2005). We have shown that astrocyte-targeted overexpression of Hsp72 not only protects CA1 neurons from transient forebrain ischemia, but also preserves GLT-1 immunoreactivity in the region (Xu et al., 2010) suggesting a possible

Multiple studies have highlighted a neuroprotective role of Hsp72 overexpression in models of ischemia (Hoehn et al., 2001; Rajdev et al., 2000; van der Weerd et al., 2005). The mechanism was initially attributed to the known chaperone functions of Hsp72 including maintaining correct protein folding and inhibiting aggregation, however, a body of work has now emerged that indicates a direct role for Hsp72 in regulation of cell death by apoptosis and potentially even necrosis (Giffard and Yenari, 2004). Mitochondria are central to both cell death pathways; severe ischemia renders mitochondria unable to produce ATP and in less extreme stress conditions, mitochondria may increase production of reactive oxygen species (ROS), lose membrane potential and undergo changes in respiratory function (Dugan and Kim-Han, 2004). Ischemia can activate mitochondrial cytochrome *c* which translocates to the cytosol where it interacts with Apaf1 to form the apoptosome and activate caspase 9, initiating a cascade leading to DNA fragmentation (Chan, 2004; Leist and Jaattela, 2001). We have shown that overexpression of Hsp72 in cultured astrocytes subjected to glucose deprivation leads to decreased formation of ROS, stabilization of the mitochondrial membrane potential and prevention of increases in state IV respiration suggesting decreased cytochrome *c* release and activation of apoptosis (Ouyang et al., 2006). Furthermore, in the MCAO model of ischemia, we have shown that transfection of Hsp72 leads to inhibition of apoptosis-inducing factor (AIF) translocation to the nucleus thereby blocking caspase-independent apoptosis (Sun et al., 2006b). This is supported by previous work by Ravagnan et al. (2001) demonstrating that Hsp72 protects Apaf -/- cells against death via an interaction with AIF. For a comprehensive review of the role of Hsp72 in cell death please see Giffard et al. (2008).

### **4.3 Mitochondrial protection and mortalin/mitochondrial Hsp70**

Mitochondrial dysfunction leading to a loss of ATP production impairs many of the energydemanding neuroprotective functions of astrocytes after ischemic injury including ion homeostasis and neurotransmitter turnover (Bambrick et al., 2004). Mortalin forms part of the mitochondrial protein import machinery by binding a translocase in the inner membrane to form an ATP-dependent motor (Voos et al., 1999) and while it is not heat inducible it has been shown to increase after a variety of other stressors including glucose deprivation, oxidative stress and focal cerebral ischemia (Hadari et al., 1997; Lee, 2001; Massa et al., 1995).

Using LXSN-mortalin-transduced astrocytes, our laboratory has shown that overexpression of mortalin produces mitochondrial protection after glucose deprivation (Voloboueva et al., 2008). Specifically, we found decreased hydroethidine fluorescence (an indicator of the accumulation of reactive oxygen species (ROS)) and preserved mitochondrial membrane potential as measured by tetramethyl rhodamine staining (TMRE), a dye whose sequestration by mitochondria depends on the mitochondrial membrane potential, in astrocytes expressing increased levels of mortalin. In addition, mortalin overexpression preserved ATP levels in astrocytes subjected to oxygen-glucose deprivation and enhanced cell survival. In a more clinically relevant model of stroke, middle cerebral artery occlusion (MCAO), we further investigated the role of mortalin in mitochondrial protection. Rats overexpressing mortalin in astrocytes and neurons by direct intraventricular injection of a DNA plasmid encoding mortalin were subjected to MCAO and found to have a reduction in infarct area, decreased ROS and lipid oxidation compared to vector-transfected controls. Similar to our *in vitro* data we showed that mortalin overexpression reduced the ischemiainduced depletion of ATP and maintained electron transport chain complex IV activity (Xu et al., 2009).

To investigate the specific role of astrocytic mitochondrial inhibition in ischemia we treated astrocyte cultures with the Krebs cycle inhibitor, fluorocitrate (Voloboueva et al., 2007).

GGlial Cells, Inflammation and Heat Shock Proteins in Cerebral Ischemia 185

pathways to inhibit astrocyte dysfunction leading to neuronal death. Microglia play an important role in the inflammatory cascade following ischemia. Activation of NFB leads to the production of pro-inflammatory cytokines which can exacerbate damage to neurons. Heat shock proteins may also have a role in inhibiting the activation of NFB, by direct interaction and stabilization of the IB:NFB complex or by inhibition of IKK preventing

phosphorylation of degradation of IB.

Fig. 1. Glial involvement in neuronal death from ischemia.

This work was supported in part by NIH grants NS053898 and GM49831 to RGG and T32

Babcock, A. A., Kuziel, W. A., Rivest, S.& Owens, T. (2003). Chemokine expression by glial

cells directs leukocytes to sites of axonal injury in the CNS. *J Neurosci,* Vol. 23, No.

Aloisi, F. (2001). Immune function of microglia. *Glia,* Vol. 36, No. 2, pp. 165-179

**6. Acknowledgment** 

21, pp. 7922-7930

GM089626 to REW.

**7. References** 

After glucose deprivation, astrocytes treated with fluorocitrate showed depletion of ATP, cell death and suppressed glutamate uptake within 3 hours. In addition, we demonstrated that inhibition of astrocytic mitochondria increased cell death in co-cultured neurons and enhanced changes in mitochondrial membrane potential in astrocytes suggesting a two-way crosstalk between these cells after injury related to energy supply and demand (Voloboueva et al., 2007).

### **4.4 The role of Grp78/BiP in calcium handling**

The endoplasmic reticulum (ER) controls several cellular processes including protein synthesis, folding and trafficking. Under conditions of physiologic stress, including ischemia, that perturb ER Ca2+ homeostasis and therefore ER protein folding, the concerted actions of multiple pathways that influence protein synthesis and folding are activated; this is termed the unfolded protein response (UPR). Grp78/BiP is an ER chaperone protein involved in protein folding, suppression of apoptosis and regulation of the UPR. It is strongly induced as part of the UPR and translocates to mitochondria and other compartments after stress where it is postulated to mediate ER-mitochondria crosstalk (Sun et al., 2006a). Several prior studies also supported a role for Grp78/BiP in protection from ischemia-induced cell death using BIX, a Grp78 inducer, prior to transient global forebrain ischemia in gerbils (Oida et al., 2008) and focal ischemia in mice (Kudo et al., 2007). Work from our laboratory further showed that overexpression of Grp78/BiP protected cultured astrocytes from OGD, suppressed the GD-induced increase in mitochondrial Ca2+ and preserved mitochondrial function (Ouyang et al., 2011).

### **5. Conclusion**

The processes leading to neuronal death following ischemia are complex and involve the integrated action of multiple pathways in a variety of cells types. Data from our laboratory, among others, has highlighted a role for dysfunction of astrocytes and microglia in the pathophysiology of cerebral ischemia. Currently, the most promising areas for intervention are ischemia-induced inflammation and oxidative stress with several drugs in clinical trials at this time aimed at suppressing cytokine release and reactive oxygen species, respectively. For example, the microglial inhibitor minocycline, which affects the release of inflammatory mediators from activated microglia is in Phase IV trials (Yenari et al., 2006), and epoetin alfa, which may be downregulated in astrocytes after ischemic injury is in Phase II/III trials (Zhao and Rempe, 2010). In this chapter we have reviewed several key functions of glial cells including control of inflammation, apoptosis and synaptic glutamate clearance as well as modulation of blood flow and mitochondrial protection (see Figure 1) that may be therapeutically targeted to protect neurons from injury. As the roles of glial cells and heat shock proteins in normal function and cerebral ischemia continue to be elucidated novel neuroprotective strategies may be developed in the future.

Astrocytes are well poised to respond to changes in blood flow by release of vasodilators such as cyclooxygenase (COX) and adenosine. In the case of ischemia from thrombus or embolus, the decrease in oxygen and glucose delivery can initiate a stress response in astrocytes including changes in morphology, increase in intermediate filaments such as GFAP (not shown), decreases in glutamate transporters and activation of mitochondrial cell death pathways. Heat shock proteins have been shown to modulate several of these

After glucose deprivation, astrocytes treated with fluorocitrate showed depletion of ATP, cell death and suppressed glutamate uptake within 3 hours. In addition, we demonstrated that inhibition of astrocytic mitochondria increased cell death in co-cultured neurons and enhanced changes in mitochondrial membrane potential in astrocytes suggesting a two-way crosstalk between these cells after injury related to energy supply and demand (Voloboueva

The endoplasmic reticulum (ER) controls several cellular processes including protein synthesis, folding and trafficking. Under conditions of physiologic stress, including ischemia, that perturb ER Ca2+ homeostasis and therefore ER protein folding, the concerted actions of multiple pathways that influence protein synthesis and folding are activated; this is termed the unfolded protein response (UPR). Grp78/BiP is an ER chaperone protein involved in protein folding, suppression of apoptosis and regulation of the UPR. It is strongly induced as part of the UPR and translocates to mitochondria and other compartments after stress where it is postulated to mediate ER-mitochondria crosstalk (Sun et al., 2006a). Several prior studies also supported a role for Grp78/BiP in protection from ischemia-induced cell death using BIX, a Grp78 inducer, prior to transient global forebrain ischemia in gerbils (Oida et al., 2008) and focal ischemia in mice (Kudo et al., 2007). Work from our laboratory further showed that overexpression of Grp78/BiP protected cultured astrocytes from OGD, suppressed the GD-induced increase in mitochondrial Ca2+ and

The processes leading to neuronal death following ischemia are complex and involve the integrated action of multiple pathways in a variety of cells types. Data from our laboratory, among others, has highlighted a role for dysfunction of astrocytes and microglia in the pathophysiology of cerebral ischemia. Currently, the most promising areas for intervention are ischemia-induced inflammation and oxidative stress with several drugs in clinical trials at this time aimed at suppressing cytokine release and reactive oxygen species, respectively. For example, the microglial inhibitor minocycline, which affects the release of inflammatory mediators from activated microglia is in Phase IV trials (Yenari et al., 2006), and epoetin alfa, which may be downregulated in astrocytes after ischemic injury is in Phase II/III trials (Zhao and Rempe, 2010). In this chapter we have reviewed several key functions of glial cells including control of inflammation, apoptosis and synaptic glutamate clearance as well as modulation of blood flow and mitochondrial protection (see Figure 1) that may be therapeutically targeted to protect neurons from injury. As the roles of glial cells and heat shock proteins in normal function and cerebral ischemia continue to be elucidated novel

Astrocytes are well poised to respond to changes in blood flow by release of vasodilators such as cyclooxygenase (COX) and adenosine. In the case of ischemia from thrombus or embolus, the decrease in oxygen and glucose delivery can initiate a stress response in astrocytes including changes in morphology, increase in intermediate filaments such as GFAP (not shown), decreases in glutamate transporters and activation of mitochondrial cell death pathways. Heat shock proteins have been shown to modulate several of these

et al., 2007).

**5. Conclusion** 

**4.4 The role of Grp78/BiP in calcium handling** 

preserved mitochondrial function (Ouyang et al., 2011).

neuroprotective strategies may be developed in the future.

pathways to inhibit astrocyte dysfunction leading to neuronal death. Microglia play an important role in the inflammatory cascade following ischemia. Activation of NFB leads to the production of pro-inflammatory cytokines which can exacerbate damage to neurons. Heat shock proteins may also have a role in inhibiting the activation of NFB, by direct interaction and stabilization of the IB:NFB complex or by inhibition of IKK preventing phosphorylation of degradation of IB.

Fig. 1. Glial involvement in neuronal death from ischemia.

### **6. Acknowledgment**

This work was supported in part by NIH grants NS053898 and GM49831 to RGG and T32 GM089626 to REW.

### **7. References**

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**10** 

**Role of Creatine Kinase – Hexokinase** 

**Nucleotides in Mitochondrial Dysfunction** 

Creatine phosphokinase (CK) (ATP: creatine phosphotransferase, ЕС 2.7.3.2.) is found in a variety of cells with high and fluctuating energy requirements. It catalyses the reversible transfer of the high-energy-N-phosphoryl group from phosphocreatine to ADP. Creatine kinase connects sites of energy production with sites of energy consumption (Dolder et. al., 2001; Focant et al., 1970; Grossmann et al., 1985; Lipskaya et al., 1989; Walzel et al., 2002;

There are know to be three cytosolic and two mitochondrial isoforms of CK. The more basic mitochondrial creatine kinase MiCKb is accumulated in mitochondria of cardiac muscle and skeletal muscle. The more acidic mitochondrial creatine kinase MiCKa was found in the

Creatine kinase can exist in two interconvertible forms: dimer and octamer (Eriksson et al., 1998; Shen et al., 2002). Creatine kinase binds to the outer leaflet of the entire inner mitochondrial membrane and is specifically enriched in the so-called contact sites where inner and outer membranes are in close proximity (Boero et al., 2003; Chen et al, 1994; Lin et

A change in the octamer/dimer ratio may influence on the association behavior of mitochondrial creatine kinase in general and thus modulate mitochondrial energy flux

Mitochondrial creatine kinase forms the functional microcompartment together with the mitochondrial porin (voltage-dependent anion channel) in the outer membrane and as well as the transmembrane protein adenine nucleotide translocase in the inner membrane (Fritz-

Hexokinase (HK) (ATP:D-hexokinase-6-phosphotransferase, EC 2.7.11) is the enzyme with

The type I isoenzyme of mammalian hexokinase is ubiquitously expressed in mammalian tissues but is found particularly at high levels in the brain where it plays an important role in regulating the rate of cerebral glucose metabolism (Schwab & Wilson, 1989; Wilson, 1985). The major portion of the hexokinase activity in the brain is associated with mitochondria. About 85% of hexokinase is bound to the outer mitochondrial membrane, forming the specific complex with porin (Magnani et al., 1982; Redker et al., 1972; Wilson, 1995). This

brain (Eppenberger-Eberhardt et al., 1991; Fridman, Roberts, 1994).

(Brdiczka, 2003; Dolder et al., 2001; Schnyder et al., 1995).

Wolf et al., 1996; Kaldis, Wallimann, 1994; Schnyder et al., 1988).

variable cellular localization (Mulichak et al., 1998; Xie & Wilson, 1990).

**1. Introduction** 

Wyss, 2000).

al., al., 1996; Wang et al., 2005).

**Complex in the Migration of Adenine** 

Elena Erlykina and Tatiana Sergeeva *Nizhny Novgorod State Medical Academy* 

*Russian Federation* 


### **Role of Creatine Kinase – Hexokinase Complex in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction**

Elena Erlykina and Tatiana Sergeeva *Nizhny Novgorod State Medical Academy Russian Federation* 

### **1. Introduction**

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Creatine phosphokinase (CK) (ATP: creatine phosphotransferase, ЕС 2.7.3.2.) is found in a variety of cells with high and fluctuating energy requirements. It catalyses the reversible transfer of the high-energy-N-phosphoryl group from phosphocreatine to ADP. Creatine kinase connects sites of energy production with sites of energy consumption (Dolder et. al., 2001; Focant et al., 1970; Grossmann et al., 1985; Lipskaya et al., 1989; Walzel et al., 2002; Wyss, 2000).

There are know to be three cytosolic and two mitochondrial isoforms of CK. The more basic mitochondrial creatine kinase MiCKb is accumulated in mitochondria of cardiac muscle and skeletal muscle. The more acidic mitochondrial creatine kinase MiCKa was found in the brain (Eppenberger-Eberhardt et al., 1991; Fridman, Roberts, 1994).

Creatine kinase can exist in two interconvertible forms: dimer and octamer (Eriksson et al., 1998; Shen et al., 2002). Creatine kinase binds to the outer leaflet of the entire inner mitochondrial membrane and is specifically enriched in the so-called contact sites where inner and outer membranes are in close proximity (Boero et al., 2003; Chen et al, 1994; Lin et al., al., 1996; Wang et al., 2005).

A change in the octamer/dimer ratio may influence on the association behavior of mitochondrial creatine kinase in general and thus modulate mitochondrial energy flux (Brdiczka, 2003; Dolder et al., 2001; Schnyder et al., 1995).

Mitochondrial creatine kinase forms the functional microcompartment together with the mitochondrial porin (voltage-dependent anion channel) in the outer membrane and as well as the transmembrane protein adenine nucleotide translocase in the inner membrane (Fritz-Wolf et al., 1996; Kaldis, Wallimann, 1994; Schnyder et al., 1988).

Hexokinase (HK) (ATP:D-hexokinase-6-phosphotransferase, EC 2.7.11) is the enzyme with variable cellular localization (Mulichak et al., 1998; Xie & Wilson, 1990).

The type I isoenzyme of mammalian hexokinase is ubiquitously expressed in mammalian tissues but is found particularly at high levels in the brain where it plays an important role in regulating the rate of cerebral glucose metabolism (Schwab & Wilson, 1989; Wilson, 1985).

The major portion of the hexokinase activity in the brain is associated with mitochondria. About 85% of hexokinase is bound to the outer mitochondrial membrane, forming the specific complex with porin (Magnani et al., 1982; Redker et al., 1972; Wilson, 1995). This

Role of Creatine Kinase – Hexokinase Complex

*Preparation of brain tissue* 

*Enzyme assay* 

protein.

animals into two groups; they were included into one group.

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 195

The animals were divided into two groups due to their physiological state after acute ischemia (severe and moderate). The severity of ischemia was estimated according to the behavior of rats, the respiratory rate and survival one. In acute severe ischemia the rats after ligation of the common carotid arteries were in severe state: they were passive, in lateral recumbent position, with agonal breathing (20-30 times per minute with respiratory arrest). In acute moderate ischemia the state of animals was satisfactory: they were active, moved in a cage, the respiratory rate was 50-70 times per minute. Due to the increase in the severity of general physiological state of rats in case of long-term ischemia we could not divide the

The mitochondrial fraction of the brain was isolated by differential centrifugation (Fonyo, Somogy, 1960; Dizhe et al., 2003). The brain tissue was homogenized at 4 0C in a medium containing 0.32 М sucrose, 10 мМ tris-НСl , 1 мМ EDTA, рН 7,4. The total tissue homogenate was centrifuge at 2000 g for 10 minutes. The resulting supernatant was collected and centrifuged further at 12 500g for 15 minutes. The pellet containing mitochondria was resuspended in 0.32 M sucrose and centrifuged at 16 500g for 15 minutes.

The mitochondria were then swollen by incubation in distilled water (at a protein concentration of 1 mg) for 30 minutes, followed by centrifugation at 20 000g for 30 minutes. The resulting supernatant was collected for further analysis. The pellet containing mitochondrial membranes was resuspended in 0.32 M sucrose with 0.25 M dithiothreitol, pH 7.4.

Creatine kinase activity was measured by the pH-stat method using ADP and creatine phosphate as substrates (Kuby, Noltman, 1962). The velocity of the creatine kinase reaction is estimated by the change in pH. The reaction mixture (3 ml) contained (final concentration): 0.25 M sucrose, 2.5 mM tris-НС1, 12 mM MgCl2, 10 mM КС1, 0.25 mM dithiothreitol, 5 mM creatine phosphate, 2 mM ADP. The reaction was started by addition of

Creatine kinase activity is expressed as 1 unit corresponds to 1 μg-equ H+/ min per 1 mg of

Hexokinase activity was measured spectrophotometrically (Felgner, Wilson 1976). The reaction mixture (3 ml) contained (final concentration): 50 mM tris-НС1, рH 8.0, 2 mM glucose, 2 mM ATP, 5 mM MgCl2, 0,25 mM NADP, 0.4 IU/min glucose-6-phosphate

Hexokinase activity is expressed as follows: 1 unit corresponds to 1 nmol of NADP

Mitochondria were resuspended in the proper (0.1M KCl; K-Na phosphate buffer 0.1-1.75 M, 0.5% (v/v) Triton X-100; 0.1% deoxycholate Na) solubilizing solution and incubated for 30 minutes. The samples were centrifuged at 40C and 20 000g, 60 minutes. Percentage of solubilization was determined as the difference of the activity before and after solubilization

Mitochondria were resuspended at a protein concentration of 0.5-1 mg/ml in 0.1M tris-HCl pH 6.6, 0.1 M KCl or 0.5% (v/v) Triton X-100. After incubation for 30 minutes on ice, with

The fraction enriched mitochondria was collected and washed by 0.32 M sucrose.

100 μg protein. Then the mixture was titrated by addition of 10 μl 0.1 N HCl.

dehydrogenase. The reaction was started by addition of 100 μg protein.

transformed/min per 1 mg of protein.

*Solubilization of creatine kinase* 

*Solubilization of hexokinase* 

of the enzyme.

physical proximity provides the basis for functional interaction between glucose phosphorylation by hexokinase and mitochondrial ATP production by oxidative phosphorylation with resulting coordination of the glycolytic and oxidative phases of glucose metabolism (Aleshin, 1998; Rosano et al., 1999). The outer mitochondrial membrane protein – porin, which forms the transmembrane channel, is responsible for specific interaction with hexokinase (Aflalo & Azoulay, 1998; Linden et al., 1982; Schlattner et al., 2001; Vyssokikh & Brdiczka, 2003).

The preferential mitochondrial localization of hexokinase in rat brain provides a predominant access to ATP, generated in mitochondria. The ADP produced by hexokinase activity is known to control both membrane potential and reactive oxygen species generation (Rose & Warms, 1967; Smith and Wilson, 1991; Viitanen et al., 1984; Wilson, 1980).

Thus both enzymes – creatine kinase and hexokinase – play an important role in dynamic compartmentation of adenine nucleotides.

Mitochondrial creatine kinase is a key enzyme of oxidative cellular energy metabolism in the brain (Bessman, 1981; Guo et al., 2003; Hemmer et al., 1994; Levin et al., 1990; Takagi et al., 2001; Wallimann et. al., 1992; Wallimnann et al., 1998). Hexokinase is an enzyme involved in the first step of glycolysis. Mitochondrial creatine kinase – hexokinase complex takes part in transport of adenine nucleotides from mitochondria to cytoplasm. Functioning of this complex depends on interaction of enzymes with the mitochondrial membrane and the oligomeric state of mitochondrial creatine kinase.

Mitochondrial dysfunction is one of the main reasons of the pathological changes in cerebral ischemia (Clostre, 2001; Delivoria-Papadopoulos et al., 2007; Fiscum, 2000; Kuznetsov, Margreiter, 2009; Mattson, Liu, 2002; Sas et al., 2007; Siesjo, 1999).

Stroke is a leading cause of disability and death in many countries. Understanding the molecular mechanisms of ischemic injury helps to find the novel therapeutic strategies for stroke. 80% of human strokes are ischemic in origin (Levine et al., 1992; Sappey-Marinier et al., 2002; Ueda et al., 2000).

Thus experimental models of cerebral ischemia have been developed in an attempt to closely mimic the changes that occur during and after human ischemic stroke. Changes in the amount and activity of enzyme proteins are critical factors in regulating intracellular metabolism under ischemic conditions (Cherubini et al., 2000; Dos Santos et al., 2004; Maulik et al., 1999; Rauchova et al., 2002).

According to modern data, membrane-associated enzyme in contrast to soluble enzyme has other catalytic properties (Beutner et al., 1998; da-Silva et al., 2004; Dolder et al., 2001; Kellershohn & Ricard, 1994; Linden et al., 1982; Lyubarev, 1997; Ovadi & Srere, 2000). The reverse adsorption on the mitochondrial membrane is controlled by ions and metabolites thus broadening the regulatory possibility of the cells under hypoxic conditions.

### **2. Materials and methods**

#### *Animals*

Experiments were performed on male outbred albino rats weighing 150-180 g.

Cerebral ischemia was produced by bilateral ligation of the common carotid arteries. The animals were anesthetized with nembutal (30 mg/kg intraperitoneally). The brain tissue was examined 30 minutes (acute ischemia), 1.5, 4, 18 hours after surgical impairment of cerebral hemodynamics.

The animals were divided into two groups due to their physiological state after acute ischemia (severe and moderate). The severity of ischemia was estimated according to the behavior of rats, the respiratory rate and survival one. In acute severe ischemia the rats after ligation of the common carotid arteries were in severe state: they were passive, in lateral recumbent position, with agonal breathing (20-30 times per minute with respiratory arrest). In acute moderate ischemia the state of animals was satisfactory: they were active, moved in a cage, the respiratory rate was 50-70 times per minute. Due to the increase in the severity of general physiological state of rats in case of long-term ischemia we could not divide the animals into two groups; they were included into one group.

### *Preparation of brain tissue*

194 Advances in the Preclinical Study of Ischemic Stroke

physical proximity provides the basis for functional interaction between glucose phosphorylation by hexokinase and mitochondrial ATP production by oxidative phosphorylation with resulting coordination of the glycolytic and oxidative phases of glucose metabolism (Aleshin, 1998; Rosano et al., 1999). The outer mitochondrial membrane protein – porin, which forms the transmembrane channel, is responsible for specific interaction with hexokinase (Aflalo & Azoulay, 1998; Linden et al., 1982; Schlattner et al.,

The preferential mitochondrial localization of hexokinase in rat brain provides a predominant access to ATP, generated in mitochondria. The ADP produced by hexokinase activity is known to control both membrane potential and reactive oxygen species generation (Rose & Warms,

Thus both enzymes – creatine kinase and hexokinase – play an important role in dynamic

Mitochondrial creatine kinase is a key enzyme of oxidative cellular energy metabolism in the brain (Bessman, 1981; Guo et al., 2003; Hemmer et al., 1994; Levin et al., 1990; Takagi et al., 2001; Wallimann et. al., 1992; Wallimnann et al., 1998). Hexokinase is an enzyme involved in the first step of glycolysis. Mitochondrial creatine kinase – hexokinase complex takes part in transport of adenine nucleotides from mitochondria to cytoplasm. Functioning of this complex depends on interaction of enzymes with the mitochondrial membrane and

Mitochondrial dysfunction is one of the main reasons of the pathological changes in cerebral ischemia (Clostre, 2001; Delivoria-Papadopoulos et al., 2007; Fiscum, 2000; Kuznetsov,

Stroke is a leading cause of disability and death in many countries. Understanding the molecular mechanisms of ischemic injury helps to find the novel therapeutic strategies for stroke. 80% of human strokes are ischemic in origin (Levine et al., 1992; Sappey-Marinier et

Thus experimental models of cerebral ischemia have been developed in an attempt to closely mimic the changes that occur during and after human ischemic stroke. Changes in the amount and activity of enzyme proteins are critical factors in regulating intracellular metabolism under ischemic conditions (Cherubini et al., 2000; Dos Santos et al., 2004; Maulik

According to modern data, membrane-associated enzyme in contrast to soluble enzyme has other catalytic properties (Beutner et al., 1998; da-Silva et al., 2004; Dolder et al., 2001; Kellershohn & Ricard, 1994; Linden et al., 1982; Lyubarev, 1997; Ovadi & Srere, 2000). The reverse adsorption on the mitochondrial membrane is controlled by ions and metabolites

Cerebral ischemia was produced by bilateral ligation of the common carotid arteries. The animals were anesthetized with nembutal (30 mg/kg intraperitoneally). The brain tissue was examined 30 minutes (acute ischemia), 1.5, 4, 18 hours after surgical impairment of

thus broadening the regulatory possibility of the cells under hypoxic conditions.

Experiments were performed on male outbred albino rats weighing 150-180 g.

1967; Smith and Wilson, 1991; Viitanen et al., 1984; Wilson, 1980).

Margreiter, 2009; Mattson, Liu, 2002; Sas et al., 2007; Siesjo, 1999).

2001; Vyssokikh & Brdiczka, 2003).

al., 2002; Ueda et al., 2000).

et al., 1999; Rauchova et al., 2002).

**2. Materials and methods** 

cerebral hemodynamics.

*Animals* 

compartmentation of adenine nucleotides.

the oligomeric state of mitochondrial creatine kinase.

The mitochondrial fraction of the brain was isolated by differential centrifugation (Fonyo, Somogy, 1960; Dizhe et al., 2003). The brain tissue was homogenized at 4 0C in a medium containing 0.32 М sucrose, 10 мМ tris-НСl , 1 мМ EDTA, рН 7,4. The total tissue homogenate was centrifuge at 2000 g for 10 minutes. The resulting supernatant was collected and centrifuged further at 12 500g for 15 minutes. The pellet containing mitochondria was resuspended in 0.32 M sucrose and centrifuged at 16 500g for 15 minutes. The fraction enriched mitochondria was collected and washed by 0.32 M sucrose.

The mitochondria were then swollen by incubation in distilled water (at a protein concentration of 1 mg) for 30 minutes, followed by centrifugation at 20 000g for 30 minutes. The resulting supernatant was collected for further analysis. The pellet containing mitochondrial membranes was resuspended in 0.32 M sucrose with 0.25 M dithiothreitol, pH 7.4.

### *Enzyme assay*

Creatine kinase activity was measured by the pH-stat method using ADP and creatine phosphate as substrates (Kuby, Noltman, 1962). The velocity of the creatine kinase reaction is estimated by the change in pH. The reaction mixture (3 ml) contained (final concentration): 0.25 M sucrose, 2.5 mM tris-НС1, 12 mM MgCl2, 10 mM КС1, 0.25 mM dithiothreitol, 5 mM creatine phosphate, 2 mM ADP. The reaction was started by addition of 100 μg protein. Then the mixture was titrated by addition of 10 μl 0.1 N HCl.

Creatine kinase activity is expressed as 1 unit corresponds to 1 μg-equ H+/ min per 1 mg of protein.

Hexokinase activity was measured spectrophotometrically (Felgner, Wilson 1976). The reaction mixture (3 ml) contained (final concentration): 50 mM tris-НС1, рH 8.0, 2 mM glucose, 2 mM ATP, 5 mM MgCl2, 0,25 mM NADP, 0.4 IU/min glucose-6-phosphate dehydrogenase. The reaction was started by addition of 100 μg protein.

Hexokinase activity is expressed as follows: 1 unit corresponds to 1 nmol of NADP transformed/min per 1 mg of protein.

### *Solubilization of creatine kinase*

Mitochondria were resuspended in the proper (0.1M KCl; K-Na phosphate buffer 0.1-1.75 M, 0.5% (v/v) Triton X-100; 0.1% deoxycholate Na) solubilizing solution and incubated for 30 minutes. The samples were centrifuged at 40C and 20 000g, 60 minutes. Percentage of solubilization was determined as the difference of the activity before and after solubilization of the enzyme.

### *Solubilization of hexokinase*

Mitochondria were resuspended at a protein concentration of 0.5-1 mg/ml in 0.1M tris-HCl pH 6.6, 0.1 M KCl or 0.5% (v/v) Triton X-100. After incubation for 30 minutes on ice, with

Role of Creatine Kinase – Hexokinase Complex

**3. Results** 

mitochondrial membrane (Table 1).

**Form of the enzyme Creatine kinase,** 

Soluble 1.78±0.10

Membrane-associated 1.40±0.07

shown to depend on the interaction with the membrane.

associated and soluble forms of the enzymes

mitochondrial membrane.

brain.

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 197

Fig. 1. The kinetic curve of the development of an induced chemiluminescence signal.

Two forms of creatine kinase and hexokinase – membrane and soluble – were found in the

The activity of membrane-associated creatine kinase equals to half of the enzyme activity in mitochondria. In contrast hexokinase activity is concentrated on the outer surface of the

**U/ mg\*min.**

n=23

n=19

Catalytic and kinetic properties of mitochondrial creatine kinase and hexokinase were

Different solubilizing agents (electrolyte, detergent and the endogenous metabolite glucose-6-phosphate) were used to analyze the character of interaction of hexokinase with the

All these agents solubilized only a third of the hexokinase activity, and only the sequence of action of electrolyte, detergent and glucose-6-phosphate removed the enzyme from the mitochondrial membrane (Fig. 2). Thus it shows the lability of protein-protein interaction

and the possibility of its regulation under the certain pathological conditions.

Table 1. The distribution of creatine kinase and hexokinase activity between membrane-

**Hexokinase, U/ mg\*min.**

11.56±0.19 n=12

1.53±0.04 n=12

occasional mixing, the samples were centrifuged at 40C and 20 000g for 30 minutes. Percentage of solubilization was determined as the difference of the activity before and after solubilization of the enzyme.

According to Wilson (2003) there are 2 types of binding sites for hexokinase on brain mitochondria. Hexokinase is readily solubilized from Type A sites by glucose-6-phosphate while hexokinase bound to Type B sites remains bound even in the presence of glucose-6 phosphate.

Mitochondria were resuspended in 2 mM glucose-6-phosphate; tris-HCl buffer, pH 8 and incubated for 30 minutes at the room temperature, and centrifuged 100 000g for 15 minutes. Aliquots of supernatant contain hexokinase Type A.

The sediment of mitochondria which contained hexokinase type B was resuspended again in 0,32 M sucrose, 0.5% (v/v) Triton X-100, 0.1M tris-HCl, pH 8. After incubation for 5 minutes on ice the samples were centrifuged at 20 000g for 10 minutes. The sediment was resuspended again in 0.32M sucrose, 0.1M KCl, 1% (v/v) Triton X-100, 0.1M tris-HCl, pH 8. After incubation for 20 minutes on ice the samples were centrifuged at 20 000g for 10 minutes. The aliquots of supernatant contained hexokinase Type B.

### *Dissociation of creatine kinase*

Mitochondrial creatine kinase was dissociated by incubation of the total mitochondrial fraction and mitochondrial membrane pellet with substrates for the transition-state analogue complex (MgCl2, ADP, KNO3, and creatine) at 4 0C for 2 hours (Lipskaya et al., 1989).

### *The free radical oxidation intensity assay*

The intensity of the free radical oxidation (FRO) was estimated by the method of H2O2, Fe2+\_-induced chemiluminescence on a BChL-07 biochemiluminometer. This method is based on the catalytic decomposition of hydrogen peroxide by ions of metal with variable valency (bivalent iron) (the Phenton reaction). The reaction mixture contained: 0,05 mM Fe2SO4, a phosphate buffer and a mitochondrial fraction. The reaction was started by addition of 2 % solution of hydrogen peroxide. Proceeding process of free radical oxidation was registered within 30 seconds. It is the time of the greatest information about its intensity. The ideal curve of the process is presented in figure 1.

The following parameters are the most informative for the estimation of the chemiluminescence intensity: the total luminescence yield (S, enables to estimate a balance between lipid peroxidation and antioxidants), maximum flash amplitude (Imax, shows a potential ability of the biological sample to free radical oxidation), and K index characterizing antioxidant potential were used as integral parameters of chemiluminescence (Kuzmina et al., 2009).

#### *The protein concentration assay*

Protein concentration was measured by the method of Bredford (Bredford & Spector, 1978).

### *Statistical analysis*

The data are expressed as mean and standard error of the mean (SEM). The results were analyzed by means of Primer of Biostatistics 4.03 (Glantz, 2005). The significance of differences between the samples was evaluated by Student's test. The level of significance was set at *p*<0.05.

Fig. 1. The kinetic curve of the development of an induced chemiluminescence signal.

### **3. Results**

196 Advances in the Preclinical Study of Ischemic Stroke

occasional mixing, the samples were centrifuged at 40C and 20 000g for 30 minutes. Percentage of solubilization was determined as the difference of the activity before and after

According to Wilson (2003) there are 2 types of binding sites for hexokinase on brain mitochondria. Hexokinase is readily solubilized from Type A sites by glucose-6-phosphate while hexokinase bound to Type B sites remains bound even in the presence of glucose-6-

Mitochondria were resuspended in 2 mM glucose-6-phosphate; tris-HCl buffer, pH 8 and incubated for 30 minutes at the room temperature, and centrifuged 100 000g for 15 minutes.

The sediment of mitochondria which contained hexokinase type B was resuspended again in 0,32 M sucrose, 0.5% (v/v) Triton X-100, 0.1M tris-HCl, pH 8. After incubation for 5 minutes on ice the samples were centrifuged at 20 000g for 10 minutes. The sediment was resuspended again in 0.32M sucrose, 0.1M KCl, 1% (v/v) Triton X-100, 0.1M tris-HCl, pH 8. After incubation for 20 minutes on ice the samples were centrifuged at 20 000g for 10

Mitochondrial creatine kinase was dissociated by incubation of the total mitochondrial fraction and mitochondrial membrane pellet with substrates for the transition-state analogue complex (MgCl2, ADP, KNO3, and creatine) at 4 0C for 2 hours (Lipskaya et al.,

The intensity of the free radical oxidation (FRO) was estimated by the method of H2O2, Fe2+\_-induced chemiluminescence on a BChL-07 biochemiluminometer. This method is based on the catalytic decomposition of hydrogen peroxide by ions of metal with variable valency (bivalent iron) (the Phenton reaction). The reaction mixture contained: 0,05 mM Fe2SO4, a phosphate buffer and a mitochondrial fraction. The reaction was started by addition of 2 % solution of hydrogen peroxide. Proceeding process of free radical oxidation was registered within 30 seconds. It is the time of the greatest information about its

The following parameters are the most informative for the estimation of the chemiluminescence intensity: the total luminescence yield (S, enables to estimate a balance between lipid peroxidation and antioxidants), maximum flash amplitude (Imax, shows a potential ability of the biological sample to free radical oxidation), and K index characterizing antioxidant potential were used as integral parameters of chemiluminescence

Protein concentration was measured by the method of Bredford (Bredford & Spector, 1978).

The data are expressed as mean and standard error of the mean (SEM). The results were analyzed by means of Primer of Biostatistics 4.03 (Glantz, 2005). The significance of differences between the samples was evaluated by Student's test. The level of significance

solubilization of the enzyme.

*Dissociation of creatine kinase* 

(Kuzmina et al., 2009).

*Statistical analysis* 

was set at *p*<0.05.

*The protein concentration assay* 

*The free radical oxidation intensity assay* 

Aliquots of supernatant contain hexokinase Type A.

minutes. The aliquots of supernatant contained hexokinase Type B.

intensity. The ideal curve of the process is presented in figure 1.

phosphate.

1989).

Two forms of creatine kinase and hexokinase – membrane and soluble – were found in the brain.

The activity of membrane-associated creatine kinase equals to half of the enzyme activity in mitochondria. In contrast hexokinase activity is concentrated on the outer surface of the mitochondrial membrane (Table 1).


Table 1. The distribution of creatine kinase and hexokinase activity between membraneassociated and soluble forms of the enzymes

Catalytic and kinetic properties of mitochondrial creatine kinase and hexokinase were shown to depend on the interaction with the membrane.

Different solubilizing agents (electrolyte, detergent and the endogenous metabolite glucose-6-phosphate) were used to analyze the character of interaction of hexokinase with the mitochondrial membrane.

All these agents solubilized only a third of the hexokinase activity, and only the sequence of action of electrolyte, detergent and glucose-6-phosphate removed the enzyme from the mitochondrial membrane (Fig. 2). Thus it shows the lability of protein-protein interaction and the possibility of its regulation under the certain pathological conditions.

Role of Creatine Kinase – Hexokinase Complex

the kinetic behavior is not changed (Fig. 4).

intact rats.

0

40

80

120

**V, U/mg\*min.**

160

200

0 0.5 1 1.5 2 2.5 3 3.5

**V, U/mg\*min.**

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 199

Fig. 4. Dependence of V0 on concentration of MgATP of membrane-associated hexokinase of

0 0.5 1 1.5 2 2.5 3 3.5

Creatine kinase has different types of kinetic behavior (Fig. 5-7). We consider that the membrane associated form of the enzyme binds by ionic interaction with the membrane and the character of the curve reveals the classical kinetic behavior (Fig. 6). The tightly bound

012345

Fig. 5. Kinetic of creatine kinase reaction in total mitochondrial fraction of intact rats.

Phosphocreatine, mM

**MgATP, mM**

Rebinding of both phosphokinases with the membrane changes their catalytic properties. Binding of hexokinase with the membrane increases the velocity of the reaction in 3 fold, but

Fig. 2. Solubilization (%) of hexokinase from mitochondrial membranes of intact rats.

Creatine kinase can not dissociate from the mitochondrial membrane even in the presence of the simultaneous action of electrolyte and detergent (Fig. 3).

So the brain creatine kinase exists in different molecular forms: the first – soluble, which is located in the intermembrane space, the second is associated, which is loosely bound to the inner mitochondrial membrane and under the certain solubilizing agents can remove into the intermembrane space, the third form (about 18%) is tightly bound with the membrane. In mitochondria from intact animals, mitochondrial creatine kinase presents as a mixture of two oligomeric forms (dimer and octamer; 65 and 35%, respectively). We consider that the tightly bound creatine kinase to exist mainly in the contact sites in the octamer form.

Fig. 3. Solubilization (%) of creatine kinase from mitochondrial membranes of intact rats.

100

Glucose-6 phosphate, KCl, Triton X-100

26

Deoxycholate

40

KCl Triton X-100 Glucose-6-

Fig. 2. Solubilization (%) of hexokinase from mitochondrial membranes of intact rats.

tightly bound creatine kinase to exist mainly in the contact sites in the octamer form.

43

Phosphate buffer Triton X-100 Phosphate buffer

Fig. 3. Solubilization (%) of creatine kinase from mitochondrial membranes of intact rats.

Creatine kinase can not dissociate from the mitochondrial membrane even in the presence of

So the brain creatine kinase exists in different molecular forms: the first – soluble, which is located in the intermembrane space, the second is associated, which is loosely bound to the inner mitochondrial membrane and under the certain solubilizing agents can remove into the intermembrane space, the third form (about 18%) is tightly bound with the membrane. In mitochondria from intact animals, mitochondrial creatine kinase presents as a mixture of two oligomeric forms (dimer and octamer; 65 and 35%, respectively). We consider that the

35

phosphate

64

+ Triton X-100

26

the simultaneous action of electrolyte and detergent (Fig. 3).

35

0

10

20

30

40

**%**

50

60

70

0

20

40

60

**%**

80

100

120

Rebinding of both phosphokinases with the membrane changes their catalytic properties. Binding of hexokinase with the membrane increases the velocity of the reaction in 3 fold, but the kinetic behavior is not changed (Fig. 4).

Fig. 4. Dependence of V0 on concentration of MgATP of membrane-associated hexokinase of intact rats.

Creatine kinase has different types of kinetic behavior (Fig. 5-7). We consider that the membrane associated form of the enzyme binds by ionic interaction with the membrane and the character of the curve reveals the classical kinetic behavior (Fig. 6). The tightly bound

Fig. 5. Kinetic of creatine kinase reaction in total mitochondrial fraction of intact rats.

Role of Creatine Kinase – Hexokinase Complex

**\*\***

0

0.5

1

1.5

**U/mg\*min.**

2

2.5

3

**U/mg\*min.**

membrane-associated enzymes are changed in acute ischemia.

hexokinase activity in cerebral ischemia (Ishibashi, 1999; Wilson et al., 2000).

**\***

intact animals severe ischemia,

solubilization by 0.1M KCl of intact rats and in 30 minutes ischemia.

**\***

intact animals severe ischemia,

before and after solubilization by phosphate buffer.

**\***

30 minutes

Fig. 9. Creatine kinase activity on the membrane of intact rats and in 30 minutes ischemia

**\*/\*\***

30 minutes

Fig. 8. Membrane associated hexokinase activity on the membrane before and after

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 201

Wang et al., 2005). As a result of this activation the properties of the mitochondrial

Severe ischemia reduced the binding of the investigated enzymes with the membrane (Fig. 8, 9). The activity of the enzymes decreased 2 fold for creatine kinase and 3 fold for hexokinase. Glucose-6-phosphate and the products of membrane degradation inhibited

**\***

**\*\***

Hexokinase activity on the membrane

Hexokinase activity on the membrane after

solubilization by 0.1M KCl

Creatine kinase activity on the membrane

Creatine kinase activity on the membrane after solubilization by phosphate buffer

moderate ischemia, 30 minutes

**\***

**\*\***

moderate ischemia, 30 minutes

form of membrane enzyme has the abnormal kinetic behavior due to ionic and hydrophobic interaction (Fig. 7). These data discribe the role of specific microenvironment in the modification of the enzyme properties.

Fig. 6. Kinetic of creatine kinase reaction on mitochondrial membrane of intact rats.

Fig. 7. Kinetic of creatine kinase reaction on mitochondrial membrane after solubilization by phosphate buffer of intact rats.

Thus the catalytic properties depend on the binding with the membrane and this process is controlled by the endogenous metabolites and the functional state of mitochondria. All forms of hypoxia and ischemia are accompanied by activation of free radical oxidation

(Ayer, Zhang, 2008; da-Silva et al., 2004; Kuznetsov, Margreiter, 2009; Meyer et al., 2006;

form of membrane enzyme has the abnormal kinetic behavior due to ionic and hydrophobic interaction (Fig. 7). These data discribe the role of specific microenvironment in the

012345

012345

Fig. 7. Kinetic of creatine kinase reaction on mitochondrial membrane after solubilization by

Thus the catalytic properties depend on the binding with the membrane and this process is

All forms of hypoxia and ischemia are accompanied by activation of free radical oxidation (Ayer, Zhang, 2008; da-Silva et al., 2004; Kuznetsov, Margreiter, 2009; Meyer et al., 2006;

controlled by the endogenous metabolites and the functional state of mitochondria.

Fig. 6. Kinetic of creatine kinase reaction on mitochondrial membrane of intact rats.

Phosphocreatine, mM

Phosphocreatine, mM

modification of the enzyme properties.

0 0.5 1 1.5 2 2.5 3 3.5

0

phosphate buffer of intact rats.

0.5 1

1.5

**V, U/mg\*min.**

2.5 3

3.5

2

**V, U/mg\*min.**

Wang et al., 2005). As a result of this activation the properties of the mitochondrial membrane-associated enzymes are changed in acute ischemia.

Severe ischemia reduced the binding of the investigated enzymes with the membrane (Fig. 8, 9). The activity of the enzymes decreased 2 fold for creatine kinase and 3 fold for hexokinase. Glucose-6-phosphate and the products of membrane degradation inhibited hexokinase activity in cerebral ischemia (Ishibashi, 1999; Wilson et al., 2000).

Fig. 8. Membrane associated hexokinase activity on the membrane before and after solubilization by 0.1M KCl of intact rats and in 30 minutes ischemia.

Fig. 9. Creatine kinase activity on the membrane of intact rats and in 30 minutes ischemia before and after solubilization by phosphate buffer.

Role of Creatine Kinase – Hexokinase Complex

membranes in moderate ischemia

0

fraction in severe ischemia.

1

2

3

0.5

1.5

**V, U/mg\*min.**

2.5

3.5

0

0.5

1

1.5

**V, U/mg\*min.**

2

2.5

3

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 203

Fig. 11. Dependence of V0 on concentration of phosphocreatine in fraction of mitochondrial

012345

0 0.5 1 1.5 2 2.5

Fig. 12. Dependence of V0 on concentration of phosphocreatine in total mitochondrial

Phosphocreatine, mM

Phosphocreatine, mM

The bar diagrams display average activities with error bars representing the standard deviation. \**p*<0.05 versus intact animals. \*\* *p*<0.05 versus initial hexokinase activity on the membrane within the same group.

The bar diagrams display average activities with error bars representing the standard deviation. *p*<0.05 versus intact animals. \*\* *p*<0.05 versus initial creatine kinase activity on the membrane within the same group.

In the second group (moderate ischemia) the activity of hexokinase was increased by 29% and by 92% for creatine kinase in comparison with intact animals. After the solubilization of the hexokinase by 0.1 M KCl the enzyme lost 38% of the initial activity. The effect of solubilization for membrane- bound creatine kinase was 69% instead of 35% for the intact rats.

The study of behavior of creatine kinase revealed the modification of its properties in acute ischemia. They differed significantly from those of the intact rats. It is connected with the realization of interconvertible transformation of oligomeric subunits of creatine kinase. This changing in kinetic behavior provides the higher sensitivity of the enzyme to the changes in substrate concentration (Fig. 10-13).

Fig. 10. Dependence of V0 on concentration of phosphocreatine in total mitochondrial fraction in moderate ischemia

The bar diagrams display average activities with error bars representing the standard deviation. \**p*<0.05 versus intact animals. \*\* *p*<0.05 versus initial hexokinase activity on the

The bar diagrams display average activities with error bars representing the standard deviation. *p*<0.05 versus intact animals. \*\* *p*<0.05 versus initial creatine kinase activity on the

In the second group (moderate ischemia) the activity of hexokinase was increased by 29% and by 92% for creatine kinase in comparison with intact animals. After the solubilization of the hexokinase by 0.1 M KCl the enzyme lost 38% of the initial activity. The effect of solubilization for membrane- bound creatine kinase was 69% instead of 35% for the intact

The study of behavior of creatine kinase revealed the modification of its properties in acute ischemia. They differed significantly from those of the intact rats. It is connected with the realization of interconvertible transformation of oligomeric subunits of creatine kinase. This changing in kinetic behavior provides the higher sensitivity of the enzyme to the changes in

0 0.5 1 1.5 2 2.5

Fig. 10. Dependence of V0 on concentration of phosphocreatine in total mitochondrial

Phosphocreatine, mM

membrane within the same group.

membrane within the same group.

substrate concentration (Fig. 10-13).

rats.

0

fraction in moderate ischemia

1

2

**V, U/mg\*min.**

3

4

Fig. 11. Dependence of V0 on concentration of phosphocreatine in fraction of mitochondrial membranes in moderate ischemia

Fig. 12. Dependence of V0 on concentration of phosphocreatine in total mitochondrial fraction in severe ischemia.

Role of Creatine Kinase – Hexokinase Complex

KCl in cerebral ischemia

Ischemia, 30 minutes

Ischemia, 1.5 hours

Ischemia, 4 hours

Ischemia, 18 hours

\**p*<0.05 versus intact animals

brain.

ischemia.

**U/mg\*min.**

**\***

intact animals severe ischemia,

estimated in various periods of ischemia (Table 2).

Intact animals 1.02±0.03

**\***

Hexokinase activity on the membrane

30 minutes

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 205

**\*/\*\***

 ischemia, 1.5 hours

Hexokinase activity on the membrane after solubilization by 0,1M KCl

Fig. 14. The activity of membrane-bound hexokinase before and after solubilization by 0.1М

The cerebral ischemia causes the imbalance between reactive oxygen species production and the level of antioxidant defense, which leads to oxidative stress. Neuronal membranes contain a considerable amount of unsaturated lipids. The low level of activity of antioxidant enzymes and formation of free radicals in neurochemical reactions provide conditions for lipid oxidation and induce enzyme modification. To evaluate the state of membranes, the intensity of free radical oxidation and antioxidant properties of the brain tissue were

**Experimental groups Imax, mV S, imp.\*30 sec. K=1/S** 

Table 2. The intensity of free radical oxidation and the activity of antioxidant system in the

Various characteristics of chemiluminescence (maximum flash amplitude and total yield of slow flash) in the mitochondrial fraction were elevated during various periods of cerebral ischemia. These changes reflect activation of free radical processes in the brain. Total yield of slow flash was 1.6-fold and 1.4-fold higher than in the intact animals in 30 min and 1.5 hours

n=8

1.89±0.07\* n=7

1.52±0.04\* n=7

1.47±0.05\* n=8

1.08±0.11 n=8

**\*/\*\***

**\*/\*\***

 ischemia, 4 hours

10.64±0.34

17.22±0.98\*

15.18±0.91\*

14.55±0.03\*

12.63±0.79\*

n=8 0.094

n=7 0.058

n=7 0.066

n=8 0.068

n=8 0.079

**\*/\*\***

**\*/\*\***

 ischemia, 18 hours

**\*/\*\***

The study of the creatine kinase reaction in the group of animals after severe ischemia showed the abnormal kinetic behavior of the enzyme, the appearance of the intermediate plateau at the low concentration (0.3-0.4 mM) of creatine phosphate, the V0 decreased 1.4-2 fold in comparison with intact rats.

Fig. 13. Dependence of V0 on concentration of phosphocreatine in fraction of mitochondrial membranes in severe ischemia.

The reversibility of these alterations has been shown during the increasing of duration of ischemia.

Increasing of the duration of cerebral ischemia to 4 and 18 hours was accompanied by changes in activity distribution for hexokinase. The activity of hexokinase progressively increased. The level of activity of the enzyme under these conditions was higher than in acute ischemia. However, the level of hexokinase activity in animals during long-term ischemia remained lower than in intact specimens (Fig. 14).

The bar diagrams display average activities with error bars representing the standard deviation. \**p*<0.05 versus intact animals. \*\* *p*<0.05 versus severe ischemia (30 minutes).

An increase in duration of cerebral ischemia influenced on the adsorption properties of hexokinase. Solubilization of hexokinase by 0.1 KCl was accompanied by decrease in the activity of the enzyme by 78% in acute cerebral ischemia. The percentage of solubilizing enzyme was 37% in 1.5 hours ischemia. It was by 11% higher than in intact specimens. The percentage of solubilizing enzyme was 30% and 27% in 4 hours and 18 hours ischemia, respectively. It did not differ from that in intact animals.

Therefore increase in the duration of cerebral ischemia was followed by an increase in the resistance of membrane structures. These changes were manifested in reduction of hexokinase desorption from the mitochondrial membrane.

The study of the creatine kinase reaction in the group of animals after severe ischemia showed the abnormal kinetic behavior of the enzyme, the appearance of the intermediate plateau at the low concentration (0.3-0.4 mM) of creatine phosphate, the V0 decreased 1.4-2

0 0.5 1 1.5 2 2.5

Fig. 13. Dependence of V0 on concentration of phosphocreatine in fraction of mitochondrial

The reversibility of these alterations has been shown during the increasing of duration of

Increasing of the duration of cerebral ischemia to 4 and 18 hours was accompanied by changes in activity distribution for hexokinase. The activity of hexokinase progressively increased. The level of activity of the enzyme under these conditions was higher than in acute ischemia. However, the level of hexokinase activity in animals during long-term

The bar diagrams display average activities with error bars representing the standard deviation. \**p*<0.05 versus intact animals. \*\* *p*<0.05 versus severe ischemia (30 minutes). An increase in duration of cerebral ischemia influenced on the adsorption properties of hexokinase. Solubilization of hexokinase by 0.1 KCl was accompanied by decrease in the activity of the enzyme by 78% in acute cerebral ischemia. The percentage of solubilizing enzyme was 37% in 1.5 hours ischemia. It was by 11% higher than in intact specimens. The percentage of solubilizing enzyme was 30% and 27% in 4 hours and 18 hours ischemia,

Therefore increase in the duration of cerebral ischemia was followed by an increase in the resistance of membrane structures. These changes were manifested in reduction of

Phosphocreatine, mM

fold in comparison with intact rats.

0

membranes in severe ischemia.

ischemia remained lower than in intact specimens (Fig. 14).

respectively. It did not differ from that in intact animals.

hexokinase desorption from the mitochondrial membrane.

0.5

1

1.5

**V, U/mg\*min.**

ischemia.

2

2.5

3

Fig. 14. The activity of membrane-bound hexokinase before and after solubilization by 0.1М KCl in cerebral ischemia

The cerebral ischemia causes the imbalance between reactive oxygen species production and the level of antioxidant defense, which leads to oxidative stress. Neuronal membranes contain a considerable amount of unsaturated lipids. The low level of activity of antioxidant enzymes and formation of free radicals in neurochemical reactions provide conditions for lipid oxidation and induce enzyme modification. To evaluate the state of membranes, the intensity of free radical oxidation and antioxidant properties of the brain tissue were estimated in various periods of ischemia (Table 2).


\**p*<0.05 versus intact animals

Table 2. The intensity of free radical oxidation and the activity of antioxidant system in the brain.

Various characteristics of chemiluminescence (maximum flash amplitude and total yield of slow flash) in the mitochondrial fraction were elevated during various periods of cerebral ischemia. These changes reflect activation of free radical processes in the brain. Total yield of slow flash was 1.6-fold and 1.4-fold higher than in the intact animals in 30 min and 1.5 hours ischemia.

Role of Creatine Kinase – Hexokinase Complex

in the Migration of Adenine Nucleotides in Mitochondrial Dysfunction 207

Mitochondrial creatine kinase is associated with mitochondrial membranes due to the forces of electrostatic and hydrophobic interaction. Cerebral ischemia was followed by changes in

The activity of associated mitochondrial creatine kinase increased in comparison with intact animals in 1.5 hours ischemia. However the activity of tightly-bound creatine kinase did not change. The activity of tightly-bound mitochondrial creatine kinase increased and the ratio between two forms of mitochondrial creatine kinase restored in 4 hours ischemia. By 18

> Ischemia, 1.5 hours

Activity of membrane-associated creatine kinase after solubilization by

Fig. 16. The activity of membrane-associated creatine kinase before and after solubilization

The bar diagrams display average activities with error bars representing the standard deviation. \**p*<0.05 versus intact animals. \*\* *p*<0.05 versus severe ischemia (30 minutes). Cerebral ischemia was shown to change the kinetic properties of mitochondrial creatine kinase (Fig. 17). Mitochondrial creatine kinase showed abnormal kinetic with the appearance of intermediate plateau. By 18 hours, the kinetic curve acquired a hyperbola

In mitochondria, mitochondrial creatine kinase is presented by two oligomeric forms (dimer and octamer). They are characterized by dynamic equilibrium (Lipskaya et al., 1989). The transition-state analogue complex of mitochondrial creatine kinase was induced to evaluate the ratio between oligomeric forms of this enzyme under conditions of cerebral circulatory

Cerebral ischemia changes the dimer/octamer ratio. This ratio is shifted toward the formation of dimers after 30-min ischemia (79%). Phospholipids serve as the structural elements of membranes that are bound to mitochondrial creatine kinase. Membrane binding properties of mitochondrial creatine kinase depend strongly on the protein dimer/octamer ratio and degree of lipid oxidation. Activation of free radical oxidation during acute ischemia is probably followed by partial dissociation of octamers to dimers. Increasing of the duration of ischemia to 18 hours was followed by an increase in the octamer ratio (53%).

Initial activity of membrane-associated creatine kinase

**\*\* \*\***

 Ischemia, 4 hours

**\*\***

 Ischemia, 18 hours

**\*\* \*\***

the activity of associated and tightly-bound mitochondrial creatine kinase (Fig. 16).

hours, the percentage of tightly-bound form of the enzyme reached 90%.

**\***

Intact animals Ischemia,

phosphate buffer

by phosphate buffer in cerebral ischemia.

0

0.5

1

1.5

**U/mg\*min.**

form.

disorders.

2

2.5

**\***

30 minutes

Parameters of free radical oxidation (Imax and S) remained practically unchanged by 4 hours ischemia. The increase in the duration of cerebral ischemia to 18 hours was accompanied by a decrease in the intensity of free radical oxidation. Parameter Imax did not differ from the corresponding parameter in intact animals.

Acute ischemia was not only followed by damage of the cell membrane structures and activation of free radical oxidation, but also induced the antioxidant system (Dziennis et al., 2008; Lai et al., 2003; Perez-Pinzon et al., 2005; Suzuki et al., 1997).

The K index serves as a criterion for the antioxidant potential of the cell. The level of the antioxidant activity of the brain tissue was elevated after ischemia for 1.5 and 4 hours. The conclusion was derived from the decrease in this index. By 18 hours ischemia, the K index did not differ from that in intact animals.

These data indicate that the prooxidant/antioxidant ratio returns to normal with increasing in the duration of cerebral circulatory disorder. The observed changes are probably related to activation of defense protein synthesis, which increases the resistance of membrane structures to the adverse effect of ischemia.

The kinetic curve for hexokinase was shown to have hyperbola form in various periods of ischemia except for 1.5 hours ischemia (Fig. 15). MgATP did not inhibit hexokinase when increasing the duration of ischemia. By 18 hours, the Km (0.13 mM) is 2-fold lower than in intact animals (0.26 mM) and is 5-fold lower than in 30 minutes ischemia (0.7 mM).

Fig. 15. Kinetic of the hexokinase reaction.

The major problem in the involvement of cell structures in the regulation of enzyme activity is the dependence of enzyme properties on the association of this enzyme with the membrane under conditions of functional changes in the organism.

Parameters of free radical oxidation (Imax and S) remained practically unchanged by 4 hours ischemia. The increase in the duration of cerebral ischemia to 18 hours was accompanied by a decrease in the intensity of free radical oxidation. Parameter Imax did not

Acute ischemia was not only followed by damage of the cell membrane structures and activation of free radical oxidation, but also induced the antioxidant system (Dziennis et al.,

The K index serves as a criterion for the antioxidant potential of the cell. The level of the antioxidant activity of the brain tissue was elevated after ischemia for 1.5 and 4 hours. The conclusion was derived from the decrease in this index. By 18 hours ischemia, the K index

These data indicate that the prooxidant/antioxidant ratio returns to normal with increasing in the duration of cerebral circulatory disorder. The observed changes are probably related to activation of defense protein synthesis, which increases the resistance of membrane

The kinetic curve for hexokinase was shown to have hyperbola form in various periods of ischemia except for 1.5 hours ischemia (Fig. 15). MgATP did not inhibit hexokinase when increasing the duration of ischemia. By 18 hours, the Km (0.13 mM) is 2-fold lower than in

The major problem in the involvement of cell structures in the regulation of enzyme activity is the dependence of enzyme properties on the association of this enzyme with the

0 0.5 1 1.5 2 2.5 3 3.5

Intact amimals Ischemia, 30 min. Ischemia, 18 h.

Km=0.13

**MgATP, mM**

Km=0.7

Km=0.26

membrane under conditions of functional changes in the organism.

intact animals (0.26 mM) and is 5-fold lower than in 30 minutes ischemia (0.7 mM).

differ from the corresponding parameter in intact animals.

did not differ from that in intact animals.

structures to the adverse effect of ischemia.

Fig. 15. Kinetic of the hexokinase reaction.

0 20

40 60

80 100

120 140

**V, U/mg\*min.**

160 180

200

2008; Lai et al., 2003; Perez-Pinzon et al., 2005; Suzuki et al., 1997).

Mitochondrial creatine kinase is associated with mitochondrial membranes due to the forces of electrostatic and hydrophobic interaction. Cerebral ischemia was followed by changes in the activity of associated and tightly-bound mitochondrial creatine kinase (Fig. 16).

The activity of associated mitochondrial creatine kinase increased in comparison with intact animals in 1.5 hours ischemia. However the activity of tightly-bound creatine kinase did not change. The activity of tightly-bound mitochondrial creatine kinase increased and the ratio between two forms of mitochondrial creatine kinase restored in 4 hours ischemia. By 18 hours, the percentage of tightly-bound form of the enzyme reached 90%.

Fig. 16. The activity of membrane-associated creatine kinase before and after solubilization by phosphate buffer in cerebral ischemia.

The bar diagrams display average activities with error bars representing the standard deviation. \**p*<0.05 versus intact animals. \*\* *p*<0.05 versus severe ischemia (30 minutes).

Cerebral ischemia was shown to change the kinetic properties of mitochondrial creatine kinase (Fig. 17). Mitochondrial creatine kinase showed abnormal kinetic with the appearance of intermediate plateau. By 18 hours, the kinetic curve acquired a hyperbola form.

In mitochondria, mitochondrial creatine kinase is presented by two oligomeric forms (dimer and octamer). They are characterized by dynamic equilibrium (Lipskaya et al., 1989). The transition-state analogue complex of mitochondrial creatine kinase was induced to evaluate the ratio between oligomeric forms of this enzyme under conditions of cerebral circulatory disorders.

Cerebral ischemia changes the dimer/octamer ratio. This ratio is shifted toward the formation of dimers after 30-min ischemia (79%). Phospholipids serve as the structural elements of membranes that are bound to mitochondrial creatine kinase. Membrane binding properties of mitochondrial creatine kinase depend strongly on the protein dimer/octamer ratio and degree of lipid oxidation. Activation of free radical oxidation during acute ischemia is probably followed by partial dissociation of octamers to dimers. Increasing of the duration of ischemia to 18 hours was followed by an increase in the octamer ratio (53%).

Role of Creatine Kinase – Hexokinase Complex

30, pp. 245-255.

69-76.

conditions.

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providing the adequate energy supply of the nervous cells due to the new adaptive

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Fig. 17. Kinetic of the membrane-bound creatine kinase reaction

Published data suggest that octameric mitochondrial creatine kinase contributes to the appearance and strengthening of contact sites, which increases the efficiency of energy formation in brain mitochondria, consolidates the membrane structure, and determines the resistance of membranes to the adverse effect of hypoxia (Gross, Wallimann, 1995; Koufen et al., 1999; Lenz et al., 2007; Meyer et al., 2006). The existence of two oligomeric forms of this enzyme probably maintains the near-equilibrium state of reaction in a wide range of physiological conditions.

### **4. Conclusion**

The results indicate that catalytic properties of mitochondrial creatine kinase and hexokinase depend on the functional interaction with mitochondrial membranes.

Acute ischemia impairs enzyme interaction with the mitochondrial membrane. Increasing in the duration of ischemia is not only followed by injury and dysfunction, but also activates the defense systems in the nervous tissue. It is manifested in the decrease in the intensity of free radical oxidation, increase in the percentage of tightly-bound mitochondrial creatine kinase, changes in kinetic properties of the enzyme and change in the dimer/octamer ratio toward the formation of octamer for the mitochondrial creatine kinase. These changes stabilize the mitochondrial creatine kinase complex. In contrast, increase in the duration of ischemia is accompanied by the decrease in the hexokinase activity on the membrane in spite the fact that it becomes higher than in acute 30 min ischemia, but the percentage of solubilizing enzyme does not differ from that in intact animals.

Therefore, the long-term ischemia leads to stabilization of the functional interaction between hexokinase and creatine kinase complex with the mitochondrial membranes at a new level, providing the adequate energy supply of the nervous cells due to the new adaptive conditions.

### **5. References**

208 Advances in the Preclinical Study of Ischemic Stroke

Km=0.29

Km=0.12

**Phosphocreatine, mM**

Km=0.035; 0.53

Published data suggest that octameric mitochondrial creatine kinase contributes to the appearance and strengthening of contact sites, which increases the efficiency of energy formation in brain mitochondria, consolidates the membrane structure, and determines the resistance of membranes to the adverse effect of hypoxia (Gross, Wallimann, 1995; Koufen et al., 1999; Lenz et al., 2007; Meyer et al., 2006). The existence of two oligomeric forms of this enzyme probably maintains the near-equilibrium state of reaction in a wide range of

012345

Intact amimals Ischemia, 30 min. Ischemia, 18 h.

The results indicate that catalytic properties of mitochondrial creatine kinase and

Acute ischemia impairs enzyme interaction with the mitochondrial membrane. Increasing in the duration of ischemia is not only followed by injury and dysfunction, but also activates the defense systems in the nervous tissue. It is manifested in the decrease in the intensity of free radical oxidation, increase in the percentage of tightly-bound mitochondrial creatine kinase, changes in kinetic properties of the enzyme and change in the dimer/octamer ratio toward the formation of octamer for the mitochondrial creatine kinase. These changes stabilize the mitochondrial creatine kinase complex. In contrast, increase in the duration of ischemia is accompanied by the decrease in the hexokinase activity on the membrane in spite the fact that it becomes higher than in acute 30 min ischemia, but the percentage of

Therefore, the long-term ischemia leads to stabilization of the functional interaction between hexokinase and creatine kinase complex with the mitochondrial membranes at a new level,

hexokinase depend on the functional interaction with mitochondrial membranes.

Fig. 17. Kinetic of the membrane-bound creatine kinase reaction

solubilizing enzyme does not differ from that in intact animals.

physiological conditions.

**4. Conclusion** 

0

0.5

1

1.5

2

2.5

**V, U/mg\*min.**

3

3.5

4


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**11** 

*Japan* 

**Diabetes-Mediated Exacerbation of Neuronal** 

Diabetes mellitus is a metabolic disorder, suffered by hundreds of millions of people throughout the world, which is characterized by hyperglycemia resulting from insufficiency of insulin secretion and/or action (Wild et al., 2004). Complications associated with diabetes affecting vessels, eyes, kidney, and peripheral nerves reduce the QOL of diabetic patients. Also, diabetes is widely recognized as a major risk factor for atherosclerotic disease such as acute brain ischemia. Indeed, diabetic patients have a higher risk of stroke compared with non-diabetic patients (Baynes 1991; Stephens et al., 2009). Additionally, they are more likely to have a poor prognosis and increased mortality after stroke (Biller et al., 1993; Vinik et al., 2002). Previous studies have demonstrated that the diabetic state increases oxidative stress in the brain and aggravates cerebral ischemic injury in both type I (Li et al., 2004; Saito et al., 2005; Rizk et al., 2005) and type II diabetic animal models (Anabela et al., 2006; Tureyen et al., 2011). In addition to neuronal damage attributed to hypoxia and ATP depletion caused by vascular obstruction in ischemic core region, cerebral injury caused by subsequent reperfusion is also involved in the pathophysiology of transient ischemia (Doyle et al., 2008; Nakka et al., 2008; Wang et al., 2010). During reperfusion, the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is enhanced by abrupt re-oxygenation (Cuzzocrea et al., 2001; Saito et al., 2005). Besides direct injurious effects to the cell membrane, proteins and DNA by

**1. Introduction** 

 \*

Corresponding Author

**Damage and Inflammation After Cerebral** 

**Medium of** *Ganoderma lucidum* **Mycelia** 

**Ischemia in Rat: Protective Effects of** 

**Water-Soluble Extract from Culture** 

*1Department of Immunobiochemistry, Josai University Graduate* 

Naohiro Iwata1, Mari Okazaki1,\*, Rika Nakano1,

Hirokazu Matsuzaki1 and Yasuhide Hibino1

Chisato Kasahara1, Shinya Kamiuchi1, Fumiko Suzuki2, Hiroshi Iizuka2,

*School of Pharmaceutical Sciences, Saitama,* 

*2Noda Shokukinkogyo Co., Ltd.* 


### **Diabetes-Mediated Exacerbation of Neuronal Damage and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water-Soluble Extract from Culture Medium of** *Ganoderma lucidum* **Mycelia**

Naohiro Iwata1, Mari Okazaki1,\*, Rika Nakano1, Chisato Kasahara1, Shinya Kamiuchi1, Fumiko Suzuki2, Hiroshi Iizuka2, Hirokazu Matsuzaki1 and Yasuhide Hibino1 *1Department of Immunobiochemistry, Josai University Graduate School of Pharmaceutical Sciences, Saitama, 2Noda Shokukinkogyo Co., Ltd. Japan* 

### **1. Introduction**

214 Advances in the Preclinical Study of Ischemic Stroke

Wilson, J.E. (2003). Isozymes of mammalian hexokinase: structure, subcellular localization

Wyss, M. & Kaddurah-Daouk, R. (2000). Creatine and creatinine metabolism. *Physiol. Rev*.,

Xie, G. & Wilson, J.E. (1990). Tetrameric structure of mitochondrially bound rat brain

hexokinase: a crosslinking study. *Arch. Biochem. Biophys*., Vol. 276, No 1, pp. 285-

and metabolic function. *The J. Exp. Biol.,* Vol. 206, pp. 2049-2057.

Vol. 80, pp. 1107-1213.

293.

Diabetes mellitus is a metabolic disorder, suffered by hundreds of millions of people throughout the world, which is characterized by hyperglycemia resulting from insufficiency of insulin secretion and/or action (Wild et al., 2004). Complications associated with diabetes affecting vessels, eyes, kidney, and peripheral nerves reduce the QOL of diabetic patients. Also, diabetes is widely recognized as a major risk factor for atherosclerotic disease such as acute brain ischemia. Indeed, diabetic patients have a higher risk of stroke compared with non-diabetic patients (Baynes 1991; Stephens et al., 2009). Additionally, they are more likely to have a poor prognosis and increased mortality after stroke (Biller et al., 1993; Vinik et al., 2002). Previous studies have demonstrated that the diabetic state increases oxidative stress in the brain and aggravates cerebral ischemic injury in both type I (Li et al., 2004; Saito et al., 2005; Rizk et al., 2005) and type II diabetic animal models (Anabela et al., 2006; Tureyen et al., 2011). In addition to neuronal damage attributed to hypoxia and ATP depletion caused by vascular obstruction in ischemic core region, cerebral injury caused by subsequent reperfusion is also involved in the pathophysiology of transient ischemia (Doyle et al., 2008; Nakka et al., 2008; Wang et al., 2010). During reperfusion, the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is enhanced by abrupt re-oxygenation (Cuzzocrea et al., 2001; Saito et al., 2005). Besides direct injurious effects to the cell membrane, proteins and DNA by

<sup>\*</sup> Corresponding Author

Diabetes-Mediated Exacerbation of Neuronal Damage

ICAM-1 and MPO in the brain during reperfusion.

**2.2 Middle cerebral artery occlusion and reperfusion** 

rats of the non-diabetic and diabetic groups, respectively.

**2. Materials and methods** 

daily for the last 2 weeks.

**2.1 Experimental diabetic animals**

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 217

reperfusion (MCAO/Re) in streptozotocin (STZ)-induced diabetic rats (Iwata et al., 2008). However, the mechanism of its cerebroprotective effect still remains unclear. Thus, we evaluated the effects of chronic oral pretreatment of MAK on the production of O2⁻· and apoptosis in STZ-rat brain after MCAO/Re. Furthermore, to clarify whether MAK suppresses inflammatory responses induced by the transient ischemia and reperfusion, we examined the effects of MAK on the expression profile of TNF-, IL-1, iNOS, COX-2,

Male Sprague Dawley rats (4-week old, weight 120-140 g; Tokyo Exp. Animal Co., Ltd., Tokyo, Japan) were purchased and housed in a temperature-controlled environment (23 ± 0.5°C) with a cycle of 12 hrs light and 12 hrs dark. The rats were given a standard rodent chow and water *ad libitum*. Animal care and the surgical procedure were performed in accordance with guidelines approved by the National Institutes of Health and the Josai University Animal Investigation Committee. A diabetic state was induced in the rats (diabetic group) by a single injection of STZ (60 mg/kg, i.p.) dissolved in 0.1 mM sodium citrate (pH 4.5), while the rats of the non-diabetic group were injected with buffer only (Iwata et al., 2008). Seven days after the STZ-injection, the plasma glucose level was determined using a glucose analyzer (Ascensia, Bayer Medical Co., Ltd., Land Nordrhein-Westfalen, Germany). Diabetes was defined by a blood glucose level greater than 300 mg/dl. Then, the diabetic and non-diabetic groups were divided into two groups respectively and were housed for an additional 6 weeks until stroke was induced by MCAO. MAK (1 g/kg; MAK group) or distilled water (control group) was administrated orally once

Focal cerebral ischemia was induced by MCAO with a standard intraluminal filament technique as previously described (Iwata et al., 2010). The animals were anesthetized with 4% halothane and maintained with 1.5% halothane and 30% oxygen under spontaneous respiration. After a midline incision at the neck, the right common carotid artery was exfoliated under an operating microscope. All branches of the external carotid artery were isolated and ligated. The tip of the 4-0 surgical nylon monofilament rounded by flame heating was inserted up through the internal carotid artery. When a small resistance was felt, insertion was stopped. The distance from bifurcation of the common carotid artery to the tip of the suture was approximately 20 mm in all rats. Cerebral blood flow was detected by laser Doppler Flowmetry (ATBF-LC1, Unique Medical Co., LTD., Tokyo) and about 50% reduction of its baseline associated with MCAO was ascertained in the rats. Rectal temperature was maintained at 37°C with a heat lamp and a heating pad during the operation. After 2 hrs of occlusion, the filament was withdrawn to allow for reperfusion. Then, the animals were permitted to recover from the anesthesia at room temperature. The rats were reperfused for 3 or 24 hrs before they were killed. The sham operation with the same manipulation without introduction of the monofilament was also performed in the 4

oxidation, ROS and RNS activate the pro-apoptotic pathway including the activation of caspase family proteases leading to DNA fragmentation in the neuronal cells of the ischemic penumbral region (Sugawara et al., 2007; Nakka et al., 2008). Hyperglycemia is assumed to be a major factor responsible for excessive generation of ROS. In the diabetic state, "glucose toxicity" caused by augmentation of the intracellular glucose oxidation process and non-enzymatic glycation of protein molecules leads to over production of ROS (Baynes, 1991; Giacco et al., 2010). Moreover, experimental transient hyperglycemia caused by intravenous infusion of glucose has been shown to increase ROS production and exacerbate brain injury after ischemia and subsequent reperfusion in rats (Tsuruta et al., 2010).

In addition to apoptotic cell death, inflammatory neurodegeneration is a crucial process contributing to cerebral damage after ischemia and reperfusion (Brown et al., 2010). ROS has been shown to activate nuclear factor-B (NF-B), which enhances transcription of the genes encoding pro-inflammatory cytokines and cell adhesion molecules, leading to neuroinflammatory responses (Saeed et al., 2007). Activation of the transcription factor NF- B by ROS in microglia and astrocytes leads to an increase in the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines including TNF-, IL-1 and IL-6, which accelerate inflammatory responses and promote neuronal cell death in the ischemic region (Brown et al., 2010). Gene expression of IL-1and IL-6 is much higher in type2-diabetic mice as compared with normoglycemic control mice (Tureyen et al., 2011). These pro-inflammatory cytokines and inflammatory mediators have been indicated to induce further inflammatory responses involving extravasation of neutrophils, macrophages/microglia through the expression of intracellular adhesion molecule 1 (ICAM-1) in endothelial cells (Wang et al., 2002; Chrissobolis et al., 2011). Myeloperoxidase (MPO) expressing in neutrophils and macrophages/ microglia, which has often been used as a histopathological marker for inflammation, generates ROS such as hypochloride and super oxide anion radical (O2⁻·) and leads to further tissue damage (Breckwoldt et al., 2008). Thus, enhanced oxidative stress and inflammatory responses induced by hyperglycemia may substantially contribute to the exacerbation of cerebral injury caused by transient ischemia and subsequent reperfusion in the diabetic state.

*Ganoderma lucidum* (*G. lucidum*), a very popular medicinal fungus, has long been known for its beneficial effects on human health and longevity in Asian countries. Its fruiting bodies and cultured mycelia are used to treat chronic hepatopathy (Shi et al., 2008), hypertension (Kabir et al., 1988), hyperglycemia (Zhang et al., 2004), and tumor (Lu et al., 2003; Kubo et al., 2005). The pharmacological activities of *G. lucidum* constituents responsible for many of its health benefits, such as antioxidant (Zhu et al., 1999), anticancer (Lu et al., 2003; Kubo et al., 2005), anti-inflammatory (Akihisa et al., 2007), and immunomodulatory activities (Lai et al., 2010) have been elucidated. The water-soluble extract from culture medium of *G. lucidum* mycelia (MAK), a commercially available nutritional supplement, is a freeze-dried powder of a hot-water extract prepared from a solid culture medium composed of bagasse and defatted-rice bran overgrown with *G. lucidum* mycelia by cultivation for about 3.5 months. In a previous study, we demonstrated that the orally administered MAK attenuated oxidative stress and relieved exacerbation of cerebral injury induced by middle cerebral artery occlusion and reperfusion (MCAO/Re) in streptozotocin (STZ)-induced diabetic rats (Iwata et al., 2008). However, the mechanism of its cerebroprotective effect still remains unclear. Thus, we evaluated the effects of chronic oral pretreatment of MAK on the production of O2⁻· and apoptosis in STZ-rat brain after MCAO/Re. Furthermore, to clarify whether MAK suppresses inflammatory responses induced by the transient ischemia and reperfusion, we examined the effects of MAK on the expression profile of TNF-, IL-1, iNOS, COX-2, ICAM-1 and MPO in the brain during reperfusion.

### **2. Materials and methods**

216 Advances in the Preclinical Study of Ischemic Stroke

oxidation, ROS and RNS activate the pro-apoptotic pathway including the activation of caspase family proteases leading to DNA fragmentation in the neuronal cells of the ischemic penumbral region (Sugawara et al., 2007; Nakka et al., 2008). Hyperglycemia is assumed to be a major factor responsible for excessive generation of ROS. In the diabetic state, "glucose toxicity" caused by augmentation of the intracellular glucose oxidation process and non-enzymatic glycation of protein molecules leads to over production of ROS (Baynes, 1991; Giacco et al., 2010). Moreover, experimental transient hyperglycemia caused by intravenous infusion of glucose has been shown to increase ROS production and exacerbate brain injury after ischemia and subsequent reperfusion in rats (Tsuruta et

In addition to apoptotic cell death, inflammatory neurodegeneration is a crucial process contributing to cerebral damage after ischemia and reperfusion (Brown et al., 2010). ROS has been shown to activate nuclear factor-B (NF-B), which enhances transcription of the genes encoding pro-inflammatory cytokines and cell adhesion molecules, leading to neuroinflammatory responses (Saeed et al., 2007). Activation of the transcription factor NF- B by ROS in microglia and astrocytes leads to an increase in the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines including TNF-, IL-1 and IL-6, which accelerate inflammatory responses and promote neuronal cell death in the ischemic region (Brown et al., 2010). Gene expression of IL-1and IL-6 is much higher in type2-diabetic mice as compared with normoglycemic control mice (Tureyen et al., 2011). These pro-inflammatory cytokines and inflammatory mediators have been indicated to induce further inflammatory responses involving extravasation of neutrophils, macrophages/microglia through the expression of intracellular adhesion molecule 1 (ICAM-1) in endothelial cells (Wang et al., 2002; Chrissobolis et al., 2011). Myeloperoxidase (MPO) expressing in neutrophils and macrophages/ microglia, which has often been used as a histopathological marker for inflammation, generates ROS such as hypochloride and super oxide anion radical (O2⁻·) and leads to further tissue damage (Breckwoldt et al., 2008). Thus, enhanced oxidative stress and inflammatory responses induced by hyperglycemia may substantially contribute to the exacerbation of cerebral injury caused by transient ischemia and subsequent reperfusion in the diabetic

*Ganoderma lucidum* (*G. lucidum*), a very popular medicinal fungus, has long been known for its beneficial effects on human health and longevity in Asian countries. Its fruiting bodies and cultured mycelia are used to treat chronic hepatopathy (Shi et al., 2008), hypertension (Kabir et al., 1988), hyperglycemia (Zhang et al., 2004), and tumor (Lu et al., 2003; Kubo et al., 2005). The pharmacological activities of *G. lucidum* constituents responsible for many of its health benefits, such as antioxidant (Zhu et al., 1999), anticancer (Lu et al., 2003; Kubo et al., 2005), anti-inflammatory (Akihisa et al., 2007), and immunomodulatory activities (Lai et al., 2010) have been elucidated. The water-soluble extract from culture medium of *G. lucidum* mycelia (MAK), a commercially available nutritional supplement, is a freeze-dried powder of a hot-water extract prepared from a solid culture medium composed of bagasse and defatted-rice bran overgrown with *G. lucidum* mycelia by cultivation for about 3.5 months. In a previous study, we demonstrated that the orally administered MAK attenuated oxidative stress and relieved exacerbation of cerebral injury induced by middle cerebral artery occlusion and

al., 2010).

state.

### **2.1 Experimental diabetic animals**

Male Sprague Dawley rats (4-week old, weight 120-140 g; Tokyo Exp. Animal Co., Ltd., Tokyo, Japan) were purchased and housed in a temperature-controlled environment (23 ± 0.5°C) with a cycle of 12 hrs light and 12 hrs dark. The rats were given a standard rodent chow and water *ad libitum*. Animal care and the surgical procedure were performed in accordance with guidelines approved by the National Institutes of Health and the Josai University Animal Investigation Committee. A diabetic state was induced in the rats (diabetic group) by a single injection of STZ (60 mg/kg, i.p.) dissolved in 0.1 mM sodium citrate (pH 4.5), while the rats of the non-diabetic group were injected with buffer only (Iwata et al., 2008). Seven days after the STZ-injection, the plasma glucose level was determined using a glucose analyzer (Ascensia, Bayer Medical Co., Ltd., Land Nordrhein-Westfalen, Germany). Diabetes was defined by a blood glucose level greater than 300 mg/dl. Then, the diabetic and non-diabetic groups were divided into two groups respectively and were housed for an additional 6 weeks until stroke was induced by MCAO. MAK (1 g/kg; MAK group) or distilled water (control group) was administrated orally once daily for the last 2 weeks.

### **2.2 Middle cerebral artery occlusion and reperfusion**

Focal cerebral ischemia was induced by MCAO with a standard intraluminal filament technique as previously described (Iwata et al., 2010). The animals were anesthetized with 4% halothane and maintained with 1.5% halothane and 30% oxygen under spontaneous respiration. After a midline incision at the neck, the right common carotid artery was exfoliated under an operating microscope. All branches of the external carotid artery were isolated and ligated. The tip of the 4-0 surgical nylon monofilament rounded by flame heating was inserted up through the internal carotid artery. When a small resistance was felt, insertion was stopped. The distance from bifurcation of the common carotid artery to the tip of the suture was approximately 20 mm in all rats. Cerebral blood flow was detected by laser Doppler Flowmetry (ATBF-LC1, Unique Medical Co., LTD., Tokyo) and about 50% reduction of its baseline associated with MCAO was ascertained in the rats. Rectal temperature was maintained at 37°C with a heat lamp and a heating pad during the operation. After 2 hrs of occlusion, the filament was withdrawn to allow for reperfusion. Then, the animals were permitted to recover from the anesthesia at room temperature. The rats were reperfused for 3 or 24 hrs before they were killed. The sham operation with the same manipulation without introduction of the monofilament was also performed in the 4 rats of the non-diabetic and diabetic groups, respectively.

Diabetes-Mediated Exacerbation of Neuronal Damage

SD relative to the sham-operated non-diabetic group.

**2.7 Real-time PCR analysis** 

**2.8 Immunohistochemistry** 

software (FV10-ASW 1.7, OLYMPUS).

**2.9 Western blotting** 

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 219

The temporal gene expression patterns of pro-inflammatory cytokines (IL-1 and TNF-) and inflammatory mediators (COX-2, iNOS and ICAM-1) were evaluated by quantitative real-time PCR analysis as described earlier (Liu et al. 2007). The rats subjected to MCAO were killed at 3 or 24 hrs of reperfusion, and the total RNA sample was obtained from the ischemic penumbral cortex of each rat. Total RNA was extracted with RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Total RNA (0.5 g) from each sample was reverse-transcribed with oligo dT and random hexamer primers using reverse transcriptase (PrimeScriptTM RT Enzyme Mix I, Takara RNA PCR Kit, Takara Biomedicals, Shiga, Japan). Real-time PCR was performed with 10 ng of cDNA and a pair of gene specific primers (Takara Biomedicals) added to the SYBR Premix EX *Taq* (Takara Biomedicals) and subjected to PCR amplification in the iCycler iQ Real-Time Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (1 cycle at 95°C for 10 sec, and 50 cycles at 95°C for 5 sec and 60°C for 34 sec). The expression of -actin was used to normalize cDNA levels. The PCR products were analyzed by a melting curve to ascertain the specificity of amplification. Data were normalized to -actin and were expressed as mean ±

Immunostaining was performed as previously described (Faraco et al., 2007). Briefly, rats were sacrificed at the indicated time points and transcardially perfused with cold saline. Brains were fixed with 4% phosphate-buffered paraformaldehyde. Coronal brain sections (8 m thick) were incubated sequentially with 3% hydrogen peroxide for 40 min at room temperature to inhibit endogenous peroxidase, followed by incubation with blocking buffer (100% block ace; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) for 2 hrs. Slides were incubated with polyclonal rabbit anti-IL-1 antibody (1:300, Santa Cruz Biotechnology, CA, USA), polyclonal rabbit anti-TNF-antibody (1:200; Rabbit mAb, Hycult Biotech, PB Uden, The Netherlands), polyclonal rabbit anti-COX-2 antibody (1:200, Cayman Chemical, Michigan, USA), polyclonal rabbit anti-iNOS antibody (1:200, Santa Cruz), monoclonal mouse anti-RECA-1 (1:200, Abcam Biotechnology, Cambridge, UK), polyclonal rabbit anti-Cleaved caspase-3 (1:100, Cell Signalling Technology, Danvers, MA, USA), and monoclonal mouse anti-MPO (1:100, Hycult Biotech) in 0.01 mol/l phosphate-buffered saline overnight at 4°C. After washing with PBS, these were correspondingly incubated with Cy3- and FITCconjugated secondary antibodies (1:200, Chemicon, California, USA) for 2 hrs at room temperature. Finally, sections were incubated with the nuclear stain TO-PRO-3 (1:10000, Invitrogen, California, USA) in phosphate-buffered saline for 10 min at room temperature with gentle agitation, washed and mounted using 70% glycerol mounting medium. Immunofluorescence was visualized using a Laser Scanning Confocal Microscope (FluoView FV1000, OLYMPUS). Fluorescence intensity was quantified using imaging

The cytosolic and nuclear extracts were prepared by the method of Meldrum (Meldrum et al., 1997; Aragno et al., 2005). Briefly, the penumbral cortex was homogenized at 10% (w/v) in a polytron homogenizer (Kinematica AG, Switzerland) using a homogenization buffer (20 mM HEPES (pH 7.9), 1 mM MgCl2, 0.5 mM EDTA, 1% Nonidet P-40, 1 mM

### **2.3 Neurological evaluation**

Post-ischemic neurological deficits were evaluated after 3 or 24 hrs of reperfusion on a 5 point scale as described as follows: grade 0:no deficit, grade 1:failure to extend right forepaw fully, grade 2:spontaneous circling or walking to contralateral side, grade 3:walking only when stimulated, grade 4:unresponsive to stimulation and a depressed level of consciousness, grade 5:death (Iwata et al., 2008; Vinik et al., 2002). Animals that did not show neurological deficits were excluded from the study.

### **2.4 Infarct and edema assessment**

After 3 or 24 hrs of reperfusion, the animals were deeply anesthetized with diethyl ether and decapitated. The brain was removed and cut into four 2-mm coronal sections using a rat brain matrix, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Wako Pure Chemicals) at 37°C for 15 min. The coronal slices were fixed in 10% formaldehyde for photography (Iwata et al., 2008; Vinik et al., 2002). Then, infarct areas were determined by using the image analysis system (Scion Image 1.62), and were added to obtain the infarct volumes per brain. Corrected infarct volume (%) = [left hemisphere volume - (right hemisphere volume - the infarct volume)] / left hemisphere volume × 100. Edema in the ischemic hemisphere was also calculated: edema (%) = (right hemisphere volume - the infarct volume) / left hemisphere volume.

#### **2.5 Detection of O2 - · generation in brain**

Detection of intracellular O2 generation in the penumbral region of the cortex following MCAO/Re was performed by staining freshly frozen brain sections (8 m thick) with the fluorescent probe dihydroethidium (DHE). The brain sections were immediately incubated with DHE (10 mol/l, Sigma) in phosphate-buffered saline for 30 min at 37°C (Muranyi et al., 2006). To determine the fluorescent intensity of oxidized DHE, three microscopic fields at the penumbral cortex regions of each hemisphere were captured using a confocal laserscanning microscope at excitation of 510 nm and emission of 580 nm. Fluorescence intensity of the oxidized DHE was quantified using imaging software (FV10-ASW 1.7, OLYMPUS Co. Ltd., Tokyo, Japan).

### **2.6 TUNEL staining**

Apoptosis in the brain tissues was measured using the Apoptosis in situ Detection Kit Wako (Wako, Laboratories, Osaka, Japan), which is based on the TUNEL (Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick end labeling) procedure, that is the addition of fluorescein-dUTP to 3'-terminals of apoptotically fragmented DNA with TdT followed by immunochemical detection using anti-fluorescein antibody conjugated with horseradish peroxidase (POD) and DAB (3-3'-diaminobenzidine tetrachloride) as a substrate. Coronal brain sections (8 m thick) were used for the assay. The slides were lightly counterstained with hematoxylin and observed under a microscope (BX51W1, OLYMPUS). Quantification of TUNEL positive cells was achieved by cell counting in areas of the penumbral cortex affected by ischemia. Three randomly chosen visual fields were counted in each region by an investigator without knowledge of the experimental conditions. The percentage of apoptotic cells was calculated by the apoptotic index, i.e. dividing the number of positive-staining nuclei by the total number of nuclei (Li et al., 2004).

### **2.7 Real-time PCR analysis**

218 Advances in the Preclinical Study of Ischemic Stroke

Post-ischemic neurological deficits were evaluated after 3 or 24 hrs of reperfusion on a 5 point scale as described as follows: grade 0:no deficit, grade 1:failure to extend right forepaw fully, grade 2:spontaneous circling or walking to contralateral side, grade 3:walking only when stimulated, grade 4:unresponsive to stimulation and a depressed level of consciousness, grade 5:death (Iwata et al., 2008; Vinik et al., 2002). Animals that did not

After 3 or 24 hrs of reperfusion, the animals were deeply anesthetized with diethyl ether and decapitated. The brain was removed and cut into four 2-mm coronal sections using a rat brain matrix, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Wako Pure Chemicals) at 37°C for 15 min. The coronal slices were fixed in 10% formaldehyde for photography (Iwata et al., 2008; Vinik et al., 2002). Then, infarct areas were determined by using the image analysis system (Scion Image 1.62), and were added to obtain the infarct volumes per brain. Corrected infarct volume (%) = [left hemisphere volume - (right hemisphere volume - the infarct volume)] / left hemisphere volume × 100. Edema in the ischemic hemisphere was also calculated: edema (%) = (right hemisphere volume - the

Detection of intracellular O2- generation in the penumbral region of the cortex following MCAO/Re was performed by staining freshly frozen brain sections (8 m thick) with the fluorescent probe dihydroethidium (DHE). The brain sections were immediately incubated with DHE (10 mol/l, Sigma) in phosphate-buffered saline for 30 min at 37°C (Muranyi et al., 2006). To determine the fluorescent intensity of oxidized DHE, three microscopic fields at the penumbral cortex regions of each hemisphere were captured using a confocal laserscanning microscope at excitation of 510 nm and emission of 580 nm. Fluorescence intensity of the oxidized DHE was quantified using imaging software (FV10-ASW 1.7, OLYMPUS Co.

Apoptosis in the brain tissues was measured using the Apoptosis in situ Detection Kit Wako (Wako, Laboratories, Osaka, Japan), which is based on the TUNEL (Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick end labeling) procedure, that is the addition of fluorescein-dUTP to 3'-terminals of apoptotically fragmented DNA with TdT followed by immunochemical detection using anti-fluorescein antibody conjugated with horseradish peroxidase (POD) and DAB (3-3'-diaminobenzidine tetrachloride) as a substrate. Coronal brain sections (8 m thick) were used for the assay. The slides were lightly counterstained with hematoxylin and observed under a microscope (BX51W1, OLYMPUS). Quantification of TUNEL positive cells was achieved by cell counting in areas of the penumbral cortex affected by ischemia. Three randomly chosen visual fields were counted in each region by an investigator without knowledge of the experimental conditions. The percentage of apoptotic cells was calculated by the apoptotic index, i.e. dividing the number of positive-staining nuclei by the total number of nuclei (Li et al., 2004).

**2.3 Neurological evaluation** 

**2.4 Infarct and edema assessment** 

infarct volume) / left hemisphere volume.

**-**

**2.5 Detection of O2**

Ltd., Tokyo, Japan).

**2.6 TUNEL staining** 

show neurological deficits were excluded from the study.

**· generation in brain** 

The temporal gene expression patterns of pro-inflammatory cytokines (IL-1 and TNF-) and inflammatory mediators (COX-2, iNOS and ICAM-1) were evaluated by quantitative real-time PCR analysis as described earlier (Liu et al. 2007). The rats subjected to MCAO were killed at 3 or 24 hrs of reperfusion, and the total RNA sample was obtained from the ischemic penumbral cortex of each rat. Total RNA was extracted with RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Total RNA (0.5 g) from each sample was reverse-transcribed with oligo dT and random hexamer primers using reverse transcriptase (PrimeScriptTM RT Enzyme Mix I, Takara RNA PCR Kit, Takara Biomedicals, Shiga, Japan). Real-time PCR was performed with 10 ng of cDNA and a pair of gene specific primers (Takara Biomedicals) added to the SYBR Premix EX *Taq* (Takara Biomedicals) and subjected to PCR amplification in the iCycler iQ Real-Time Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (1 cycle at 95°C for 10 sec, and 50 cycles at 95°C for 5 sec and 60°C for 34 sec). The expression of -actin was used to normalize cDNA levels. The PCR products were analyzed by a melting curve to ascertain the specificity of amplification. Data were normalized to -actin and were expressed as mean ± SD relative to the sham-operated non-diabetic group.

### **2.8 Immunohistochemistry**

Immunostaining was performed as previously described (Faraco et al., 2007). Briefly, rats were sacrificed at the indicated time points and transcardially perfused with cold saline. Brains were fixed with 4% phosphate-buffered paraformaldehyde. Coronal brain sections (8 m thick) were incubated sequentially with 3% hydrogen peroxide for 40 min at room temperature to inhibit endogenous peroxidase, followed by incubation with blocking buffer (100% block ace; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) for 2 hrs. Slides were incubated with polyclonal rabbit anti-IL-1 antibody (1:300, Santa Cruz Biotechnology, CA, USA), polyclonal rabbit anti-TNF-antibody (1:200; Rabbit mAb, Hycult Biotech, PB Uden, The Netherlands), polyclonal rabbit anti-COX-2 antibody (1:200, Cayman Chemical, Michigan, USA), polyclonal rabbit anti-iNOS antibody (1:200, Santa Cruz), monoclonal mouse anti-RECA-1 (1:200, Abcam Biotechnology, Cambridge, UK), polyclonal rabbit anti-Cleaved caspase-3 (1:100, Cell Signalling Technology, Danvers, MA, USA), and monoclonal mouse anti-MPO (1:100, Hycult Biotech) in 0.01 mol/l phosphate-buffered saline overnight at 4°C. After washing with PBS, these were correspondingly incubated with Cy3- and FITCconjugated secondary antibodies (1:200, Chemicon, California, USA) for 2 hrs at room temperature. Finally, sections were incubated with the nuclear stain TO-PRO-3 (1:10000, Invitrogen, California, USA) in phosphate-buffered saline for 10 min at room temperature with gentle agitation, washed and mounted using 70% glycerol mounting medium. Immunofluorescence was visualized using a Laser Scanning Confocal Microscope (FluoView FV1000, OLYMPUS). Fluorescence intensity was quantified using imaging software (FV10-ASW 1.7, OLYMPUS).

### **2.9 Western blotting**

The cytosolic and nuclear extracts were prepared by the method of Meldrum (Meldrum et al., 1997; Aragno et al., 2005). Briefly, the penumbral cortex was homogenized at 10% (w/v) in a polytron homogenizer (Kinematica AG, Switzerland) using a homogenization buffer (20 mM HEPES (pH 7.9), 1 mM MgCl2, 0.5 mM EDTA, 1% Nonidet P-40, 1 mM

Diabetes-Mediated Exacerbation of Neuronal Damage

control diabetic rats.

**Control**

**MAK**

**non-DM**

**(A)**

**Time after reperfusion**

**5 mm**

Fig. 1. Effects of MAK on infarction induced by MCAO/Re in non-diabetic (DM) and

hemispheres of the DM and non-DM groups after MCAO/Re by TTC staining (B).

Representative coronal brain section photographs of the DM and non-DM rats stained by TTC at 3 or 24 hrs of reperfusion after 2 hrs MCAO (A). Infarct volume in ischemic

**Infarct volume**

**(B)**

**( % ipsilateral**

**0**

**a**

**20**

**<sup>a</sup> <sup>c</sup> <sup>b</sup>**

**(n=3-5)**

*P***<0.01 vs. non-DM**

**<sup>b</sup>***P***<0.01 vs. DM <sup>c</sup>***P***<0.05 vs. DM**

**a**

**non-DM non-DM + MAK**

**DM DM + MAK**

**40**

**60**

**a**

**3Re 24Re**

**80**

**100**

**hemisphere )**

**3Re 24Re Sham**

**MAK**

DM rat brains.

**Control DM**

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 221

showing viable (red) and dead (white) tissues are in (A). In the sham operated animals, there was no apparent damage in any brain region. The infarct area in the control nondiabetic rats after 24 hrs of reperfusion extended to the corpus striatum and cortex, whereas only a small striatal infarct was observed after 3 hrs of reperfusion. In the control diabetic group, brain injury induced by MCAO/Re was remarkably exacerbated. The cerebral infarct was produced within 3 hrs of reperfusion and the infarct region was extended to a large part of the left striatum and cortex in the diabetic rats. In contrast, the ischemic damage in the MAK-pretreated non-diabetic and diabetic animal brain was smaller as compared to those of the respective controls. Quantitative determination of infarct volume (B) indicated that the cerebral infarct volume was increased and associated with reperfusion time, which was markedly accelerated in the control diabetic group. The infarct volume evaluated at 3 hrs after reperfusion in the control diabetic group was significantly increased about 5-fold as compared with the non-diabetic group. Brain edema also tended to be exacerbated by diabetes, whereas no significant difference was detected between the non-diabetic and diabetic groups (data not shown). Consistent with the result of brain infarct volume, neurological deficits were exacerbated by reperfusion and diabetes (Fig. 2). MAK-pretreated diabetic rats showed significant alleviation in the neurological deficits compared to the

EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 4 g/ml pepstatin A, and 4 g/ml leupeptin). Homogenates were centrifuged at 1,000×g for 5 min at 4°C. Supernatants were removed and centrifuged at 105,000×g at 4°C for 40 min to obtain the cytosolic fraction. The pelleted nuclei were resuspended in extraction buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 20% glycerol, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 4 g/ml pepstatin A, and 4 g/ml leupeptin). The suspensions were incubated on ice for 30 min for high-salt extraction followed by centrifugation at 15,000×g for 20 min at 4°C. The samples were stored at -80℃ until used. Brain tissues were homogenized in the SDS sample buffer (125 mM Tris (pH 6.8), 4% SDS, 10% sucrose, 0.01% bromophenol blue, and 10% 2 mercaptoethanol) and boiled for 1 min. Protein concentration was quantified by using the Bradford method (Protein Assay Reagent Kit, Bio-Rad Laboratories). The samples (40 g) were separated by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, England) through a semidry-type blotting (Bio-Rad) method, blocked by 5% nonfat dry milk in PBS with Tween-20 (PBS-T) (137 mM NaCl, 8.10 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.1% Tween-20), and incubated with appropriate antibodies as described below. The filters were incubated with each primary antibody overnight at 4°C, with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hr at room temperature in 5% nonfat dry milk/PBS-T. Finally, the target molecules were visualized through an enhanced chemiluminescence western blotting detection system (Amersham Biosciences) on the X-ray film (Amersham Biosciences). The following primary and secondary antibodies used in this study were NF-B (rabbit, 1:2,000 Santa Cruz) and -actin (mouse, 1:10,000, Sigma), anti-rabbit IgG HRP antibody (1:5,000, Amersham Biosciences), and anti-mouse IgG HRP antibody (1:10,000, Amersham Biosciences).

### **2.10 Statistical analysis**

Statistical analysis was performed with a two-way ANOVA followed by post hoc Tukey's multiple-comparison test. Neurological deficit scores were analyzed by the Kruskal-Wallis test followed by the Mann-Whitney *U* test. In all cases a *P*-value of <0.05 was regarded as statistically significant.

### **3. Results**

### **3.1 Physiological characteristic parameter**

The control diabetic group had typical characteristics of type 1 diabetes such as a decrease in the body weight (296.6 ± 33.3 g) and hyperglycemia (535.4 ± 104.0 mg/dl) compared with the control non-diabetic group (340.0 ± 22.4 g, 119.8 ± 17.1 mg/dl), which were similar to previous reports (Iwata et al., 2008; 2010). Chronic treatment of MAK for 2 weeks showed a slight but significant decrease in blood glucose of the diabetic rats (404.4 ± 109.2 mg/dl, *P*<0.01), whereas MAK had little effect on the body weight (266.6 ± 33.3 g).

### **3.2 Infarct volume and neurological deficits after transient MCAO with reperfusion**

Figure 1 shows the effects of MAK on the brain infarction by MCAO/Re in the non-diabetic and diabetic groups. Representative coronal brain sections stained by TTC after MCAO/Re

EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 4 g/ml pepstatin A, and 4 g/ml leupeptin). Homogenates were centrifuged at 1,000×g for 5 min at 4°C. Supernatants were removed and centrifuged at 105,000×g at 4°C for 40 min to obtain the cytosolic fraction. The pelleted nuclei were resuspended in extraction buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 300 mM NaCl, 0.2 mM EDTA, 20% glycerol, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 4 g/ml pepstatin A, and 4 g/ml leupeptin). The suspensions were incubated on ice for 30 min for high-salt extraction followed by centrifugation at 15,000×g for 20 min at 4°C. The samples were stored at -80℃ until used. Brain tissues were homogenized in the SDS sample buffer (125 mM Tris (pH 6.8), 4% SDS, 10% sucrose, 0.01% bromophenol blue, and 10% 2 mercaptoethanol) and boiled for 1 min. Protein concentration was quantified by using the Bradford method (Protein Assay Reagent Kit, Bio-Rad Laboratories). The samples (40 g) were separated by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, England) through a semidry-type blotting (Bio-Rad) method, blocked by 5% nonfat dry milk in PBS with Tween-20 (PBS-T) (137 mM NaCl, 8.10 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 0.1% Tween-20), and incubated with appropriate antibodies as described below. The filters were incubated with each primary antibody overnight at 4°C, with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hr at room temperature in 5% nonfat dry milk/PBS-T. Finally, the target molecules were visualized through an enhanced chemiluminescence western blotting detection system (Amersham Biosciences) on the X-ray film (Amersham Biosciences). The following primary and secondary antibodies used in this study were NF-B (rabbit, 1:2,000 Santa Cruz) and -actin (mouse, 1:10,000, Sigma), anti-rabbit IgG HRP antibody (1:5,000, Amersham Biosciences), and anti-mouse IgG HRP antibody (1:10,000, Amersham

Statistical analysis was performed with a two-way ANOVA followed by post hoc Tukey's multiple-comparison test. Neurological deficit scores were analyzed by the Kruskal-Wallis test followed by the Mann-Whitney *U* test. In all cases a *P*-value of <0.05 was regarded as

The control diabetic group had typical characteristics of type 1 diabetes such as a decrease in the body weight (296.6 ± 33.3 g) and hyperglycemia (535.4 ± 104.0 mg/dl) compared with the control non-diabetic group (340.0 ± 22.4 g, 119.8 ± 17.1 mg/dl), which were similar to previous reports (Iwata et al., 2008; 2010). Chronic treatment of MAK for 2 weeks showed a slight but significant decrease in blood glucose of the diabetic rats (404.4 ± 109.2 mg/dl,

**3.2 Infarct volume and neurological deficits after transient MCAO with reperfusion**  Figure 1 shows the effects of MAK on the brain infarction by MCAO/Re in the non-diabetic and diabetic groups. Representative coronal brain sections stained by TTC after MCAO/Re

*P*<0.01), whereas MAK had little effect on the body weight (266.6 ± 33.3 g).

Biosciences).

**3. Results** 

**2.10 Statistical analysis** 

statistically significant.

**3.1 Physiological characteristic parameter** 

showing viable (red) and dead (white) tissues are in (A). In the sham operated animals, there was no apparent damage in any brain region. The infarct area in the control nondiabetic rats after 24 hrs of reperfusion extended to the corpus striatum and cortex, whereas only a small striatal infarct was observed after 3 hrs of reperfusion. In the control diabetic group, brain injury induced by MCAO/Re was remarkably exacerbated. The cerebral infarct was produced within 3 hrs of reperfusion and the infarct region was extended to a large part of the left striatum and cortex in the diabetic rats. In contrast, the ischemic damage in the MAK-pretreated non-diabetic and diabetic animal brain was smaller as compared to those of the respective controls. Quantitative determination of infarct volume (B) indicated that the cerebral infarct volume was increased and associated with reperfusion time, which was markedly accelerated in the control diabetic group. The infarct volume evaluated at 3 hrs after reperfusion in the control diabetic group was significantly increased about 5-fold as compared with the non-diabetic group. Brain edema also tended to be exacerbated by diabetes, whereas no significant difference was detected between the non-diabetic and diabetic groups (data not shown). Consistent with the result of brain infarct volume, neurological deficits were exacerbated by reperfusion and diabetes (Fig. 2). MAK-pretreated diabetic rats showed significant alleviation in the neurological deficits compared to the control diabetic rats.

Fig. 1. Effects of MAK on infarction induced by MCAO/Re in non-diabetic (DM) and DM rat brains.

Representative coronal brain section photographs of the DM and non-DM rats stained by TTC at 3 or 24 hrs of reperfusion after 2 hrs MCAO (A). Infarct volume in ischemic hemispheres of the DM and non-DM groups after MCAO/Re by TTC staining (B).

Diabetes-Mediated Exacerbation of Neuronal Damage

**3.4 Apoptosis after transient MCAO with reperfusion** 

activation induced by MCAO/Re in these groups.

**3Re 24Re non-DM DM non-DM DM**

> **100 m**

Fig. 4. Effects of MAK on neuronal apoptosis induced by MCAO/Re in non-DM and DM rat

Representative photographs of apoptotic cells detected by TUNEL staining in the cortex coronal sections of non-DM and DM rats (A). Quantitative analysis of TUNEL-positive cells

**Apoptosis index (%)**

**(B)**

**b**

**3Re 24Re**

**a***P***<0.01 vs. non-DM**

**<sup>b</sup>***P***<0.01 vs. DM <sup>c</sup>***P***<0.05 vs. non-DM**

**b c**

**non-DM non-DM + MAK DM DM + MAK**

**(n=3)**

**a**

**MAK**

brains.

index in the cortex (B).

**Control**

**(A)**

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 223

Representative histological images of TUNEL staining and cleaved caspase-3 activity in the control non-diabetic, MAK-pretreated non-diabetic, control diabetic and MAKpretreated diabetic groups subjected to MCAO and 3 or 24 of hrs reperfusion are observed in Figs. 4 and 5. TUNEL staining of the ischemic penumbral region of the cortex was performed to determine nucleosomal DNA fragmentation accompanied by apoptotic cell death. In sham-operated non-diabetic and diabetic rats without MCAO/Re, no TUNEL positive cells were detected in the brain sections. TUNEL positive cells were increased in both the control non-diabetic and diabetic rats especially by MCAO and 3 hrs of reperfusion, which was remarkably suppressed by the pretreatment of MAK. The activation level of caspase-3, which directly activates DNase in the apoptotic final process was determined by immunostaining for cleaved caspase-3 in the ischemic penumbral cortex of non-diabetic and diabetic rats. Similar to the result of TUNEL staining, the number of cells expressing cleaved caspase-3, an activated form of this enzyme, was remarkably increased by MCAO/Re in the control diabetic group as compared with the control non-diabetic group. Pretreatment of MAK significantly inhibited the caspase-3

Fig. 2. Effects of MAK on neurological deficits induced by MCAO/Re in non-DM and DM rats.

Post-ischemic neurological deficits were evaluated at 3 or 24 hrs of reperfusion after 2 hrs of MCAO by a 5-point scale as described in the table.

#### **3.3 O2 - · generation after transient MCAO with reperfusion**

Intracellular O2⁻· generation in the ischemic penumbral region of the cortex induced by MCAO/Re was detected using the fluorescent probe DHE (Fig. 3). O2 - generation was increased by diabetes and MCAO/Re. No fluorescence of DHE was detected in the cortex of sham operated non-diabetic rats, whereas DHE fluorescence was present in the cortex of sham-operated diabetic rats. MCAO and subsequent following 24 hrs of reperfusion induced DHE fluorescence in the ischemic region of both the non-diabetic and diabetic cortex, which was remarkably augmented in the diabetic brain. Pretreatment of MAK reduced the O2⁻· generation in the cortex enhanced by MCAO/Re and diabetes.

Fig. 3. Effects of MAK on production of superoxide after MCAO/Re in non-DM and DM rat brains.

Representative photographs of superoxide production detected by DHE staining in the cortex coronal sections of the non-DM and DM rats (A). Quantitative analysis of DHE fluorescence intensity in the cortex (B).

### **3.4 Apoptosis after transient MCAO with reperfusion**

222 Advances in the Preclinical Study of Ischemic Stroke

Fig. 2. Effects of MAK on neurological deficits induced by MCAO/Re in non-DM and DM

Post-ischemic neurological deficits were evaluated at 3 or 24 hrs of reperfusion after 2 hrs of

Intracellular O2⁻· generation in the ischemic penumbral region of the cortex induced by MCAO/Re was detected using the fluorescent probe DHE (Fig. 3). O2- generation was increased by diabetes and MCAO/Re. No fluorescence of DHE was detected in the cortex of sham operated non-diabetic rats, whereas DHE fluorescence was present in the cortex of sham-operated diabetic rats. MCAO and subsequent following 24 hrs of reperfusion induced DHE fluorescence in the ischemic region of both the non-diabetic and diabetic cortex, which was remarkably augmented in the diabetic brain. Pretreatment of MAK

**grade 0 : no deficit Neurological evaluation**

**grade 5 : death**

**grade 1 : failure to extend right forepaw fully grade 2 : spontaneous circling or walking to contralateral side grade 3 : walking only when stimulated grade 4 : unresponsive to stimulation and a depressed level of consciousness**

**0**

rats.

**3.3 O2 -**

**Control**

**MAK**

brains.

**(A)**

**a**

**1**

**2**

**a**

**b**

**a**

**3Re 24Re**

*P***<0.05 vs. non-DM b***P***<0.05 vs. DM (n=8-12)**

**non-DM DM**

fluorescence intensity in the cortex (B).

**b**

MCAO by a 5-point scale as described in the table.

**sham 24Re**

**a**

**non-DM non-DM + MAK**

**· generation after transient MCAO with reperfusion** 

reduced the O2⁻· generation in the cortex enhanced by MCAO/Re and diabetes.

**non-DM DM**

**100 m**

Fig. 3. Effects of MAK on production of superoxide after MCAO/Re in non-DM and DM rat

Representative photographs of superoxide production detected by DHE staining in the cortex coronal sections of the non-DM and DM rats (A). Quantitative analysis of DHE

**0**

**1**

**2**

**Fluorescence of DHE relative to** 

**(B)**

**sham-operated non-DM**

**3**

**4**

**sham 24Re**

**b**

**a**

**b**

**non-DM non-DM + MAK DM DM + MAK**

**a**

**a**

**<sup>a</sup>***P***<0.01 vs. non-DM (n=3-4) <sup>b</sup>***P***<0.01 vs. DM**

**DM DM + MAK**

**3**

**Neurological score**

**4**

**5**

Representative histological images of TUNEL staining and cleaved caspase-3 activity in the control non-diabetic, MAK-pretreated non-diabetic, control diabetic and MAKpretreated diabetic groups subjected to MCAO and 3 or 24 of hrs reperfusion are observed in Figs. 4 and 5. TUNEL staining of the ischemic penumbral region of the cortex was performed to determine nucleosomal DNA fragmentation accompanied by apoptotic cell death. In sham-operated non-diabetic and diabetic rats without MCAO/Re, no TUNEL positive cells were detected in the brain sections. TUNEL positive cells were increased in both the control non-diabetic and diabetic rats especially by MCAO and 3 hrs of reperfusion, which was remarkably suppressed by the pretreatment of MAK. The activation level of caspase-3, which directly activates DNase in the apoptotic final process was determined by immunostaining for cleaved caspase-3 in the ischemic penumbral cortex of non-diabetic and diabetic rats. Similar to the result of TUNEL staining, the number of cells expressing cleaved caspase-3, an activated form of this enzyme, was remarkably increased by MCAO/Re in the control diabetic group as compared with the control non-diabetic group. Pretreatment of MAK significantly inhibited the caspase-3 activation induced by MCAO/Re in these groups.

Fig. 4. Effects of MAK on neuronal apoptosis induced by MCAO/Re in non-DM and DM rat brains.

Representative photographs of apoptotic cells detected by TUNEL staining in the cortex coronal sections of non-DM and DM rats (A). Quantitative analysis of TUNEL-positive cells index in the cortex (B).

Diabetes-Mediated Exacerbation of Neuronal Damage

**(A)**

**DM**

non-DM and DM rat brains.

**Control**

**MAK**

**Control**

**MAK**

**(B)**

**IL-/TO-PRO-3**

**0**

**0.2**

**0.4**

**0.6**

**0.8**

**1.0**

both these cytokines.

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 225

expression of TNF- mRNA as compared with the control non-diabetic rats. MCAO/Re increased the expression of TNF-mRNA, which was remarkably potentiated in the diabetic group. Immunohistochemistry for these cytokines confirmed an up-regulation in biosynthesis of IL-1 and TNF-by MCAO/Re and diabetes (Figs. 7 and 8). The expression of these pro-inflammatory cytokines was markedly accelerated in the ischemic diabetic rat cortex, whereas pretreatment of MAK significantly suppressed the augmented expression of

#2 #3 **non-DM**

<sup>+</sup> ++

**++** *<sup>P</sup>***<0.01 vs. non-DM**

**\*\*** *<sup>P</sup>***<0.01 vs. DM <sup>+</sup>** *<sup>P</sup>***<0.05 vs. non-DM \*** *P***<0.05 vs. DM**

Fig. 7. Effects of MAK on expression of IL-1 in the penumbral cortex after MCAO/Re in

Representative photographs of IL-1 immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A).

**\***

Quantitative analysis of IL-1 fluorescence intensity in the cortex (B).

**\*\***

++

**sham 3Re 24Re**

++

**non-DM + H2O non-DM + MAK DM + H2O DM + MAK**

**\***

**(n=3)**

**IL-1 TO-PRO-3Merge IL-1 Merge IL-1 Merge sham 3Re 24Re**

**TO-PRO-3 TO-PRO-3**

**100 m**

Fig. 5. Effects of MAK on cleaved caspase-3 after MCAO/Re in non-DM and DM rat brains. Representative photographs of cleaved caspase-3 staining in the cortex coronal sections of non-DM and DM rats (A). Quantitative analysis of cleaved caspase-3 positive cells fluorescence intensity in the cortex (B).

### **3.5 Expression of IL1- and TNF- in the cortex**

Figure 6 shows the effects of MAK on the expression of mRNA of IL-1 (A) and TNF- (B) in the non-diabetic and diabetic rat penumbral cortex after MCAO/Re. The control diabetic group had a 5.6-fold increase in the level of basal expression of IL-1 mRNA as compared with the control non-diabetic group. MCAO/Re increased the expression level of IL-1 mRNA in the ischemic cortex of both groups, which was remarkably accelerated and augmented by diabetes. The expression level of IL-1 mRNA in the control diabetic group reached maximum in an early period of reperfusion and at 3 hrs of reperfusion was about 10-fold higher than that of the control non-diabetic rats, which was significantly suppressed by pretreatment of MAK. Also, the control diabetic group had an increased level of the basal

**100 m**

non-DM and DM rats (A). Quantitative analysis of cleaved caspase-3 positive cells

Fig. 5. Effects of MAK on cleaved caspase-3 after MCAO/Re in non-DM and DM rat brains. Representative photographs of cleaved caspase-3 staining in the cortex coronal sections of

Figure 6 shows the effects of MAK on the expression of mRNA of IL-1 (A) and TNF- (B) in the non-diabetic and diabetic rat penumbral cortex after MCAO/Re. The control diabetic group had a 5.6-fold increase in the level of basal expression of IL-1 mRNA as compared with the control non-diabetic group. MCAO/Re increased the expression level of IL-1 mRNA in the ischemic cortex of both groups, which was remarkably accelerated and augmented by diabetes. The expression level of IL-1 mRNA in the control diabetic group reached maximum in an early period of reperfusion and at 3 hrs of reperfusion was about 10-fold higher than that of the control non-diabetic rats, which was significantly suppressed by pretreatment of MAK. Also, the control diabetic group had an increased level of the basal

**0**

**+ \***

+

*P***<0.05 vs. non-DM** *P***<0.05 vs. DM (n=3-4)**

**\***

**sham 3Re 24Re**

+

**non-DM non-DM + MAK**

**DM DM + MAK**

**4**

**TNF-**

Fig. 6. Effects of MAK on expression of pro-inflammatory cytokines mRNA in the

The expression levels of IL-1 (A) and TNF- mRNA (B) in the non-DM and DM rat penumbral cortex after MCAO/Re were determined by real-time PCR analysis.

**mRNA relative to** 

**(B)**

**non-DM sham cortex** 

**8**

**12**

**16**

**20**

**0**

**20**

**Cleaved Caspase-3-**

**(B)**

**Positive Cells**

 **(%)**

**40**

**60**

++

+

**\*\***

**3Re 24Re**

**++***P***<0.01 vs. non-DM**

**\*\****P***<0.01 vs. DM +***P***<0.05 vs. non-DM**

**80**

++

++

**\*\***

**non-DM non-DM + MAK DM DM + MAK**

**(n=3)**

**100**

**3Re 24Re non-DM DM non-DM DM**

fluorescence intensity in the cortex (B).

+

**<sup>80</sup> sham 3Re 24Re**

**\***

**<sup>+</sup>***P***<0.01 vs. non-DM \****P***<0.01 vs. DM (n=3-4)**

+

**non-DM non-DM + MAK**

**DM DM + MAK**

penumbral cortex after MCAO/Re in non-DM and DM rat brains.

+

**3.5 Expression of IL1- and TNF- in the cortex** 

**MAK**

**IL-1**

**0**

**20**

**40**

**60**

**mRNA relative to non-DM sham cortex** 

**(A)**

**(A)**

**Control**

expression of TNF- mRNA as compared with the control non-diabetic rats. MCAO/Re increased the expression of TNF-mRNA, which was remarkably potentiated in the diabetic group. Immunohistochemistry for these cytokines confirmed an up-regulation in biosynthesis of IL-1 and TNF-by MCAO/Re and diabetes (Figs. 7 and 8). The expression of these pro-inflammatory cytokines was markedly accelerated in the ischemic diabetic rat cortex, whereas pretreatment of MAK significantly suppressed the augmented expression of both these cytokines.

Fig. 7. Effects of MAK on expression of IL-1 in the penumbral cortex after MCAO/Re in non-DM and DM rat brains.

Representative photographs of IL-1 immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A). Quantitative analysis of IL-1 fluorescence intensity in the cortex (B).

Diabetes-Mediated Exacerbation of Neuronal Damage

**3.6 Activation of NF-B in the cortex** 

and DM rat cortex (C: control, M: MAK).

pretreatment of MAK (Fig. 14).

**3.7 Expression of inflammatory mediators in the cortex** 

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 227

The expression of pro-inflammatory cytokines and inflammatory mediators such as COX-2, iNOS and ICAM-1 in the cells is controlled by the transcription factor NF-B. Thus, the activation level of NF-B in the cortex tissue was estimated by western blotting (Fig. 9). Translocation of NF-B from cytosol to nucleus was not detected in the cortex tissue of sham-operated non-diabetic rats. In the control non-diabetic rats submitted to MCAO/Re, there was a significant increase in NF-B translocation. In contrast, the translocation of NF-B was observed even in the sham-operated diabetic rat cortex and was significantly enhanced by MCAO and subsequent reperfusion. Pretreatment of MAK markedly inhibited

Fig. 9. Effects of MAK on NF-B expression after MCAO/Re in non-DM and DM rat brains. Representative western blot of NF-B in cytoplasm and nucleus extracts from the non-DM

Figure 10 shows the effects of MAK on MCAO/Re-induced gene expression of COX-2, iNOS and ICAM-1 in the non-diabetic and diabetic rat cortex. After MCAO/Re, no distinct increase in the expression level of COX-2, iNOS and ICAM-1 mRNA was observed in the control non-diabetic group, whereas the gene expression of these inflammatory mediators was strongly enhanced in the control diabetic group. Immunohistochemistry revealed that the control diabetic rats had an increased basal expression level of COX-2 and iNOS as compared with the control non-diabetic rats (Figs. 11 and 12). The expression of ICAM-1 in endothelial cells, which indicates increased extravasation of neutrophils and macrophages/microglia into brain parenchyma, was determined using double immunostaining for ICAM-1 and endothelial cell antibody (RECA-1) (Fig. 13). The cortex of sham-operated diabetic rats had a significant upregulated level of ICAM-1 in endothelial cells as compared with the sham-operated nondiabetic rats, which had no ICAM-1-like immunoreactive cells. MCAO/Re increased the expression of ICAM-1 both in the non-diabetic and diabetic groups, which was remarkably enhanced in the diabetic rats during 24 hrs of reperfusion. Pretreatment of MAK significantly reduced the expression of ICAM-1 both in the non-diabetic and diabetic rat cortex. Similarly, the MPO activity in the cortex was progressively increased during reperfusion, which was enhanced by diabetes and was significantly suppressed by

the activation of NF-B both in the non-diabetic and diabetic groups.

Fig. 8. Effects of MAK on expression of TNF- in the penumbral cortex after MCAO/Re in non-DM and DM rat brains.

Representative photographs of TNF- immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A). Quantitative analysis of TNF- fluorescence intensity in the cortex (B).

### **3.6 Activation of NF-B in the cortex**

226 Advances in the Preclinical Study of Ischemic Stroke

**TNF- Merge TNF- Merge TNF- Merge sham 3Re 24Re**

**TO-PRO-3 TO-PRO-3 TO-PRO-3**

++

\*

**non-DM non-DM + MAK**

**DM DM + MAK**

**MAK**

**DM**

**non-DM**

**Control**

**(A)**

**MAK**

**(B)**

**TNF-**

non-DM and DM rat brains.

**0**

**0.2**

**0.4**

**0.6**

**0.8**

**1.0**

+

Quantitative analysis of TNF- fluorescence intensity in the cortex (B).

**/TO-PRO-3**

++

**sham 3Re 24Re**

\*\*

**++** *<sup>P</sup>***<0.01 vs. non-DM**

**\*\*** *<sup>P</sup>***<0.01 vs. DM <sup>+</sup>** *<sup>P</sup>***<0.05 vs. non-DM \*** *P***<0.05 vs. DM**

++

**(n=3)**

Fig. 8. Effects of MAK on expression of TNF- in the penumbral cortex after MCAO/Re in

Representative photographs of TNF- immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A).

**Control**

#3

**100 m** The expression of pro-inflammatory cytokines and inflammatory mediators such as COX-2, iNOS and ICAM-1 in the cells is controlled by the transcription factor NF-B. Thus, the activation level of NF-B in the cortex tissue was estimated by western blotting (Fig. 9). Translocation of NF-B from cytosol to nucleus was not detected in the cortex tissue of sham-operated non-diabetic rats. In the control non-diabetic rats submitted to MCAO/Re, there was a significant increase in NF-B translocation. In contrast, the translocation of NF-B was observed even in the sham-operated diabetic rat cortex and was significantly enhanced by MCAO and subsequent reperfusion. Pretreatment of MAK markedly inhibited the activation of NF-B both in the non-diabetic and diabetic groups.

Fig. 9. Effects of MAK on NF-B expression after MCAO/Re in non-DM and DM rat brains. Representative western blot of NF-B in cytoplasm and nucleus extracts from the non-DM and DM rat cortex (C: control, M: MAK).

### **3.7 Expression of inflammatory mediators in the cortex**

Figure 10 shows the effects of MAK on MCAO/Re-induced gene expression of COX-2, iNOS and ICAM-1 in the non-diabetic and diabetic rat cortex. After MCAO/Re, no distinct increase in the expression level of COX-2, iNOS and ICAM-1 mRNA was observed in the control non-diabetic group, whereas the gene expression of these inflammatory mediators was strongly enhanced in the control diabetic group. Immunohistochemistry revealed that the control diabetic rats had an increased basal expression level of COX-2 and iNOS as compared with the control non-diabetic rats (Figs. 11 and 12). The expression of ICAM-1 in endothelial cells, which indicates increased extravasation of neutrophils and macrophages/microglia into brain parenchyma, was determined using double immunostaining for ICAM-1 and endothelial cell antibody (RECA-1) (Fig. 13). The cortex of sham-operated diabetic rats had a significant upregulated level of ICAM-1 in endothelial cells as compared with the sham-operated nondiabetic rats, which had no ICAM-1-like immunoreactive cells. MCAO/Re increased the expression of ICAM-1 both in the non-diabetic and diabetic groups, which was remarkably enhanced in the diabetic rats during 24 hrs of reperfusion. Pretreatment of MAK significantly reduced the expression of ICAM-1 both in the non-diabetic and diabetic rat cortex. Similarly, the MPO activity in the cortex was progressively increased during reperfusion, which was enhanced by diabetes and was significantly suppressed by pretreatment of MAK (Fig. 14).

Diabetes-Mediated Exacerbation of Neuronal Damage

**(A)**

**MAK**

**DM**

**non-DM**

**Control**

**MAK**

**COX-2/ TOPRO3**

non-DM and DM rat brains.

**0**

**0.2**

**0.4**

+

**0.6 (B) sham 3Re 24Re**

++

**\***

**++** *<sup>P</sup>***<0.01 vs. non-DM**

**\*\*** *<sup>P</sup>***<0.01 vs. DM <sup>+</sup>** *<sup>P</sup>***<0.05 vs. non-DM \*** *P***<0.05 vs. DM**

Fig. 11. Effects of MAK on expression of COX-2 in the penumbral cortex after MCAO/Re in

Representative photographs of COX-2 immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A).

+

**(n=3)**

**\*\***

+

**non-DM non-DM + MAK**

**DM DM + MAK**

**\***

Quantitative analysis of COX-2 fluorescence intensity in the cortex (B).

**Control**

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 229

**COX-2 Merge COX-2 Merge COX-2 Merge sham 3Re 24Re**

**TO-PRO-3 TO-PRO-3 TO-PRO-3**

#3

**100 m**

Fig. 10. Effects of MAK on expression of mRNA of inflammatory mediators in the penumbral cortex after MCAO/Re in non-DM and DM rat brains. Expression levels of COX-2 (A), iNOS (B) and ICAM-1 mRNA (C) were determined by realtime PCR analysis in the non-DM and DM rat penumbral cortex after MCAO/Re.

Fig. 10. Effects of MAK on expression of mRNA of inflammatory mediators in the

time PCR analysis in the non-DM and DM rat penumbral cortex after MCAO/Re.

Expression levels of COX-2 (A), iNOS (B) and ICAM-1 mRNA (C) were determined by real-

penumbral cortex after MCAO/Re in non-DM and DM rat brains.

**100 m**

Fig. 11. Effects of MAK on expression of COX-2 in the penumbral cortex after MCAO/Re in non-DM and DM rat brains.

Representative photographs of COX-2 immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A). Quantitative analysis of COX-2 fluorescence intensity in the cortex (B).

Diabetes-Mediated Exacerbation of Neuronal Damage

**non-DM**

**Control**

**(A)**

**MAK**

**MAK**

**(B)**

**ICAM-1/RECA-1**

in non-DM and DM rat brains.

**0** 

analysis of ICAM-1 fluorescence intensity in the cortex (B).

+

**\***

**<sup>+</sup>***P***<0.01 vs. non-DM \****P***<0.01 vs. DM (n=3)**

Fig. 13. Effects of MAK on expression of ICAM-1 in the penumbral cortex after MCAO/Re

Representative photographs of ICAM-1 immunostaining (red fluorescence) and RECA-1 (green fluorescence) in the cortex coronal sections of non-DM and DM rats. (A). Quantitative

**sham 3Re 24Re**

**0.4** 

**0.8** 

**1.2** 

**1.6** 

**Control**

**DM**

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 231

**ICAM-1 RECA-1 Merge ICAM-1 RECA-1 Merge ICAM-1 RECA-1 Merge**

+

**non-DM non-DM + MAK**

**DM DM + MAK**

**\***

**sham 3Re 24Re**

**250 m**

Fig. 12. Effects of MAK on expression of iNOS in the penumbral cortex after MCAO/Re in non-DM and DM rat brains.

Representative photographs of iNOS immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A). Quantitative analysis of iNOS fluorescence intensity in the cortex (B).

**iNOS Merge iNOS Merge iNOS Merge** #3 **sham 3Re 24Re**

> **100 m**

**TO-PRO-3 TO-PRO-3 TO-PRO-3**

+

**(n=3)**

Fig. 12. Effects of MAK on expression of iNOS in the penumbral cortex after MCAO/Re in

Representative photographs of iNOS immunostaining (red fluorescence) and nuclei by TO-PRO-3 (blue fluorescence) in the cortex coronal sections of non-DM and DM rats. (A).

**\***

**non-DM non-DM + MAK**

**DM DM + MAK**

++

++

**++** *<sup>P</sup>***<0.01 vs. non-DM**

**\*\*** *<sup>P</sup>***<0.01 vs. DM <sup>+</sup>** *<sup>P</sup>***<0.05 vs. non-DM \*** *P***<0.05 vs. DM**

**sham 3Re 24Re**

**\*\***

**(A)**

**MAK**

**DM**

**non-DM**

**Control**

**MAK**

**iNOS/TO-PRO-3**

non-DM and DM rat brains.

**0.5 (B)**

++

Quantitative analysis of iNOS fluorescence intensity in the cortex (B).

**0**

**0.1**

**0.2**

**0.3**

**0.4**

**Control**

Fig. 13. Effects of MAK on expression of ICAM-1 in the penumbral cortex after MCAO/Re in non-DM and DM rat brains.

**<sup>+</sup>***P***<0.01 vs. non-DM \****P***<0.01 vs. DM (n=3)**

Representative photographs of ICAM-1 immunostaining (red fluorescence) and RECA-1 (green fluorescence) in the cortex coronal sections of non-DM and DM rats. (A). Quantitative analysis of ICAM-1 fluorescence intensity in the cortex (B).

Diabetes-Mediated Exacerbation of Neuronal Damage

**4. Discussion** 

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 233

The present study shows that the STZ-induced diabetic state enhanced brain infarction and neurological dysfunction caused by transient focal ischemia and subsequent reperfusion in rats. Diabetes spontaneously enhanced ROS generation and expression of pro-inflammatory cytokines and inflammatory mediators via NF-B activation in the brain, which is accelerated after cerebral ischemia and subsequent reperfusion leading to neuronal apoptosis and inflammatory neurodegeneration. Chronic oral treatment of MAK alleviated the exacerbation of cerebral injury and neurological deficits in the diabetic state, which

ROS-induced oxidative stress is considered to be involved in the pathogenesis of transient cerebral ischemic injury (Fiskum et al., 2004; Saito et al., 2005; Niizuma et al., 2009). In particular, reperfusion after a long period of vessel occlusion triggers explosive generation of ROS, which causes cell death by peroxidative damage of lipids, proteins and nucleic acids (Warner et al., 2004; Anabela et al., 2006). In addition to the early necrotic cell death in the ischemic core region, ROS triggers apoptosis, a delayed death of cells, in the ischemic penumbra (Nakka et al., 2008; Niizuma et al., 2010). Furthermore, ROS induces rupture of blood brain barrier through transcriptional activation of matrix metalloproteinase and pro-inflammatory cytokines resulting in the extension of cerebral infarction and exacerbation of brain edema (Cunningham et al., 2005; Zhao et al., 2006). In this study, we observed that exacerbation of damage in the brain of diabetic rats was accelerated in a time-dependent manner during the reperfusion phase. MCAO and 3 hrs of reperfusion in the control non-diabetic rats showed little infarction in the brain and moderate neurological deficits, whereas the diabetic rats subjected to MCAO and 3 hrs of reperfusion had a large infarction that was similar in size to that in the non-diabetic rats after 24 hrs of reperfusion and severe neurological deficits. Additionally, a histochemical study revealed that generation of O2⁻· and the occurrence of apoptosis in the ischemic penumbra were markedly increased in the brain of diabetic rats. A large amount of ROS locally generated by cerebral ischemia/reperfusion induces free radical chain reactions (Saito et al., 2005), which may be enhanced by increased oxidative stress in the diabetic state. Evidence is being accumulated that oxidative stress is enhanced by hyperglycemia in the diabetic state (Kusaka et al., 2004; Rizk et al., 2005; Tsuruta et al., 2010). In the diabetic state, "glucose toxicity" caused by augmentation of intracellular glucose oxidation process and nonenzymatic glycation of protein molecules leads to over production of ROS and damage of neurons and endothelial cells (Baynes 1991). Previous studies have demonstrated that the hyperglycemic condition in cerebral ischemia without diabetes exacerbates brain injury due to enhanced production of ROS in the brain (Anabela et al., 2006; Tsuruta et al., 2010). Augmented oxidative stress involving increased ROS generation, augmented lipid peroxidation and reduction of antioxidants has been indicated in the brain, kidney, pancreas and liver of STZ-induced diabetic rats (Muralikrishna Adibhatla et al., 2006). Actually, the occurrence of apoptosis in the ischemic penumbral region has been shown to be enhanced by diabetes and correlate with serum glucose (Li et al., 1999). Antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), which scavenge ROS, are considered to contribute to the neuronal protection against ischemia/reperfusion (Warner et al.,

could be attributed to its antioxidant activity and anti-inflammatory effects.

Fig. 14. Effects of MAK on MPO expression after MCAO/Re in non-DM and DM rat brains. Representative photographs of leukocytic infiltrate by MPO staining in the cortex coronal sections of non-DM and DM rats (A). Quantitative analysis of MPO fluorescence intensity in the cortex (B).

### **4. Discussion**

232 Advances in the Preclinical Study of Ischemic Stroke

**3Re non-DM DM**

**\*\***

**(n=3)**

**non-DM + H2O non-DM + MAK DM + H2O DM + MAK**

++

**sham 3Re 24Re**

**++***<sup>p</sup>* **< 0.01 vs. non-DM + H2O group.**

Fig. 14. Effects of MAK on MPO expression after MCAO/Re in non-DM and DM rat brains. Representative photographs of leukocytic infiltrate by MPO staining in the cortex coronal sections of non-DM and DM rats (A). Quantitative analysis of MPO fluorescence intensity in

**\*\****p* **< 0.01 vs. DM + H2O group. +***p* **< 0.05 vs. non-DM + H2O group.**

**sham non-DM DM**

**MAK**

**fluores c enc e intens**

the cortex (B).

**0**

+

**2**

**4**

**6**

**8**

**10**

 **ity**

**(B)**

**Control**

**(A)**

**24Re non-DM DM**

> **100 m**

The present study shows that the STZ-induced diabetic state enhanced brain infarction and neurological dysfunction caused by transient focal ischemia and subsequent reperfusion in rats. Diabetes spontaneously enhanced ROS generation and expression of pro-inflammatory cytokines and inflammatory mediators via NF-B activation in the brain, which is accelerated after cerebral ischemia and subsequent reperfusion leading to neuronal apoptosis and inflammatory neurodegeneration. Chronic oral treatment of MAK alleviated the exacerbation of cerebral injury and neurological deficits in the diabetic state, which could be attributed to its antioxidant activity and anti-inflammatory effects.

ROS-induced oxidative stress is considered to be involved in the pathogenesis of transient cerebral ischemic injury (Fiskum et al., 2004; Saito et al., 2005; Niizuma et al., 2009). In particular, reperfusion after a long period of vessel occlusion triggers explosive generation of ROS, which causes cell death by peroxidative damage of lipids, proteins and nucleic acids (Warner et al., 2004; Anabela et al., 2006). In addition to the early necrotic cell death in the ischemic core region, ROS triggers apoptosis, a delayed death of cells, in the ischemic penumbra (Nakka et al., 2008; Niizuma et al., 2010). Furthermore, ROS induces rupture of blood brain barrier through transcriptional activation of matrix metalloproteinase and pro-inflammatory cytokines resulting in the extension of cerebral infarction and exacerbation of brain edema (Cunningham et al., 2005; Zhao et al., 2006). In this study, we observed that exacerbation of damage in the brain of diabetic rats was accelerated in a time-dependent manner during the reperfusion phase. MCAO and 3 hrs of reperfusion in the control non-diabetic rats showed little infarction in the brain and moderate neurological deficits, whereas the diabetic rats subjected to MCAO and 3 hrs of reperfusion had a large infarction that was similar in size to that in the non-diabetic rats after 24 hrs of reperfusion and severe neurological deficits. Additionally, a histochemical study revealed that generation of O2⁻· and the occurrence of apoptosis in the ischemic penumbra were markedly increased in the brain of diabetic rats. A large amount of ROS locally generated by cerebral ischemia/reperfusion induces free radical chain reactions (Saito et al., 2005), which may be enhanced by increased oxidative stress in the diabetic state. Evidence is being accumulated that oxidative stress is enhanced by hyperglycemia in the diabetic state (Kusaka et al., 2004; Rizk et al., 2005; Tsuruta et al., 2010). In the diabetic state, "glucose toxicity" caused by augmentation of intracellular glucose oxidation process and nonenzymatic glycation of protein molecules leads to over production of ROS and damage of neurons and endothelial cells (Baynes 1991). Previous studies have demonstrated that the hyperglycemic condition in cerebral ischemia without diabetes exacerbates brain injury due to enhanced production of ROS in the brain (Anabela et al., 2006; Tsuruta et al., 2010). Augmented oxidative stress involving increased ROS generation, augmented lipid peroxidation and reduction of antioxidants has been indicated in the brain, kidney, pancreas and liver of STZ-induced diabetic rats (Muralikrishna Adibhatla et al., 2006). Actually, the occurrence of apoptosis in the ischemic penumbral region has been shown to be enhanced by diabetes and correlate with serum glucose (Li et al., 1999). Antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), which scavenge ROS, are considered to contribute to the neuronal protection against ischemia/reperfusion (Warner et al.,

Diabetes-Mediated Exacerbation of Neuronal Damage

further investigated.

stress by its anti-oxidant effects.

**6. Acknowledgements** 

**5. Conclusion** 

and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water–Soluble Extract... 235

animals. However, the mechanistic basis and active ingredients responsible for its pharmacological effects have not been well defined. Previously, we showed that MAK inhibits the generation of O2⁻· and lipid peroxidation in a concentration dependent manner *in vitro* (Okazaki et al., 2008). Furthermore, oral administration of MAK to STZ-induced diabetic animals significantly reduced the blood glucose level and lipid peroxidation, and suppressed impairment of SOD, CAT and GPx in the brain (Iwata et al., 2008), liver and kidney (Okazaki et al., 2008). Collectively, MAK can act as an antioxidant *in vivo* and shows anti-diabetic effects by relieving diabetic-induced oxidative stress. We observed that the oral pretreatment of MAK with diabetic rats decreased the cerebral O2⁻· generation, apoptosis and subsequent inflammatory responses induced by MCAO/Re, which could be as a result of improved antioxidant status in the diabetic state. MAK had a slight effect on the elevated blood glucose level in the diabetic rats, confirming that the cerebroprotective effect of MAK could be due to its antioxidant activity. Recently, polysaccharides (Lin et al., 2010) and triterpenes (Dudhgaonkar et al., 2009), which are two major active constituents of *G. lucidum*, have been reported to suppress lipopolysaccharide-induced expression of inflammatory mediators via down-regulation of MAP kinase signaling cascade and NF-B activity both *in vitro* and *in vivo*, whereas the mechanism of their anti-inflammatory effects remains unclear. As MAK is assumed to contain similar polysaccharides and triterpenes, it might inhibit inflammatory responses directly via its immunomodulatory effects. There are a number of factors that may explain the severe symptom of brain ischemia in the diabetes. For example, the ischemic cerebral injury in diabetic state may be aggravated by acidosis, activation of aldose reductase and NAD(P)H oxidase, enhanced production of advanced glycation end-products, protein kinase C activation induced by excessive Ca2+ influx, etc. The effects of MAK on these factors and identification of its active ingredients need to be

In this study, we demonstrated that the STZ-induced diabetic state markedly aggravated MCAO/reperfusion-induced neurological deficits, infarction and apoptosis in the rat brain. Furthermore, we elucidated that the levels of O2⁻· generation and pro-inflammatory cytokines (IL-1 and TNF-) and inflammatory mediators (COX-2 and iNOS) expression via NF-B activation were up-regulated in the diabetic cortex, which were remarkably enhanced during reperfusion after ischemia. Post-ischemic activation of neutrophil and macrophage/microglia and extravasation estimated by ICAM-1 and MPO expression were also enhanced by diabetes. Chronic pretreatment of MAK protected the diabetic rats against the exacerbation in cerebral ischemic injury and inflammatory responses. These results suggest that daily intake of MAK relieves the exacerbation of cerebral ischemic injury in the diabetic state, which may be mainly attributed to the improvement of augmented oxidative

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society

for the Promotion of Science (No. 19590700, 22590663 and 23790750).

2004). Indeed, overexpression of SOD in transgenic mice has been shown to have a reduced infarction volume and edema after transient ischemia and reperfusion (Tsubokawa et al., 2007). Impairment of these antioxidant enzymes caused by nonenzymatic glycation is responsible for the increase of lipid peroxidation in the brain of diabetic animals. In a previous study, we also observed that the level of lipid peroxidation marker TBARS (thiobarbituric acid reactive substances) was elevated and activity of SOD, CAT and GPx was decreased in STZ-induced diabetic rat brain, which were reversed by chronic treatment of ascorbic acid (Iwata et al., 2010). Thus, the enhanced oxidative stress in diabetes is considered to cause functional impairment of antioxidant enzymes and the resulting diminution in antioxidative defense leads to further enhancement of radical reactions and other dysfunctions in the brain.

Inflammatory mechanisms that are activated after cerebral ischemia play an important role in the pathogenesis of brain injury (Caso et al., 2007; Ridder et al., 2009). The transcription factor NF-B assumes a key role in many biological processes including cellular stress responses and regulates apoptosis and inflammation (Ridder et al., 2009). Activation of NF-B is crucial for the inflammatory responses leading to gene expression of pro-inflammatory cytokines and mediators in immunocytes (Hu et al., 2005; Ridder et al., 2009). In this study, we demonstrated that the diabetic rats had an increased basal level of the gene expression of pro-inflammatory cytokines IL-1 and TNF-, and inflammatory mediators COX-2 and iNOS as compared to that of non-diabetic rats. MCAO/Re increased the gene expression of these cytokines and enzymes, which was remarkably accelerated and augmented by diabetes. The result from immunoblot analysis of NF-B in the diabetic rat cortex confirmed that the increased production of these cytokines and enzymes was mediated by the enhanced activation of NF-B. Furthermore, we observed that the post-ischemic induction of MPO and ICAM-1, which are hallmarks of neutrophil and macrophage/microglia activation and extravasation, was significantly increased in the diabetic rat brain indicating exaggeration of inflammatory responses in ischemic injury. Our present data are in agreement with other studies describing the exacerbated inflammation in diabetic model animals (Li et al., 2004; Tsuruta et al. 2010; Tureyen et al., 2011). For example, a model of type-2 diabetes *db/db* mouse that has a point mutation of the leptin receptor showed increased brain damage and higher expression levels of IL-1, IL-6, ICAM-1, MPO and other inflammatory markers as compared with the *db/+* control after MCAO/Re (Tureyen et al., 2011). A recent study using an electrochemical O2⁻· sensor has shown that experimental transient hyperglycemia induced by intravenous infusion of glucose increased local O2⁻· generation and exacerbated brain injury after ischemia and subsequent reperfusion in the rats (Tsuruta et al., 2010). Interestingly, high-mobility group box-1 and ICAM-1 in brain and plasma, which are induced in early inflammation and enhance inflammatory responses, were correlated with total O2⁻· generation during ischemia and reperfusion. Nevertheless, the hyperglycemia-induced overproduction of ROS may be mainly attributed to exacerbated inflammatory responses and cerebral damage after ischemia and reperfusion in diabetes. MAK, a nutritional supplement, is a water-soluble extract from a solid culture medium

composed of bagasse and defatted-rice bran overgrown with *G. lucidum* mycelia. MAK is used as a health supplement and revitalizer, and a number of studies have demonstrated its anti-tumor (Kubo et al., 2005) and immunomodulating activities (Nakagawa et al., 1999) in animals. However, the mechanistic basis and active ingredients responsible for its pharmacological effects have not been well defined. Previously, we showed that MAK inhibits the generation of O2⁻· and lipid peroxidation in a concentration dependent manner *in vitro* (Okazaki et al., 2008). Furthermore, oral administration of MAK to STZ-induced diabetic animals significantly reduced the blood glucose level and lipid peroxidation, and suppressed impairment of SOD, CAT and GPx in the brain (Iwata et al., 2008), liver and kidney (Okazaki et al., 2008). Collectively, MAK can act as an antioxidant *in vivo* and shows anti-diabetic effects by relieving diabetic-induced oxidative stress. We observed that the oral pretreatment of MAK with diabetic rats decreased the cerebral O2⁻· generation, apoptosis and subsequent inflammatory responses induced by MCAO/Re, which could be as a result of improved antioxidant status in the diabetic state. MAK had a slight effect on the elevated blood glucose level in the diabetic rats, confirming that the cerebroprotective effect of MAK could be due to its antioxidant activity. Recently, polysaccharides (Lin et al., 2010) and triterpenes (Dudhgaonkar et al., 2009), which are two major active constituents of *G. lucidum*, have been reported to suppress lipopolysaccharide-induced expression of inflammatory mediators via down-regulation of MAP kinase signaling cascade and NF-B activity both *in vitro* and *in vivo*, whereas the mechanism of their anti-inflammatory effects remains unclear. As MAK is assumed to contain similar polysaccharides and triterpenes, it might inhibit inflammatory responses directly via its immunomodulatory effects. There are a number of factors that may explain the severe symptom of brain ischemia in the diabetes. For example, the ischemic cerebral injury in diabetic state may be aggravated by acidosis, activation of aldose reductase and NAD(P)H oxidase, enhanced production of advanced glycation end-products, protein kinase C activation induced by excessive Ca2+ influx, etc. The effects of MAK on these factors and identification of its active ingredients need to be further investigated.

### **5. Conclusion**

234 Advances in the Preclinical Study of Ischemic Stroke

2004). Indeed, overexpression of SOD in transgenic mice has been shown to have a reduced infarction volume and edema after transient ischemia and reperfusion (Tsubokawa et al., 2007). Impairment of these antioxidant enzymes caused by nonenzymatic glycation is responsible for the increase of lipid peroxidation in the brain of diabetic animals. In a previous study, we also observed that the level of lipid peroxidation marker TBARS (thiobarbituric acid reactive substances) was elevated and activity of SOD, CAT and GPx was decreased in STZ-induced diabetic rat brain, which were reversed by chronic treatment of ascorbic acid (Iwata et al., 2010). Thus, the enhanced oxidative stress in diabetes is considered to cause functional impairment of antioxidant enzymes and the resulting diminution in antioxidative defense leads to further enhancement of radical

Inflammatory mechanisms that are activated after cerebral ischemia play an important role in the pathogenesis of brain injury (Caso et al., 2007; Ridder et al., 2009). The transcription factor NF-B assumes a key role in many biological processes including cellular stress responses and regulates apoptosis and inflammation (Ridder et al., 2009). Activation of NF-B is crucial for the inflammatory responses leading to gene expression of pro-inflammatory cytokines and mediators in immunocytes (Hu et al., 2005; Ridder et al., 2009). In this study, we demonstrated that the diabetic rats had an increased basal level of the gene expression of pro-inflammatory cytokines IL-1 and TNF-, and inflammatory mediators COX-2 and iNOS as compared to that of non-diabetic rats. MCAO/Re increased the gene expression of these cytokines and enzymes, which was remarkably accelerated and augmented by diabetes. The result from immunoblot analysis of NF-B in the diabetic rat cortex confirmed that the increased production of these cytokines and enzymes was mediated by the enhanced activation of NF-B. Furthermore, we observed that the post-ischemic induction of MPO and ICAM-1, which are hallmarks of neutrophil and macrophage/microglia activation and extravasation, was significantly increased in the diabetic rat brain indicating exaggeration of inflammatory responses in ischemic injury. Our present data are in agreement with other studies describing the exacerbated inflammation in diabetic model animals (Li et al., 2004; Tsuruta et al. 2010; Tureyen et al., 2011). For example, a model of type-2 diabetes *db/db* mouse that has a point mutation of the leptin receptor showed increased brain damage and higher expression levels of IL-1, IL-6, ICAM-1, MPO and other inflammatory markers as compared with the *db/+* control after MCAO/Re (Tureyen et al., 2011). A recent study using an electrochemical O2⁻· sensor has shown that experimental transient hyperglycemia induced by intravenous infusion of glucose increased local O2⁻· generation and exacerbated brain injury after ischemia and subsequent reperfusion in the rats (Tsuruta et al., 2010). Interestingly, high-mobility group box-1 and ICAM-1 in brain and plasma, which are induced in early inflammation and enhance inflammatory responses, were correlated with total O2⁻· generation during ischemia and reperfusion. Nevertheless, the hyperglycemia-induced overproduction of ROS may be mainly attributed to exacerbated inflammatory responses and cerebral damage after ischemia and reperfusion in diabetes. MAK, a nutritional supplement, is a water-soluble extract from a solid culture medium composed of bagasse and defatted-rice bran overgrown with *G. lucidum* mycelia. MAK is used as a health supplement and revitalizer, and a number of studies have demonstrated its anti-tumor (Kubo et al., 2005) and immunomodulating activities (Nakagawa et al., 1999) in

reactions and other dysfunctions in the brain.

In this study, we demonstrated that the STZ-induced diabetic state markedly aggravated MCAO/reperfusion-induced neurological deficits, infarction and apoptosis in the rat brain. Furthermore, we elucidated that the levels of O2⁻· generation and pro-inflammatory cytokines (IL-1 and TNF-) and inflammatory mediators (COX-2 and iNOS) expression via NF-B activation were up-regulated in the diabetic cortex, which were remarkably enhanced during reperfusion after ischemia. Post-ischemic activation of neutrophil and macrophage/microglia and extravasation estimated by ICAM-1 and MPO expression were also enhanced by diabetes. Chronic pretreatment of MAK protected the diabetic rats against the exacerbation in cerebral ischemic injury and inflammatory responses. These results suggest that daily intake of MAK relieves the exacerbation of cerebral ischemic injury in the diabetic state, which may be mainly attributed to the improvement of augmented oxidative stress by its anti-oxidant effects.

### **6. Acknowledgements**

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 19590700, 22590663 and 23790750).

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**12** 

*Slovak Republic* 

**Mechanisms of Ischemic Induced Neuronal** 

Stroke is the second leading cause of death and the primary cause of disability in humans. The phenomenon of ischemic tolerance perfectly describes the quote: "What does not kill you makes you stronger.'' Ischemic pre- or post- conditioning is actually the strongest known procedure to prevent or reverse delayed neuronal death. It works specifically in sensitive vulnerable neuronal populations, which are represented by pyramidal neurons in the hippocampal CA1 region. However, tolerance is effective in other brain cell populations as well. Although, its nomenclature is **''ischemic'' tolerance (IT)** ", the tolerant phenotype can also be induced by other stimuli that lead to delayed neuronal death (intoxication). Recent data have proven further that this phenomenon is not only limited to application of sublethal stimuli before the lethal stress (**preconditioning)** but also that reversed arrangement of events, sublethal stress after lethal insult (**postconditioning),** are equally effective. Another very important term is **''cross conditioning,''** or the capability of one stressor to induce tolerance against another. Delayed neuronal death is the slow development of post-ischemic neuro-degeneration. This delay allows a therapeutic window of opportunity lasting 2–3 days to reverse the cellular death process. It seems therefore that the mechanisms of ischemic tolerance-delayed post-conditioning could be of use not only

This paper summarizes results of experimental studies which have shown that acute *in vivo*  forebrain ischemia as well as ischemic/reperfusion injury (IRI) both alter, the expression, function and kinetic parameters of Ca2+ transporters as well as the physical membrane environment. Furthermore, that IRI leads to the inhibition of mitochondrial respiratory complexes I and IV. Also, that conversely, ischemic preconditioning (IPC) acts at the level of both initiation and execution of IRI-induced mitochondrial apoptosis and activates

Evidence is presented to show that endoplasmic reticulum (ER) is the site of complex processes such as calcium storage, synthesis and folding of proteins as well as cell response to stress. ER function is impaired in IRI which in turn induces depletion of stored calcium, the conserved stress responses linked with delayed neuronal death. In addition, IRI initiates time dependent differences in endoplasmic reticular (ER) gene expression of the key

after ischemia but also in some other processes leading up to apoptosis.

inhibition of p53 translocation to mitochondria.

**1. Introduction** 

**Death and Ischemic Tolerance** 

*Comenius University, Jessenius Faculty of Medicine, Department of Medical Biochemistry, Martin,* 

Jan Lehotsky, Martina Pavlikova, Stanislav Straka, Maria Kovalska, Peter Kaplan and Zuzana Tatarkova


### **Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance**

Jan Lehotsky, Martina Pavlikova, Stanislav Straka, Maria Kovalska, Peter Kaplan and Zuzana Tatarkova *Comenius University, Jessenius Faculty of Medicine, Department of Medical Biochemistry, Martin, Slovak Republic* 

### **1. Introduction**

240 Advances in the Preclinical Study of Ischemic Stroke

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Stroke is the second leading cause of death and the primary cause of disability in humans. The phenomenon of ischemic tolerance perfectly describes the quote: "What does not kill you makes you stronger.'' Ischemic pre- or post- conditioning is actually the strongest known procedure to prevent or reverse delayed neuronal death. It works specifically in sensitive vulnerable neuronal populations, which are represented by pyramidal neurons in the hippocampal CA1 region. However, tolerance is effective in other brain cell populations as well. Although, its nomenclature is **''ischemic'' tolerance (IT)** ", the tolerant phenotype can also be induced by other stimuli that lead to delayed neuronal death (intoxication). Recent data have proven further that this phenomenon is not only limited to application of sublethal stimuli before the lethal stress (**preconditioning)** but also that reversed arrangement of events, sublethal stress after lethal insult (**postconditioning),** are equally effective. Another very important term is **''cross conditioning,''** or the capability of one stressor to induce tolerance against another. Delayed neuronal death is the slow development of post-ischemic neuro-degeneration. This delay allows a therapeutic window of opportunity lasting 2–3 days to reverse the cellular death process. It seems therefore that the mechanisms of ischemic tolerance-delayed post-conditioning could be of use not only after ischemia but also in some other processes leading up to apoptosis.

This paper summarizes results of experimental studies which have shown that acute *in vivo*  forebrain ischemia as well as ischemic/reperfusion injury (IRI) both alter, the expression, function and kinetic parameters of Ca2+ transporters as well as the physical membrane environment. Furthermore, that IRI leads to the inhibition of mitochondrial respiratory complexes I and IV. Also, that conversely, ischemic preconditioning (IPC) acts at the level of both initiation and execution of IRI-induced mitochondrial apoptosis and activates inhibition of p53 translocation to mitochondria.

Evidence is presented to show that endoplasmic reticulum (ER) is the site of complex processes such as calcium storage, synthesis and folding of proteins as well as cell response to stress. ER function is impaired in IRI which in turn induces depletion of stored calcium, the conserved stress responses linked with delayed neuronal death. In addition, IRI initiates time dependent differences in endoplasmic reticular (ER) gene expression of the key

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 243

The mechanisms underlying ischemic tolerance are rather complex and not yet fully understood. Two windows have been identified in all multiple paradigms for IPC. One that represents very rapid and short-lasting post-translational changes and a second, which develops slowly (over days) after initial insult as a robust and long lasting transcriptional changes which culminate in prolonged neuroprotection (Dirnagl et al., 2009; Obrenovitch, 2008; Yenari et al., 2008). Differences in intensity, duration, and frequency of specific inducer/stressor determine the spectrum of responses to noxious stimuli. In other words, when the stimulus is too weak to induce any response, when it is sufficient to serve as a tolerance trigger, or when it is too strong and harmful, resulting in apoptotic or necrotic

It is symptomatic that there are no clear boundaries between acquisition of tolerance and cellular apoptosis/necrosis (Dirnagl et al., 2009). Rodent and cell culture models serve as a basis for the study of the tolerance phenomenon. Mother nature presents the perfect model to help understand this better. In nature, we ubiquitously find adaptation to extreme environmental conditions, for example, the hypoxic or anoxic tolerance. Hibernation is another example of inherent adaptation to extreme low-blood perfusion in animals. As such, ischemic tolerance can be conceived as an evolutionary conserved form of cerebral plasticity (Dirnagl et al., 2009,). It is not surprising therefore that different animal species have evolved different molecular strategies to cope with anoxia and severe metabolic stress. This

A number of common mechanisms with different relevance features can be recognized

At first pass, the patient population that suffers from cerebral ischemic injury due to unpredictable focal stroke, cardiac arrest, or subarachnoid hemorrhage represents, by definition, one that is unlikely to derive benefit from preconditioning research. However, the novel endogenous survival pathways identified in preclinical IT studies may ultimately become **targets for drugs** that protect the brain even when acutely administered after the precipitating event. Importantly, a significant number of other patients—those in which we can anticipate a period of cerebral ischemia following transient ischemic attack, aneurysm clipping, subarachnoid hemorrhage, carotid endarterectomy or stenting, asymptomatic carotid stenosis, coronary bypass, and cardiac valve replacement—represent defined at-risk populations ideally suited for translational **therapeutic preconditioning.** The candidate drugs that might underpin clinical trials for this latter group of patients actually comprise a relatively long and therefore promising list, particularly if the current foundation of

The concept of IPC in the heart was introduced in the late 80s by Murry et al. (1986) and later on in the brain by Schurr et al. (1986) and Kitagawa et al. (1991). Most stressors, including preischemia/hypoxia, induce both rapid and delayed tolerance phenotypes (Gidday, 2006). Mechanisms that are prominent during the first phases of acute ischemic insults such as excitoxicity are presumed to be induced during rapid IT. In particular,

leads to the trigger of the neuroprotective tolerance state.



preclinical studies is expanded with intention.

damage.

(Lehotsky et al., 2009b):


unfolded protein response (UPR), or proteins at both the mRNA and protein levels. Moreover, gene expression of the UPR proteins is affected by pre-ischemic (IPC) treatment caused by the increased expression of Ca2+ binding protein, GRP 78 and transcriptional factor ATF6 in reperfusion times. Thus, IPC exerts a role in the attenuation of ER stress response, which might, in turn, be involved in the neuroprotective phenomenon of ischemic tolerance. Hippocampal cells respond to the IRI by the specific expression pattern of the secretory pathways Ca2+ pump (SPCA1) and this pattern is affected by preischemic challenge. IPC also incompletely suppresses lipid and protein oxidation of hippocampal membranes and leads to partial recovery of the ischemic-induced depression of SPCA activity. The data suggests a correlation of SPCA function with the role of secretory pathways (Golgi apparatus) in response to preischemic challenge.

### **2. Ischemic stroke**

**Ischemic stroke** arises in humans as a consequence of a cardiac arrest, the stoppage of blood flow to the brain due to embolic or thrombic occlusion of arteries. Global or focal ischemia is very severe pathogenic event with multiple, parallel, and sequential pathogenesis. Global forebrain ischemia leads to selective cell death of vulnerable pyramidal neurons in the hippocampal CA1 region. It also leads to death of cerebral cortex neurons (layers 3, 5, and 6) and the dorsolateral striatum. When blood flow decreases during focal ischemia, the area surrounding the necrotic core of ischemia, also known as ''penumbra'' is perfused by collateral vessels. It also undergoes fatal apoptosis of neurons (Endres et al., 2008).

Despite decades of intense research, no effective neuroprotective drugs are available to treat acute stroke or cardiac arrest. For this reason, recent attention has shifted to defining the brain's own evolutionarily conserved endogenous neuroprotective mechanisms, which occurs in **ischemic tolerance (IT) or after ischemic preconditioning (IPC).** IT induced by several paradigms represents an important phenomenon of the central nervous system (CNS) including adaptation to sublethal short-term ischemia. This results in increased tolerance of CNS to lethal ischemia (Kirino, 2002; Dirnagl et al., 2003; Gidday, 2006). The molecular mechanisms underlying IT are not yet fully understood because of its extreme complexity, involving many signaling pathways and alterations in gene expression. Additionally, a metabolic depression has also been suggested to play an important role in IT (Yenari et al., 2008).

### **2.1 Ischemic tolerance as a possible neuroprotective strategy**

A transient, ischemia-resistant phenotype known as **"ischemic tolerance (IT)"** can be established in brain in a rapid or delayed fashion by a preceding non-injurious "preconditioning" stimulus. Thus, **ischemic preconditioning (IPC)** as one of the inducers, represents a phenomenon which eventually leads to an increase in the **tolerance** of CNS to the lethal ischemia (Dirnagl et al., 2009; Obrenovitch, 2008). Initial pre-clinical studies of this phenomenon relied primarily on brief periods of ischemia or hypoxia as the IPC stimuli, but it was later realized that many other stressors, including pharmacological agents, are also effective. Although considerably more experimentation is needed to thoroughly validate the efficacy of any already identified preconditioning agent to protect ischemic brain, the fact that some of these agents are already clinically used implies that the growing enthusiasm for translational success in the field of pharmacologic preconditioning may be well justified.

The mechanisms underlying ischemic tolerance are rather complex and not yet fully understood. Two windows have been identified in all multiple paradigms for IPC. One that represents very rapid and short-lasting post-translational changes and a second, which develops slowly (over days) after initial insult as a robust and long lasting transcriptional changes which culminate in prolonged neuroprotection (Dirnagl et al., 2009; Obrenovitch, 2008; Yenari et al., 2008). Differences in intensity, duration, and frequency of specific inducer/stressor determine the spectrum of responses to noxious stimuli. In other words, when the stimulus is too weak to induce any response, when it is sufficient to serve as a tolerance trigger, or when it is too strong and harmful, resulting in apoptotic or necrotic damage.

It is symptomatic that there are no clear boundaries between acquisition of tolerance and cellular apoptosis/necrosis (Dirnagl et al., 2009). Rodent and cell culture models serve as a basis for the study of the tolerance phenomenon. Mother nature presents the perfect model to help understand this better. In nature, we ubiquitously find adaptation to extreme environmental conditions, for example, the hypoxic or anoxic tolerance. Hibernation is another example of inherent adaptation to extreme low-blood perfusion in animals. As such, ischemic tolerance can be conceived as an evolutionary conserved form of cerebral plasticity (Dirnagl et al., 2009,). It is not surprising therefore that different animal species have evolved different molecular strategies to cope with anoxia and severe metabolic stress. This leads to the trigger of the neuroprotective tolerance state.

A number of common mechanisms with different relevance features can be recognized (Lehotsky et al., 2009b):


242 Advances in the Preclinical Study of Ischemic Stroke

unfolded protein response (UPR), or proteins at both the mRNA and protein levels. Moreover, gene expression of the UPR proteins is affected by pre-ischemic (IPC) treatment caused by the increased expression of Ca2+ binding protein, GRP 78 and transcriptional factor ATF6 in reperfusion times. Thus, IPC exerts a role in the attenuation of ER stress response, which might, in turn, be involved in the neuroprotective phenomenon of ischemic tolerance. Hippocampal cells respond to the IRI by the specific expression pattern of the secretory pathways Ca2+ pump (SPCA1) and this pattern is affected by preischemic challenge. IPC also incompletely suppresses lipid and protein oxidation of hippocampal membranes and leads to partial recovery of the ischemic-induced depression of SPCA activity. The data suggests a correlation of SPCA function with the role of secretory

**Ischemic stroke** arises in humans as a consequence of a cardiac arrest, the stoppage of blood flow to the brain due to embolic or thrombic occlusion of arteries. Global or focal ischemia is very severe pathogenic event with multiple, parallel, and sequential pathogenesis. Global forebrain ischemia leads to selective cell death of vulnerable pyramidal neurons in the hippocampal CA1 region. It also leads to death of cerebral cortex neurons (layers 3, 5, and 6) and the dorsolateral striatum. When blood flow decreases during focal ischemia, the area surrounding the necrotic core of ischemia, also known as ''penumbra'' is perfused by

Despite decades of intense research, no effective neuroprotective drugs are available to treat acute stroke or cardiac arrest. For this reason, recent attention has shifted to defining the brain's own evolutionarily conserved endogenous neuroprotective mechanisms, which occurs in **ischemic tolerance (IT) or after ischemic preconditioning (IPC).** IT induced by several paradigms represents an important phenomenon of the central nervous system (CNS) including adaptation to sublethal short-term ischemia. This results in increased tolerance of CNS to lethal ischemia (Kirino, 2002; Dirnagl et al., 2003; Gidday, 2006). The molecular mechanisms underlying IT are not yet fully understood because of its extreme complexity, involving many signaling pathways and alterations in gene expression. Additionally, a metabolic depression has also been suggested to play an important role in IT

A transient, ischemia-resistant phenotype known as **"ischemic tolerance (IT)"** can be established in brain in a rapid or delayed fashion by a preceding non-injurious "preconditioning" stimulus. Thus, **ischemic preconditioning (IPC)** as one of the inducers, represents a phenomenon which eventually leads to an increase in the **tolerance** of CNS to the lethal ischemia (Dirnagl et al., 2009; Obrenovitch, 2008). Initial pre-clinical studies of this phenomenon relied primarily on brief periods of ischemia or hypoxia as the IPC stimuli, but it was later realized that many other stressors, including pharmacological agents, are also effective. Although considerably more experimentation is needed to thoroughly validate the efficacy of any already identified preconditioning agent to protect ischemic brain, the fact that some of these agents are already clinically used implies that the growing enthusiasm for translational success in the field of pharmacologic pre-

collateral vessels. It also undergoes fatal apoptosis of neurons (Endres et al., 2008).

pathways (Golgi apparatus) in response to preischemic challenge.

**2.1 Ischemic tolerance as a possible neuroprotective strategy** 

**2. Ischemic stroke** 

(Yenari et al., 2008).

conditioning may be well justified.


At first pass, the patient population that suffers from cerebral ischemic injury due to unpredictable focal stroke, cardiac arrest, or subarachnoid hemorrhage represents, by definition, one that is unlikely to derive benefit from preconditioning research. However, the novel endogenous survival pathways identified in preclinical IT studies may ultimately become **targets for drugs** that protect the brain even when acutely administered after the precipitating event. Importantly, a significant number of other patients—those in which we can anticipate a period of cerebral ischemia following transient ischemic attack, aneurysm clipping, subarachnoid hemorrhage, carotid endarterectomy or stenting, asymptomatic carotid stenosis, coronary bypass, and cardiac valve replacement—represent defined at-risk populations ideally suited for translational **therapeutic preconditioning.** The candidate drugs that might underpin clinical trials for this latter group of patients actually comprise a relatively long and therefore promising list, particularly if the current foundation of preclinical studies is expanded with intention.

The concept of IPC in the heart was introduced in the late 80s by Murry et al. (1986) and later on in the brain by Schurr et al. (1986) and Kitagawa et al. (1991). Most stressors, including preischemia/hypoxia, induce both rapid and delayed tolerance phenotypes (Gidday, 2006). Mechanisms that are prominent during the first phases of acute ischemic insults such as excitoxicity are presumed to be induced during rapid IT. In particular,

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 245

depletion, ion imbalance, as well as other biochemical changes, such as an increase of free radicals, mitochondrial dysfunction, lactic acidosis, and inhibition of proteosynthesis as a

The endoplasmic reticulum of eukaryotic cell reacts to ischemic injury by **the unfolded protein response (UPR),** which can be highly variable, depending on dosage and duration of ischemic treatment (Imaizumi et al., 2001), and intensity of UPR signals (Yoshida et al., 2003). However, when ER stress is too severe and prolonged, apoptosis is induced. Various enzymes and transcription factors including the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) (Harding et al., 1999), the transcription factors ATF4 and ATF6 (activating transcription factor 6) and the inositol-requiring enzyme IRE1 (Shen et al., 2001) are involved in the UPR. In the physiological state, PERK, ATF6, and IRE1 activity is suppressed by binding of the ER chaperone: glucose regulated protein 78 (GRP78). Morimoto et al. (2007) reported that induction of GRP78 prevents neuronal damage induced by ER stress, and the increase in GRP78 (BiP) expression may correlate with the degree of

**Statins**, inhibitors of sterol synthesis, have been shown to reduce cerebrovascular events by their pleiotropic effects independent of the cholesterol lowering mechanism. Nagotani et al. (2005) found that simvastatin was the most effective statin against spontaneous stroke in human and animals. Strong liposolubility of statins may result in high permeability through the blood–brain barrier to the parenchyma, thereby protecting the neurons against ROSinduced lipid peroxidation and DNA oxidation. The neuroprotective properties of **simvastatin** in experimental stroke have been evaluated by using several rodent-simulated models of cerebral ischemia (Shabanzadeh et al., 2005; Hayashi et al., 2005). As shown by previous studies, the changes of the UPR gene expression induced by transient ischemia occur mostly during the first 24 h (Paschen 2003b) or the first few days after the insult (Qi et al., 2004). In line with this, Urban et al. (2009) have decided to measure changes in mRNA and protein levels of GRP78, ATF6, and XBP1 after 15 min of global ischemia and 1, 3, and 24 h reperfusion (UPR reaction). In addition, they have focused their attention on the effect of simvastatin pretreatment on the stress reaction of endoplasmic reticulum induced by

Adult male Wistar rats were used as animal model for the experiment. Global forebrain ischemia was induced by the standard four-vessel occlusion model (Lehotský et al., 2004; Sivonova et al., 2008; Uríkova et al., 2006). For maximal proof of changes in mRNA levels, authors used real-time PCR. Cortexes from sham control, ischemic and simvastatin-treated animals were homogenized, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and level of levels of ER stress gene proteins was analyzed by Western blotting after ischemic/ reperfusion damage (I/R) in naive rats and rats pretreated with simvastatin (20 mg/kg for 14 days). In the non-treated I/R animals, the mRNA level was significantly maximal in ischemic period (43 ± 3.2% in comparison to control), followed by rapid significant decrease from the first hour of reperfusion to a minimum value reached at the third hour (57 ± 7.8% lower than control). The mRNA level at 24 h of reperfusion reached control values. The level of XBP1 protein in non-treated animals showed only slight, not significant, differences compared to controls, mainly at later reperfusion periods (3 and 24 h). The influence of simvastatin on mRNA level was significant only in the first and the third hours of reperfusion compared to control I/R animals (about 32.8 ± 4.1% lower and two times higher in I3R, respectively). The changes in mRNA levels were not projected onto

consequence of endoplasmic reticular (ER) stress (DeGracia et al., 2002).

neuroprotection.

ischemic/reperfusion insult.

elevation of adenosine and activation of adenosine receptor with the modulation of ATP sensitive K+ channel are paralleled by the activation of protein kinase C and other kinases in rapid tolerance. A critical role for nitric oxide signaling pathways in IPC and tolerance was also suggested (Nandagopal et al., 2001). As was recently shown by Meller et al. (2008), the selective ubiquitin–proteasome degradation of a cell death-associated protein, Bcl-2 interacting mediator of cell death (Bim) with the reduced activation of programmed cell death-associated caspases (caspase 3) could play an important role in rapid tolerance to ischemia. As mentioned earlier, IT can be induced by various stimuli that are not necessarily ischemic or hypoxic.

Fig. 1**.** Ischemic insult without any maneuvers leads to ischemic/reperfusion injured phenotype. Cerebroprotection can be induced by different types of preconditioning or postconditioning maneuvers/stimuli (ischemic, immunological, pharmacological and anesthetic). Temporary defined responses during therapeutical window may induce protective response with which subsequent ischemia serve as basis of the ischemic-tolerant phenotype. Adapted from Lehotsky et al. (2009b).

Thus, the phenomenon of cross-tolerance implies that noxious stress can initiate cellular tolerance to subsequent stress that is different in nature from the first one. Therefore, one stressor can promote cross-tolerance to another; however, the efficacy of this tolerance may be more modest, and it appears to vary with the nature and intensity of the first challenge. Additionally, the window of evolved IT may also be shifted. However, the nature of the stimulus may determine the specific protective or in worse meaning the reduced damage epiphenotype.

### **3. Prophylactic treatment with statins: Effect on ischemic damage**

Neuronal ischemic/reperfusion damage in the brain occurs rapidly. However, significant structural changes are observed over a course of hours or days in the form of delayed neuronal death. Interruption of blood flow initiates high-energy metabolism failure, ATP

elevation of adenosine and activation of adenosine receptor with the modulation of ATP sensitive K+ channel are paralleled by the activation of protein kinase C and other kinases in rapid tolerance. A critical role for nitric oxide signaling pathways in IPC and tolerance was also suggested (Nandagopal et al., 2001). As was recently shown by Meller et al. (2008), the selective ubiquitin–proteasome degradation of a cell death-associated protein, Bcl-2 interacting mediator of cell death (Bim) with the reduced activation of programmed cell death-associated caspases (caspase 3) could play an important role in rapid tolerance to ischemia. As mentioned earlier, IT can be induced by various stimuli that are not necessarily

Fig. 1**.** Ischemic insult without any maneuvers leads to ischemic/reperfusion injured phenotype. Cerebroprotection can be induced by different types of preconditioning or postconditioning maneuvers/stimuli (ischemic, immunological, pharmacological and anesthetic). Temporary defined responses during therapeutical window may induce protective response with which subsequent ischemia serve as basis of the ischemic-tolerant

**3. Prophylactic treatment with statins: Effect on ischemic damage** 

Thus, the phenomenon of cross-tolerance implies that noxious stress can initiate cellular tolerance to subsequent stress that is different in nature from the first one. Therefore, one stressor can promote cross-tolerance to another; however, the efficacy of this tolerance may be more modest, and it appears to vary with the nature and intensity of the first challenge. Additionally, the window of evolved IT may also be shifted. However, the nature of the stimulus may determine the specific protective or in worse meaning the reduced damage

Neuronal ischemic/reperfusion damage in the brain occurs rapidly. However, significant structural changes are observed over a course of hours or days in the form of delayed neuronal death. Interruption of blood flow initiates high-energy metabolism failure, ATP

phenotype. Adapted from Lehotsky et al. (2009b).

ischemic or hypoxic.

epiphenotype.

depletion, ion imbalance, as well as other biochemical changes, such as an increase of free radicals, mitochondrial dysfunction, lactic acidosis, and inhibition of proteosynthesis as a consequence of endoplasmic reticular (ER) stress (DeGracia et al., 2002).

The endoplasmic reticulum of eukaryotic cell reacts to ischemic injury by **the unfolded protein response (UPR),** which can be highly variable, depending on dosage and duration of ischemic treatment (Imaizumi et al., 2001), and intensity of UPR signals (Yoshida et al., 2003). However, when ER stress is too severe and prolonged, apoptosis is induced. Various enzymes and transcription factors including the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) (Harding et al., 1999), the transcription factors ATF4 and ATF6 (activating transcription factor 6) and the inositol-requiring enzyme IRE1 (Shen et al., 2001) are involved in the UPR. In the physiological state, PERK, ATF6, and IRE1 activity is suppressed by binding of the ER chaperone: glucose regulated protein 78 (GRP78). Morimoto et al. (2007) reported that induction of GRP78 prevents neuronal damage induced by ER stress, and the increase in GRP78 (BiP) expression may correlate with the degree of neuroprotection.

**Statins**, inhibitors of sterol synthesis, have been shown to reduce cerebrovascular events by their pleiotropic effects independent of the cholesterol lowering mechanism. Nagotani et al. (2005) found that simvastatin was the most effective statin against spontaneous stroke in human and animals. Strong liposolubility of statins may result in high permeability through the blood–brain barrier to the parenchyma, thereby protecting the neurons against ROSinduced lipid peroxidation and DNA oxidation. The neuroprotective properties of **simvastatin** in experimental stroke have been evaluated by using several rodent-simulated models of cerebral ischemia (Shabanzadeh et al., 2005; Hayashi et al., 2005). As shown by previous studies, the changes of the UPR gene expression induced by transient ischemia occur mostly during the first 24 h (Paschen 2003b) or the first few days after the insult (Qi et al., 2004). In line with this, Urban et al. (2009) have decided to measure changes in mRNA and protein levels of GRP78, ATF6, and XBP1 after 15 min of global ischemia and 1, 3, and 24 h reperfusion (UPR reaction). In addition, they have focused their attention on the effect of simvastatin pretreatment on the stress reaction of endoplasmic reticulum induced by ischemic/reperfusion insult.

Adult male Wistar rats were used as animal model for the experiment. Global forebrain ischemia was induced by the standard four-vessel occlusion model (Lehotský et al., 2004; Sivonova et al., 2008; Uríkova et al., 2006). For maximal proof of changes in mRNA levels, authors used real-time PCR. Cortexes from sham control, ischemic and simvastatin-treated animals were homogenized, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and level of levels of ER stress gene proteins was analyzed by Western blotting after ischemic/ reperfusion damage (I/R) in naive rats and rats pretreated with simvastatin (20 mg/kg for 14 days). In the non-treated I/R animals, the mRNA level was significantly maximal in ischemic period (43 ± 3.2% in comparison to control), followed by rapid significant decrease from the first hour of reperfusion to a minimum value reached at the third hour (57 ± 7.8% lower than control). The mRNA level at 24 h of reperfusion reached control values. The level of XBP1 protein in non-treated animals showed only slight, not significant, differences compared to controls, mainly at later reperfusion periods (3 and 24 h).

The influence of simvastatin on mRNA level was significant only in the first and the third hours of reperfusion compared to control I/R animals (about 32.8 ± 4.1% lower and two times higher in I3R, respectively). The changes in mRNA levels were not projected onto

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 247

induced ER dysfunction (Kumar et al., 2001; Paschen, 2003a) and possibly leading to a pro apoptotic phenotype (De-Gracia and Montie, 2004). Similarly, in experiments of Urban et al. (2009), authors did not detected any significant changes in the protein level of XBP1 neither in ischemic period nor in the first 24 h of reperfusion. The results from measurements of XBP1 mRNA in simvastatin-treated animals did not show any significant changes in comparison to naive ischemic animals, i.e., the maximal differences were detected in the first and third hour of reperfusion (about 32.8 ± 4.1% lower in I1R and two times higher in I3R, respectively). A bit surprisingly, the protein level of XBP1 was generally decreased in pretreated animals (mainly in ischemic and I1R phase than non treated group), and did not reach control levels. Recently, a novel action of statins was proven in neurons, involving cell growth and signaling as well as down-regulation of proinflammatory gene expression attenuating neurogenic inflammation (Johnson-Anuna et al., 2005; Bucelli et al., 2008). The results of real-time PCR measurement showed an increased mRNA level of GRP78 in ischemic time and at later phases of reperfusion in non-treated animals. Probable reason is that GRP78 is a member of the 70-kDa heat shock protein family that acts as a molecular chaperone in the folding and assembly of newly synthesized proteins within the ER. Yu et al. (1999) reported that suppression of GRP78 expression enhanced apoptosis and disruption of cellular calcium homeostasis in hippocampal neurons exposed to excitotoxic and oxidative insults. This indicates that a raised level of GRP78 makes cells more resistant to

In experiments of Urban et al. (2009), authors did not find any significant changes in protein levels of GRP78 neither in simvastatin-treated nor in non-treated group of animals. They have just found maximum at third hour of reperfusion in statin group and small decrease at 24 h of reperfusion in both groups. Those results are similar to the findings of Burda et al. (2003), who failed to find any differences in GRP78 protein levels at any of the reperfusion times considered (max 4 h), either in rats with or without acquired ischemic tolerance. However, in a model of ischemic preconditioning in rats (Hayashi et al., 2003; Garca et al., 2004) an increase in GRP78 expression was detected after 2 days of preconditioning. Authors proposed that development of tolerance includes changes in PERK/GRP78 association, which were responsible for the decrease in eIF2a phosphorylation induced by preconditioning. Other studies using distinct ischemic models also failed to detect increased

The results of Urban et al, (2009) also showed an increased mRNA expression of ATF6, however, only in ischemic time. Consequently levels of mRNA for GRP78 were increased only slightly compared to controls. The minimum level of mRNA for ATF6 was observed at third hour of reperfusion followed by increase till 24 h of reperfusion. This minimum was probably due to pro-survival mechanism through inhibition of proapoptotic protein GADD153, which usually acts as a transcription factor of UPR genes. GADD153 protein decreased during reperfusion, until the minimum was reached at the third hour of reperfusion (Kumar et al., 2003). Urban et al. (2009) also showed significant higher levels of ATF6 mRNA in statin-treated animals in comparison to non-statin animals at ischemic period and at third hour of reperfusion (about 35.2 ± 6.6% and 42 ± 2.6% higher level), which was also translated into the higher protein level, whose values had significant maximum at

third hour of reperfusion (about 60% higher level than in non-treated animals).

The experimental results altogether indicate that global ischemia/reperfusion initiates timedependent differences in endoplasmic reticular gene expression at both the mRNA and

the stressful conditions (Aoki et al., 2001).

levels of GRP78 protein (Paschen 2003a).

protein levels, which, in contrast to control I/R animals, was found to be significantly lower (about 37.4 ± 2.2% in ischemic phase and about 36.3 ± 5.7% lower in first hour of reperfusion). In this paper, Urban et al (2009) were interested in finding whether global ischemia induced by four vessel occlusion followed by reperfusion at different time points would initiate the unfolded protein response of ER in cortical neurons. In addition, they have proved that prophylactic simvastatin therapy affects expression of gene coding for the main proteins involved in UPR.

Clinical trials demonstrated that 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors or statins exert beneficial effects when used as stroke prophylactic agents (Byington et al., 2001; Vaughan et al., 2001). These studies showed that statins reduce the incidence of both first and secondary events by 25– 30% and prevention is believed to be achieved mainly through their activity on blood vessel wall function. However, in addition to exerting anti-atherosclerotic and anti-thrombotic effects, statins also possess antiinflammatory and neuroprotective actions, which have been identified as cholesterolindependent or pleiotropic effects (Vaughan and Delanty, 1999; Takemoto and Liao, 2001). The findings indicated that administration of simvastatin or other statins reduced the size of brain damage (Sironi et al., 2003; Amin-Hanjan et al., 2001). The beneficial effect of simvastatin is achieved only when the drug is administered before the ischemic insult; therefore, acting as a prophylactic agent (Balduini et al., 2003). In the model of focal ischemia induced by middle carotid artery occlusion (MCAO), the size of the damaged tissue increased by 47% after 24 h and by 83% after 48 h as compared to the infarct size detected at 2 h. This time-dependent enhancement of the damage was abolished in animals pre-treated with simvastatin, as the volume of infarct was never larger than the volume reported 2 h after MCAO. (Cimino et al., 2005).

In general, I/R injury initiates suppression of global proteosynthesis (de la Vega et al., 2001; Paschen, 2003a). Ischemia is one of the strongest stimuli of gene induction in the brain. Different gene systems related to reperfusion processes of brain injury, repair, and recovery are up-regulated (Gidday, 2006). Focal ischemia shorter than 3,5 minutes and seven days of reperfusion usually causes degeneration of 75% of the neurons in the hippocampal CA1 region (Ohtsuki et al., 1996). On the other hand 6–10-minutes long global ischemia and three days of reperfusion caused death of almost all pyramidal neurons in the same hippocampal area (Coimbra and Wieloch, 1994). Urban et al., (2009) showed a significant increase of XBP1 mRNA level in ischemic phase in comparison to control (about 43% more). These findings are similar to those observed by Paschen (2003a), which showed a marked increase in processed XBP1 mRNA levels using semi-quantitative RT-PCR after focal ischemia. These changes were most pronounced in the cerebral cortex, where high levels were found throughout the entire observation period. Urban et al (2009) obtained similar results; however, the differences were smaller probably due to the different ischemic model. The rapid increase of mRNA level of XBP1 along with other genes in ischemic phase of non-treated animals was probably due to forthcoming dissociation of protein GRP78, which reached a maximum at ischemic phase and first hour of reperfusion, from bounds with sensors of UPR which quickly (ATF6) or slowly (XBP1) started transcription of effector's genes.

In simvastatin-treated animals, rapid increase of mRNA in ischemic phase was mainly a consequence of transcription factor ATF6. It has been proposed that the strong inhibition of translation induced after transient cerebral ischemia prevents the expression of key effector UPR proteins such as the XBP1, GRP78, or ATF4, thereby hindering recovery from ischemia-

protein levels, which, in contrast to control I/R animals, was found to be significantly lower (about 37.4 ± 2.2% in ischemic phase and about 36.3 ± 5.7% lower in first hour of reperfusion). In this paper, Urban et al (2009) were interested in finding whether global ischemia induced by four vessel occlusion followed by reperfusion at different time points would initiate the unfolded protein response of ER in cortical neurons. In addition, they have proved that prophylactic simvastatin therapy affects expression of gene coding for the

Clinical trials demonstrated that 3-hydroxy-3- methylglutaryl coenzyme A reductase inhibitors or statins exert beneficial effects when used as stroke prophylactic agents (Byington et al., 2001; Vaughan et al., 2001). These studies showed that statins reduce the incidence of both first and secondary events by 25– 30% and prevention is believed to be achieved mainly through their activity on blood vessel wall function. However, in addition to exerting anti-atherosclerotic and anti-thrombotic effects, statins also possess antiinflammatory and neuroprotective actions, which have been identified as cholesterolindependent or pleiotropic effects (Vaughan and Delanty, 1999; Takemoto and Liao, 2001). The findings indicated that administration of simvastatin or other statins reduced the size of brain damage (Sironi et al., 2003; Amin-Hanjan et al., 2001). The beneficial effect of simvastatin is achieved only when the drug is administered before the ischemic insult; therefore, acting as a prophylactic agent (Balduini et al., 2003). In the model of focal ischemia induced by middle carotid artery occlusion (MCAO), the size of the damaged tissue increased by 47% after 24 h and by 83% after 48 h as compared to the infarct size detected at 2 h. This time-dependent enhancement of the damage was abolished in animals pre-treated with simvastatin, as the volume of infarct was never larger than the volume

In general, I/R injury initiates suppression of global proteosynthesis (de la Vega et al., 2001; Paschen, 2003a). Ischemia is one of the strongest stimuli of gene induction in the brain. Different gene systems related to reperfusion processes of brain injury, repair, and recovery are up-regulated (Gidday, 2006). Focal ischemia shorter than 3,5 minutes and seven days of reperfusion usually causes degeneration of 75% of the neurons in the hippocampal CA1 region (Ohtsuki et al., 1996). On the other hand 6–10-minutes long global ischemia and three days of reperfusion caused death of almost all pyramidal neurons in the same hippocampal area (Coimbra and Wieloch, 1994). Urban et al., (2009) showed a significant increase of XBP1 mRNA level in ischemic phase in comparison to control (about 43% more). These findings are similar to those observed by Paschen (2003a), which showed a marked increase in processed XBP1 mRNA levels using semi-quantitative RT-PCR after focal ischemia. These changes were most pronounced in the cerebral cortex, where high levels were found throughout the entire observation period. Urban et al (2009) obtained similar results; however, the differences were smaller probably due to the different ischemic model. The rapid increase of mRNA level of XBP1 along with other genes in ischemic phase of non-treated animals was probably due to forthcoming dissociation of protein GRP78, which reached a maximum at ischemic phase and first hour of reperfusion, from bounds with sensors of UPR which quickly (ATF6) or slowly

In simvastatin-treated animals, rapid increase of mRNA in ischemic phase was mainly a consequence of transcription factor ATF6. It has been proposed that the strong inhibition of translation induced after transient cerebral ischemia prevents the expression of key effector UPR proteins such as the XBP1, GRP78, or ATF4, thereby hindering recovery from ischemia-

main proteins involved in UPR.

reported 2 h after MCAO. (Cimino et al., 2005).

(XBP1) started transcription of effector's genes.

induced ER dysfunction (Kumar et al., 2001; Paschen, 2003a) and possibly leading to a pro apoptotic phenotype (De-Gracia and Montie, 2004). Similarly, in experiments of Urban et al. (2009), authors did not detected any significant changes in the protein level of XBP1 neither in ischemic period nor in the first 24 h of reperfusion. The results from measurements of XBP1 mRNA in simvastatin-treated animals did not show any significant changes in comparison to naive ischemic animals, i.e., the maximal differences were detected in the first and third hour of reperfusion (about 32.8 ± 4.1% lower in I1R and two times higher in I3R, respectively). A bit surprisingly, the protein level of XBP1 was generally decreased in pretreated animals (mainly in ischemic and I1R phase than non treated group), and did not reach control levels. Recently, a novel action of statins was proven in neurons, involving cell growth and signaling as well as down-regulation of proinflammatory gene expression attenuating neurogenic inflammation (Johnson-Anuna et al., 2005; Bucelli et al., 2008).

The results of real-time PCR measurement showed an increased mRNA level of GRP78 in ischemic time and at later phases of reperfusion in non-treated animals. Probable reason is that GRP78 is a member of the 70-kDa heat shock protein family that acts as a molecular chaperone in the folding and assembly of newly synthesized proteins within the ER. Yu et al. (1999) reported that suppression of GRP78 expression enhanced apoptosis and disruption of cellular calcium homeostasis in hippocampal neurons exposed to excitotoxic and oxidative insults. This indicates that a raised level of GRP78 makes cells more resistant to the stressful conditions (Aoki et al., 2001).

In experiments of Urban et al. (2009), authors did not find any significant changes in protein levels of GRP78 neither in simvastatin-treated nor in non-treated group of animals. They have just found maximum at third hour of reperfusion in statin group and small decrease at 24 h of reperfusion in both groups. Those results are similar to the findings of Burda et al. (2003), who failed to find any differences in GRP78 protein levels at any of the reperfusion times considered (max 4 h), either in rats with or without acquired ischemic tolerance. However, in a model of ischemic preconditioning in rats (Hayashi et al., 2003; Garca et al., 2004) an increase in GRP78 expression was detected after 2 days of preconditioning. Authors proposed that development of tolerance includes changes in PERK/GRP78 association, which were responsible for the decrease in eIF2a phosphorylation induced by preconditioning. Other studies using distinct ischemic models also failed to detect increased levels of GRP78 protein (Paschen 2003a).

The results of Urban et al, (2009) also showed an increased mRNA expression of ATF6, however, only in ischemic time. Consequently levels of mRNA for GRP78 were increased only slightly compared to controls. The minimum level of mRNA for ATF6 was observed at third hour of reperfusion followed by increase till 24 h of reperfusion. This minimum was probably due to pro-survival mechanism through inhibition of proapoptotic protein GADD153, which usually acts as a transcription factor of UPR genes. GADD153 protein decreased during reperfusion, until the minimum was reached at the third hour of reperfusion (Kumar et al., 2003). Urban et al. (2009) also showed significant higher levels of ATF6 mRNA in statin-treated animals in comparison to non-statin animals at ischemic period and at third hour of reperfusion (about 35.2 ± 6.6% and 42 ± 2.6% higher level), which was also translated into the higher protein level, whose values had significant maximum at third hour of reperfusion (about 60% higher level than in non-treated animals).

The experimental results altogether indicate that global ischemia/reperfusion initiates timedependent differences in endoplasmic reticular gene expression at both the mRNA and

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 249

Mitochondrial dysfunction and oxidative stress were often implicated in pathophysiology of neurodegenerative diseases, including cerebral ischemia (Lin and Beal, 2006). Inhibition of complex I itself or in combination with elevated Ca2+ led to enhanced ROS production in different *in vitro* and *in vivo* systems (Yadava and Nicholls, 2007). Importantly, an enhanced production of ROS and consequent induction of p53-dependent apoptosis due to damage to neuronal DNA has also been documented after inhibition of complex I. A recent study showed that spare respiratory capacity rather then oxidative stress is involved in excitotoxic

As shown by experimental and clinical studies, IRI –induced mitochondrial pathway of apoptosis is an important event leading to neuronal cell death after blood flow arrest. Impact of IRI and ischemic preconditioning on the level of apoptotic and anti-apoptotic proteins was assessed in both cortical and hippocampal mitochondria by Western blot analysis of p53, bax, and bcl-x (Racay et al., 2007, 2009b). Remarkably, IRI led to increase of p53 level in hippocampal mitochondria, with significant differences after 3 h (217.1 ± 42.2% of control), 24 h (286.8 ± 65% of control), and 72 h (232.9 ± 37.3% of control) of reperfusion. Interestingly, translocation of p53 to mitochondria was observed in hippocampus but not in cerebral cortex. However, levels of both the apoptotic proteins bax and the anti-apoptotic bcl-xl were unchanged in both hippocampal and cortical mitochondria. Ischemia-induced translocation of p53 to mitochondria was completely abolished by IPC since no significant changes in mitochondrial p53 level were observed after preconditioned ischemia. Similar to naive ischemia, the levels of both bax and bcl-xl were not affected by IPC. In addition, IPC had significant protective effect on ischemia-induced DNA fragmentation, as well as on number of positive Fluoro-Jade C staining cells. Thus, it indicates that IPC abolished almost completely both initiation and execution of mitochondrial apoptosis induced by global brain

ischemia in vulnerable CA1 layer of rat hippocampus (Racay et al., 2007, 2009b).

The studies showed that ischemia induced inhibition of mitochondrial complexes I and IV, however inhibition is not accompanied by a decrease of mitochondrial Ca2+ uptake rate apparently due to the excess capacity of the complex I and complex IV. On the other hand, depressed activities of complex I and IV are conditions favourable of initiation of cell degenerative pathways, e.g. opening of mitochondrial permeability transition pore, ROS generation and apoptosis initiation, and might represent important mechanism of ischemic

Accordingly, ischemic preconditioning acts at the level of both initiation and execution of ischemia-induced mitochondrial apoptosis affording protection from ischemia associated changes in integrity of mitochondrial membranes. IPC also activates inhibition of p53 translocation to mitochondria. Inhibition of the mitochondrial p53 pathway thus might provide a potentially important mechanism of neuronal survival in the face of ischemic

Ischemic tolerance can be developed by prior ischemic non-injurious stimulus or preconditioning. The molecular mechanisms underlying ischemic tolerance are not yet fully understood yet. Therefore a series of papers (Urban et al., 2009; Lehotsky et al., 2009; Pavlikova et al.,2009) have focused attention at the mRNA and protein levels of **the ER stress** genes after **ischemic/reperfusion damage (IRI)** in naive and preconditioned groups

**5. Stress reaction of neuronal endoplasmic reticulum after IRI and IPC** 

cell death (Yadava and Nicholls, 2007).

damage to neurons.

of rats.

brain damage (Otani, 2008).

protein levels and these authors also found the generally enhanced level of mRNA in simvastatin pre-treated animals. The maximal differences between naive ischemic and pretreated ischemic animals authors detected in protein levels of proteins ATF6 and XBP1. The level of ATF6 was 60% higher in simvastatin pre-treated animals, which might suggest that ATF6 is one of the main proteins targeted to enhance neuroprotective effect at the ER gene level during first two hours of reperfusion.

In conclusion, these data indicate that statins, in addition to their cholesterol-lowering effect may exert a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

### **4. Impact of IRI and IPC on mitochondrial calcium transport, p53 translocation and neuronal apoptosis**

**Mitochondria** are important regulators of neuronal cell life and death through their role in metabolic energy production and involvement in apoptosis (Yuan and Yanker, 2000). Remarkably, mitochondrial dysfunction is considered to be one of the key events linking ischemic/recirculation insult with neuronal cell death (Berridge et al., 2003). In addition, mitochondria play a dual role in intracellular calcium. They are involved in the normal control of neuronal Ca2+ homeostasis (Berridge et al., 2003), such as Ca2+ signaling, Ca2+ dependent exocytosis and stimulation of oxidative metabolism and ATP production (Rizzuto, 2001; Gunter et al., 2004).

Conversely, mitochondrial Ca2+ overload and dysfunction, due to excitotoxic activation of glutamate receptors, is a crucial early event which follows ischemic or traumatic brain injury (Nicholls et al., 2007). Evidence for mitochondrial Ca2+ accumulation after excitotoxic stimulation comes from experimental studies which support the idea that mitochondrial depolarization during glutamate exposure is neuroprotective (Pivovarova et al., 2004), while its reduction correlates with excitotoxicity (Ward et al., 2007). In addition, activation of apoptosis has been documented after brain ischemia in several studies (Cao et al., 2003; Endo et al., 2006), and that this phenomenon might be closely linked to mitochondrial dysfunction. In fact, mitochondrial dysfunction provoked activation of apoptotic machinery by direct triggering of cytochrome c release (Clayton et al., 2005), or induction of Bax-dependent neuronal apoptosis through mitochondrial oxidative damage (Endo et al., 2006).

Mitochondria are involved in the control of neuronal Ca2+ homeostasis and neuronal Ca2+ signaling. In a series of recent papers (Racay et al., 2007, 2009a,b,c), authors have studied the effect of global cerebral ischemia/reperfusion injury (IRI) and ischemic tolerance developed by prior ischemic non-injurious stimulus – preconditioning- ischemic preconditioning (IPC) on mitochondrial **Ca2+homeostasis** and mitochondrial way of **apoptosis**. As documented by Racay et al. (2007, 2009a), global ischemia led to progressive decrease of complex I activity after IRI to 65.7% of control at 24 h after reperfusion. In preconditioned animals, the activity of complex I was also significantly inhibited after ischemia (to 65.4% of control) and ischemia/reperfusion for 1, 3, and 24 h (62-78% of control). Although the values in preconditioned animals were significantly smaller compared to naive ischemia, IPC did not protect complex I from ischemia induced inhibition. On the other hand, activity of the terminal enzyme complex of respiratory chain, complex IV were slightly protected by IPC and the net effect of IPC was the shift of its minimal activity from 1 h to 3 h after reperfusion (Racay et al., 2009c).

protein levels and these authors also found the generally enhanced level of mRNA in simvastatin pre-treated animals. The maximal differences between naive ischemic and pretreated ischemic animals authors detected in protein levels of proteins ATF6 and XBP1. The level of ATF6 was 60% higher in simvastatin pre-treated animals, which might suggest that ATF6 is one of the main proteins targeted to enhance neuroprotective effect at the ER gene

In conclusion, these data indicate that statins, in addition to their cholesterol-lowering effect may exert a neuroprotective role in the attenuation of ER stress response after acute

**Mitochondria** are important regulators of neuronal cell life and death through their role in metabolic energy production and involvement in apoptosis (Yuan and Yanker, 2000). Remarkably, mitochondrial dysfunction is considered to be one of the key events linking ischemic/recirculation insult with neuronal cell death (Berridge et al., 2003). In addition, mitochondria play a dual role in intracellular calcium. They are involved in the normal control of neuronal Ca2+ homeostasis (Berridge et al., 2003), such as Ca2+ signaling, Ca2+ dependent exocytosis and stimulation of oxidative metabolism and ATP production

Conversely, mitochondrial Ca2+ overload and dysfunction, due to excitotoxic activation of glutamate receptors, is a crucial early event which follows ischemic or traumatic brain injury (Nicholls et al., 2007). Evidence for mitochondrial Ca2+ accumulation after excitotoxic stimulation comes from experimental studies which support the idea that mitochondrial depolarization during glutamate exposure is neuroprotective (Pivovarova et al., 2004), while its reduction correlates with excitotoxicity (Ward et al., 2007). In addition, activation of apoptosis has been documented after brain ischemia in several studies (Cao et al., 2003; Endo et al., 2006), and that this phenomenon might be closely linked to mitochondrial dysfunction. In fact, mitochondrial dysfunction provoked activation of apoptotic machinery by direct triggering of cytochrome c release (Clayton et al., 2005), or induction of Bax-dependent neuronal apoptosis through mitochondrial

Mitochondria are involved in the control of neuronal Ca2+ homeostasis and neuronal Ca2+ signaling. In a series of recent papers (Racay et al., 2007, 2009a,b,c), authors have studied the effect of global cerebral ischemia/reperfusion injury (IRI) and ischemic tolerance developed by prior ischemic non-injurious stimulus – preconditioning- ischemic preconditioning (IPC) on mitochondrial **Ca2+homeostasis** and mitochondrial way of **apoptosis**. As documented by Racay et al. (2007, 2009a), global ischemia led to progressive decrease of complex I activity after IRI to 65.7% of control at 24 h after reperfusion. In preconditioned animals, the activity of complex I was also significantly inhibited after ischemia (to 65.4% of control) and ischemia/reperfusion for 1, 3, and 24 h (62-78% of control). Although the values in preconditioned animals were significantly smaller compared to naive ischemia, IPC did not protect complex I from ischemia induced inhibition. On the other hand, activity of the terminal enzyme complex of respiratory chain, complex IV were slightly protected by IPC and the net effect of IPC was the shift of its minimal activity from 1 h to 3 h after reperfusion

**4. Impact of IRI and IPC on mitochondrial calcium transport, p53** 

level during first two hours of reperfusion.

**translocation and neuronal apoptosis** 

ischemic/reperfusion insult.

(Rizzuto, 2001; Gunter et al., 2004).

oxidative damage (Endo et al., 2006).

(Racay et al., 2009c).

Mitochondrial dysfunction and oxidative stress were often implicated in pathophysiology of neurodegenerative diseases, including cerebral ischemia (Lin and Beal, 2006). Inhibition of complex I itself or in combination with elevated Ca2+ led to enhanced ROS production in different *in vitro* and *in vivo* systems (Yadava and Nicholls, 2007). Importantly, an enhanced production of ROS and consequent induction of p53-dependent apoptosis due to damage to neuronal DNA has also been documented after inhibition of complex I. A recent study showed that spare respiratory capacity rather then oxidative stress is involved in excitotoxic cell death (Yadava and Nicholls, 2007).

As shown by experimental and clinical studies, IRI –induced mitochondrial pathway of apoptosis is an important event leading to neuronal cell death after blood flow arrest. Impact of IRI and ischemic preconditioning on the level of apoptotic and anti-apoptotic proteins was assessed in both cortical and hippocampal mitochondria by Western blot analysis of p53, bax, and bcl-x (Racay et al., 2007, 2009b). Remarkably, IRI led to increase of p53 level in hippocampal mitochondria, with significant differences after 3 h (217.1 ± 42.2% of control), 24 h (286.8 ± 65% of control), and 72 h (232.9 ± 37.3% of control) of reperfusion. Interestingly, translocation of p53 to mitochondria was observed in hippocampus but not in cerebral cortex. However, levels of both the apoptotic proteins bax and the anti-apoptotic bcl-xl were unchanged in both hippocampal and cortical mitochondria. Ischemia-induced translocation of p53 to mitochondria was completely abolished by IPC since no significant changes in mitochondrial p53 level were observed after preconditioned ischemia. Similar to naive ischemia, the levels of both bax and bcl-xl were not affected by IPC. In addition, IPC had significant protective effect on ischemia-induced DNA fragmentation, as well as on number of positive Fluoro-Jade C staining cells. Thus, it indicates that IPC abolished almost completely both initiation and execution of mitochondrial apoptosis induced by global brain ischemia in vulnerable CA1 layer of rat hippocampus (Racay et al., 2007, 2009b).

The studies showed that ischemia induced inhibition of mitochondrial complexes I and IV, however inhibition is not accompanied by a decrease of mitochondrial Ca2+ uptake rate apparently due to the excess capacity of the complex I and complex IV. On the other hand, depressed activities of complex I and IV are conditions favourable of initiation of cell degenerative pathways, e.g. opening of mitochondrial permeability transition pore, ROS generation and apoptosis initiation, and might represent important mechanism of ischemic damage to neurons.

Accordingly, ischemic preconditioning acts at the level of both initiation and execution of ischemia-induced mitochondrial apoptosis affording protection from ischemia associated changes in integrity of mitochondrial membranes. IPC also activates inhibition of p53 translocation to mitochondria. Inhibition of the mitochondrial p53 pathway thus might provide a potentially important mechanism of neuronal survival in the face of ischemic brain damage (Otani, 2008).

### **5. Stress reaction of neuronal endoplasmic reticulum after IRI and IPC**

Ischemic tolerance can be developed by prior ischemic non-injurious stimulus or preconditioning. The molecular mechanisms underlying ischemic tolerance are not yet fully understood yet. Therefore a series of papers (Urban et al., 2009; Lehotsky et al., 2009; Pavlikova et al.,2009) have focused attention at the mRNA and protein levels of **the ER stress** genes after **ischemic/reperfusion damage (IRI)** in naive and preconditioned groups of rats.

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 251

proteins and hinders recovery from ischemia-induced ER dysfunction (Kumar et al., 2001; Paschen et al., 2003a) which possibly leads to a pro-apoptotic phenotype (DeGracia and Montie, 2004). Similarly, Thuerauf et al. (2006) found that myocardial ischemia activates UPR with the increased expression of XBP1 protein and XBP1-inducible protein. They contribute to protection of the myocardium during hypoxia. Also the results of Paschen et al. (2003a) using semi-quantitative RT-PCR showed a marked increase in XBP1 mRNA

Preischemia induced elevation of mRNA and protein GRP78 levels in reperfusion periods. GRP78 is a member of the 70kDa heat shock protein family that acts as a molecular chaperone in the folding and assembly of newly synthesized proteins within the ER. As shown by Yu et al. (1999) the suppression of GRP78 expression enhances apoptosis and disruption of cellular calcium homeostasis in hippocampal neurons that are exposed to excitotoxic and oxidative insults. This indicates that a raised level of GRP78 makes cells more resistant to the stressful conditions (Aoki et al. 2001). Similar results were obtained by Morimoto et al. (2007) in the focal ischemia model. Also Hayashi et al. (2003) and Garcia et al. (2004), who demonstrated an increase in GRP78 expression after 2 days of preconditioning proposed that the development of tolerance includes changes in PERK/GRP78 association, which were responsible for the decrease in eIF2a phosphorylation induced by preconditioning. On the other hand, Burda et al. (2003), failed to find any differences in the level of GRP78 protein in rats with or without acquired ischemic tolerance. This was probably due to exposure to very short reperfusion times. ATF6 is an ERmembrane-bound transcription factor activated by ER stress, which is specialized in the regulation of ER quality control proteins (Adachi et al., 2008). Haze et al. (1999) found that the overexpression of full-length ATF6 activates transcription of the GRP78 gene. Explanation of generally higher levels of protein p90ATF6 in preischemic group is probably connected to an increased promotor activity of GADD153 to UPR genes (Oyadomari et al.,

The data from these experiments (Urban et al. 2009; Lehotsky et al. 2009) suggest that IRI initiates time dependent differences in **endoplasmic reticular gene expression** at both the mRNA and protein levels and that endoplasmic gene expression is affected by preischemic treatment. These data and recent experiments of Bickler et al. (2009) also suggest that preconditioning paradigm (preischemia) may exert a role in the attenuation of ER stress response and that InsP3 receptor mediated Ca2+ signaling is an important mediator in the neuroprotective phenomenon of acquired ischemic tolerance. Changes in gene expression of the key proteins provide an insight into ER stress pathways. It also might suggest possible targets of future therapeutic interventions to enhance recovery after stroke (Yenari et al.,

**6. Effect of ischemic preconditioning on secretory pathways Ca2+-ATPase** 

The Golgi apparatus, as a part of **secretory pathways (SP)** in neural cells, represents a dynamic Ca2+ store. Ca2+ ions play an active role in processes such as secretion of neurotransmitters and secretory proteins for the growth/ reorganization of neuronal circuits, synaptic transmission, neural plasticity, and remodeling of dendrites (Michelangeli et al., 2005). In addition, SP are involved in the stress sensing, neuronal aging, and transduction of apoptotic signals (Maag et al., 2003; Sepulveda et al., 2008). On the other

levels after focal ischemia in the cerebral cortex.

2004).

2008; Pignataro et al., 2009).

**gene expression** 

In the UPR response, an activated IRE1 specifically cuts out the coding region of X-box protein 1 (XBP1) mRNA (Calfon et al., 2002) which after translation functions as a transcription factor specific for ER stress genes including GRP78 and GRP94. In these experiments, the hippocampal mRNA for XBP1 showed elevated levels in the naive IRI group of animals during the ischemic phase (about 43% ) as well as persistent nonsignificant changes in all other analyzed periods (Urban et al., 2009; Lehotsky et al., 2009).

**Preischemic treatment (IPC)** induces the level of hippocampal mRNA in ischemic phase only slight but not significant differences compared to controls, followed by significant decreases at 24 hours of reperfusion (by about 12.8 ± 1.4% compared to controls). When analyzed the translational product, the hippocampal **XBP1 protein** level in naive IRI animal group showed significant differences in ischemic phase (39.2 ± 1.6% compared to controls) and the levels were significantly elevated at later reperfusion periods (3 and 24 h) (82 ± 2.4% and 24.1± 1.6% respectively compared to controls). The influence of preischemia (IPC) on protein levels was significant mainly in later ischemic times. The protein level reached a maximum at 3 h of reperfusion (about 230% of controls) and stayed elevated in the later reperfusion (40.3 ± 4.9% compared to controls) (Urban et al., 2009; Lehotsky et al., 2009).

Endoplasmic reticular chaperone, the Ca2+ binding, **glucose regulated protein 78 (GRP78)** was shown to prevent neuronal damage (Morimoto et al., 2007). Under ER dysfunction and GRP78 dissociation it subsequently induced expression of ER stress genes. At the level of mRNA for GRP78 in hippocampus from naive IRI group of animals, the authors observed that maximal differences appeared in later reperfusion phases. Preischemic pretreatment (IPC) led to elevated mRNA hippocampal levels in the reperfusion period by about 11.7 ± 3.6 during the first hour and by about 8.7± 1.8% the next 24 hours of reperfusion in comparison to mRNA levels in corresponding ischemic/reperfusion times. Remarkably, the level of GRP78 protein in naive IRI showed rapid increases in ischemic time (by about 217% of controls) and remained elevated throughout 3 to 24 hours of reperfusion (about 213% and 43%, respectively, compared to controls). Increased mRNA values in preconditioned animals also corresponded with the significant increase of the levels of GRP78 protein. The changes are documented in the ischemic phase and also in all reperfusion times (by about 250% of controls and about 50% of corresponding ischemic/reperfusion times) (Urban et al., 2009; Lehotsky et al., 2009).

**ATF6** works as a key transcription factor in the resolution of the mammalian UPR (Yoshida et al. 2001). As shown in this experiment, the mRNA level for ATF6 in naive IRI animals showed gradual significant increases up to 24 hours of reperfusion (9.2 ± 4 % higher than control) and preconditioning (IPC) did not significantly alter mRNA levels in all analyzed periods. Similarl to mRNA levels, the hippocampal ATF6 protein level in naive IRI animals followed the same patterns. IPC on the other hand, induced remarkable changes in the protein levels at ischemic phase achieving significant increased levels (about 170%) in comparison to controls and stayed elevated in earlier reperfusion times (about 37 and 62 % higher than in controls) and later reperfusion time (about 15% of controls).

In general, IRI initiates suppression of global proteosynthesis, which is practically recovered in the reperfusion period with the exception of the most vulnerable neurons, such as pyramidal cells of CA1 hippocampal region (de la Vega et al., 2001). Ischemia is one of the strongest stimuli of gene induction in the brain. Different gene systems related to reperfusion processes of brain injury, repair and recovery are modulated (Gidday, 2006). In fact, IRI induces transient inhibition of translation, which prevents the expression of UPR

In the UPR response, an activated IRE1 specifically cuts out the coding region of X-box protein 1 (XBP1) mRNA (Calfon et al., 2002) which after translation functions as a transcription factor specific for ER stress genes including GRP78 and GRP94. In these experiments, the hippocampal mRNA for XBP1 showed elevated levels in the naive IRI group of animals during the ischemic phase (about 43% ) as well as persistent nonsignificant changes in all other analyzed periods (Urban et al., 2009; Lehotsky et al., 2009). **Preischemic treatment (IPC)** induces the level of hippocampal mRNA in ischemic phase only slight but not significant differences compared to controls, followed by significant decreases at 24 hours of reperfusion (by about 12.8 ± 1.4% compared to controls). When analyzed the translational product, the hippocampal **XBP1 protein** level in naive IRI animal group showed significant differences in ischemic phase (39.2 ± 1.6% compared to controls) and the levels were significantly elevated at later reperfusion periods (3 and 24 h) (82 ± 2.4% and 24.1± 1.6% respectively compared to controls). The influence of preischemia (IPC) on protein levels was significant mainly in later ischemic times. The protein level reached a maximum at 3 h of reperfusion (about 230% of controls) and stayed elevated in the later reperfusion (40.3 ± 4.9% compared to controls) (Urban et al., 2009; Lehotsky et al., 2009). Endoplasmic reticular chaperone, the Ca2+ binding, **glucose regulated protein 78 (GRP78)** was shown to prevent neuronal damage (Morimoto et al., 2007). Under ER dysfunction and GRP78 dissociation it subsequently induced expression of ER stress genes. At the level of mRNA for GRP78 in hippocampus from naive IRI group of animals, the authors observed that maximal differences appeared in later reperfusion phases. Preischemic pretreatment (IPC) led to elevated mRNA hippocampal levels in the reperfusion period by about 11.7 ± 3.6 during the first hour and by about 8.7± 1.8% the next 24 hours of reperfusion in comparison to mRNA levels in corresponding ischemic/reperfusion times. Remarkably, the level of GRP78 protein in naive IRI showed rapid increases in ischemic time (by about 217% of controls) and remained elevated throughout 3 to 24 hours of reperfusion (about 213% and 43%, respectively, compared to controls). Increased mRNA values in preconditioned animals also corresponded with the significant increase of the levels of GRP78 protein. The changes are documented in the ischemic phase and also in all reperfusion times (by about 250% of controls and about 50% of corresponding ischemic/reperfusion times) (Urban et al.,

**ATF6** works as a key transcription factor in the resolution of the mammalian UPR (Yoshida et al. 2001). As shown in this experiment, the mRNA level for ATF6 in naive IRI animals showed gradual significant increases up to 24 hours of reperfusion (9.2 ± 4 % higher than control) and preconditioning (IPC) did not significantly alter mRNA levels in all analyzed periods. Similarl to mRNA levels, the hippocampal ATF6 protein level in naive IRI animals followed the same patterns. IPC on the other hand, induced remarkable changes in the protein levels at ischemic phase achieving significant increased levels (about 170%) in comparison to controls and stayed elevated in earlier reperfusion times (about 37 and 62 %

In general, IRI initiates suppression of global proteosynthesis, which is practically recovered in the reperfusion period with the exception of the most vulnerable neurons, such as pyramidal cells of CA1 hippocampal region (de la Vega et al., 2001). Ischemia is one of the strongest stimuli of gene induction in the brain. Different gene systems related to reperfusion processes of brain injury, repair and recovery are modulated (Gidday, 2006). In fact, IRI induces transient inhibition of translation, which prevents the expression of UPR

higher than in controls) and later reperfusion time (about 15% of controls).

2009; Lehotsky et al., 2009).

proteins and hinders recovery from ischemia-induced ER dysfunction (Kumar et al., 2001; Paschen et al., 2003a) which possibly leads to a pro-apoptotic phenotype (DeGracia and Montie, 2004). Similarly, Thuerauf et al. (2006) found that myocardial ischemia activates UPR with the increased expression of XBP1 protein and XBP1-inducible protein. They contribute to protection of the myocardium during hypoxia. Also the results of Paschen et al. (2003a) using semi-quantitative RT-PCR showed a marked increase in XBP1 mRNA levels after focal ischemia in the cerebral cortex.

Preischemia induced elevation of mRNA and protein GRP78 levels in reperfusion periods. GRP78 is a member of the 70kDa heat shock protein family that acts as a molecular chaperone in the folding and assembly of newly synthesized proteins within the ER. As shown by Yu et al. (1999) the suppression of GRP78 expression enhances apoptosis and disruption of cellular calcium homeostasis in hippocampal neurons that are exposed to excitotoxic and oxidative insults. This indicates that a raised level of GRP78 makes cells more resistant to the stressful conditions (Aoki et al. 2001). Similar results were obtained by Morimoto et al. (2007) in the focal ischemia model. Also Hayashi et al. (2003) and Garcia et al. (2004), who demonstrated an increase in GRP78 expression after 2 days of preconditioning proposed that the development of tolerance includes changes in PERK/GRP78 association, which were responsible for the decrease in eIF2a phosphorylation induced by preconditioning. On the other hand, Burda et al. (2003), failed to find any differences in the level of GRP78 protein in rats with or without acquired ischemic tolerance. This was probably due to exposure to very short reperfusion times. ATF6 is an ERmembrane-bound transcription factor activated by ER stress, which is specialized in the regulation of ER quality control proteins (Adachi et al., 2008). Haze et al. (1999) found that the overexpression of full-length ATF6 activates transcription of the GRP78 gene. Explanation of generally higher levels of protein p90ATF6 in preischemic group is probably connected to an increased promotor activity of GADD153 to UPR genes (Oyadomari et al., 2004).

The data from these experiments (Urban et al. 2009; Lehotsky et al. 2009) suggest that IRI initiates time dependent differences in **endoplasmic reticular gene expression** at both the mRNA and protein levels and that endoplasmic gene expression is affected by preischemic treatment. These data and recent experiments of Bickler et al. (2009) also suggest that preconditioning paradigm (preischemia) may exert a role in the attenuation of ER stress response and that InsP3 receptor mediated Ca2+ signaling is an important mediator in the neuroprotective phenomenon of acquired ischemic tolerance. Changes in gene expression of the key proteins provide an insight into ER stress pathways. It also might suggest possible targets of future therapeutic interventions to enhance recovery after stroke (Yenari et al., 2008; Pignataro et al., 2009).

### **6. Effect of ischemic preconditioning on secretory pathways Ca2+-ATPase gene expression**

The Golgi apparatus, as a part of **secretory pathways (SP)** in neural cells, represents a dynamic Ca2+ store. Ca2+ ions play an active role in processes such as secretion of neurotransmitters and secretory proteins for the growth/ reorganization of neuronal circuits, synaptic transmission, neural plasticity, and remodeling of dendrites (Michelangeli et al., 2005). In addition, SP are involved in the stress sensing, neuronal aging, and transduction of apoptotic signals (Maag et al., 2003; Sepulveda et al., 2008). On the other

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 253

functional post-ischemic disturbances of neuronal ion transport mechanisms (Lipton, 1999; Lehotsky et al., 2002a; Obrenovitch, 2008) and inhibition of global proteosynthesis (Burda et al., 2003), which both are implicated in neuronal cell damage and/or recovery from ischemic

IPC caused significant reductions of LPO products and it reduced protein oxidative changes induced by ischemia in the hippocampal membranes in both the ischemic time and in reperfusion period. One of the possible explanations comes from the studies describing upregulation of defense mechanisms (antioxidant enzymes) against oxidative stress due to the preconditioning challenge (Danielisova et al., 2005; Gidday, 2006; Obrenovitch, 2008). In addition, forebrain ischemia causes small but significant drops in **the SPCA-associated Ca2+-ATPase activity** (by about 9%). The activity increases in early reperfusion times. However, it did not reach the control level and reached the highest depression after 24 h reperfusion to 88% of control. In the experiments, the IPC had a partial protective effect on the SPCA-associated Ca2+-ATPase activity. Ischemic insult after IPC pretreatment initiate only non-significant inhibition of Ca2+-ATPase activity compared to preconditioned control. After 1 and 3 h of reperfusion, the activity exceeded the control levels and reached it again after 24 h of reperfusion. However, the changes were not statistically significant at any reperfusion time. As shown in earlier studies, preconditioning upregulates defense mechanisms against oxidative stress (Danielisova et al., 2005; Gidday, 2006; Obrenovitch, 2008), which might partially restore the depression of enzyme activity. Additionally, as shown in the study by Western blot analysis, IPC induced an elevation of SPCA protein

In summary, the experiments conclusively showed that cerebral IRI-induced depression of SPCA activity and lipid and protein oxidation in rat hippocampal membranes. IRI also activates induction of SPCA1 gene expression in later reperfusion periods. IPC partially suppresses oxidative changes in hippocampal membranes and also partially restores the

In addition, IPC initiates earlier cellular response to the injury by the significant elevation of mRNA expression (to 142% comparing to 1 h of corresponding reperfusion) and to 154 and 111% comparing to 3 and 24 h of corresponding reperfusion, respectively. Similar patterns were observed on the translational level by Western blot analysis. Results of Pavlikova et al. (2009) indicate the specific SPCA1 expression pattern in injured ischemic hippocampus and might serve to understand the molecular mechanisms involved in the structural integrity and function of the Golgi complex after ischemic challenge. They also suggest for the correlation of SPCA function with the role of SP in response to preischemic challenge. Collective studies confirm, that reactive oxygen species (ROS) contribute to neuronal cell injuries secondary to ischemia and reperfusion (Lehotsky et al., 2004; Burda et al., 2005; Danielisova et al., 2005; Shi and Liu, 2007) and might initiate cell death signaling pathways after cerebral ischemia and parallels with selective post-ischemic vulnerability of the brain (Valko et al., 2007; Shi and Liu, 2007; Otani, 2008; Dirnagl et al., 2009). As shown by measurement of steady state fluorescence of ANS in hippocampal mitochondria (Racay et al., 2007, 2009a), naive IRI induced significant increase in ANS flurescence (it binds to hydrophobic part of membrane lipids and proteins) of the forebrain in both ischemic and reperfusion periods. These results support data from previous experiments (Lehotsky et al., 2004; Babusikova et al., 2008), which showed that IRI induced structural changes on hippocampal membrane lipids and both, the lipoperoxidation dependent and the direct

level in comparison to corresponding naive ischemic control.

ischemic-induced depression of SPCA activity.

insult.

hand, a high luminal Ca2+ concentration, and Mn2+, is required in the Golgi apparatus for the optimal activity of many enzymes and for post-translational processing and trafficking of the newly formed proteins. For both cytosolic and Golgi Ca2+ and Mn2+homeostasis, **the secretory pathway Ca2+-ATPases (SPCAs)** play an important role.

The SPCAs represent a subfamily of P-type ATPases related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and the plasma-membrane Ca2+-ATPase (PMCA) (Van Baelen et al., 2004; Murin et al., 2006). Two isoforms sharing 64% of sequence identity, namely SPCA1 and SPCA2, are expressed in mammalian cells (Wootton et al., 2004; Xiang et al., 2005). While SPCA2 expression seems to be more restricted to specific cell types, the SPCA1 is considered as a house-keeping isoform with pronounced expression in neural cells (Wootton et al., 2004; Murin et al., 2006; Sepulveda et al., 2008). The higher expression levels of SPCA1 in the brain coincide with a relatively high ratio of SPCA activity (thapsigargin insensitive) to the total activity of Ca2+-dependent ATPases. Therefore, implying a significant role of SPCA-facilitated transport of Ca2+ for calcium storage within the brain (Wootton et al., 2004).

As shown by previous studies, the SPCA plays a pivotal role in normal neural development, neural migration, and morphogenesis (Sepulveda et al., 2007, 2008). In addition, as shown in SPCA1 knockout mice, SPCA1 deficiency caused alteration in neural tube development and Golgi stress. These animals presented structural changes in the Golgi such as dilatation and the reduction in the number of stacked leaflets (Okunade et al., 2007). In apoptosis, a morphological change in the Golgi complex, for example its fragmentation, represents an early causative step rather than a secondary event, and it is very commonly associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, corticobasal degeneration, Alzheimer's and Creutzfeldt-Jacob diseases, and spinocerebelar ataxia type 2 (Gonatas et al., 2006).

### **6.1 Effect of oxidative damage on SPCA1**

Collective studies confirm that reactive oxygen species contribute to neuronal cell injuries secondary to ischemia and reperfusion (Lehotsky et al., 2004; Burda et al., 2005; Danielisova et al., 2005; Shi and Liu, 2007). Oxidative burst lasting several minutes upon the onset of reperfusion is followed by dysregulation of antioxidant mechanism and moderate but persistently elevated production of oxygen radicals which might initiate cell death signaling pathways after cerebral ischemia and parallels with selective postischemic vulnerability of the brain (Valko et al., 2007; Shi and Liu, 2007).

One of the main aims of the study of Pavlikova et al. (2009) was to determine whether IRI and IPC would affect the physical and functional properties of hippocampal membrane vesicles including Golgi SP. Neuronal microsomes are vulnerable to physical and functional oxidative damage (Lehotsky et al. 1999, 2002a; Urikova et al. 2006). The nature of the effect of free radicals on SPCA1 protein is not yet known. Authors show here for the first time that SPCA activity is also selectively damaged by free radicals in vitro, the property which is similar to other P-type ATPase such as SERCA and PMCA (Lehotsky et al. 2002b). In the study, authors showed that transient ischemia for 15 min induces considerable LPO and protein oxidation in hippocampal membranes. Protein oxidation pursues disturbances in oxidant/antioxidant balance and depression of enzymatic activities of main antioxidant enzymes detected at later stages after the ischemic insult (Lehotsky et al., 2002a; Urkova et al., 2006). Thus, oxidative alterations detected after IRI may at least partially explain

hand, a high luminal Ca2+ concentration, and Mn2+, is required in the Golgi apparatus for the optimal activity of many enzymes and for post-translational processing and trafficking of the newly formed proteins. For both cytosolic and Golgi Ca2+ and Mn2+homeostasis, **the** 

The SPCAs represent a subfamily of P-type ATPases related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and the plasma-membrane Ca2+-ATPase (PMCA) (Van Baelen et al., 2004; Murin et al., 2006). Two isoforms sharing 64% of sequence identity, namely SPCA1 and SPCA2, are expressed in mammalian cells (Wootton et al., 2004; Xiang et al., 2005). While SPCA2 expression seems to be more restricted to specific cell types, the SPCA1 is considered as a house-keeping isoform with pronounced expression in neural cells (Wootton et al., 2004; Murin et al., 2006; Sepulveda et al., 2008). The higher expression levels of SPCA1 in the brain coincide with a relatively high ratio of SPCA activity (thapsigargin insensitive) to the total activity of Ca2+-dependent ATPases. Therefore, implying a significant role of SPCA-facilitated transport of Ca2+ for calcium storage within the brain

As shown by previous studies, the SPCA plays a pivotal role in normal neural development, neural migration, and morphogenesis (Sepulveda et al., 2007, 2008). In addition, as shown in SPCA1 knockout mice, SPCA1 deficiency caused alteration in neural tube development and Golgi stress. These animals presented structural changes in the Golgi such as dilatation and the reduction in the number of stacked leaflets (Okunade et al., 2007). In apoptosis, a morphological change in the Golgi complex, for example its fragmentation, represents an early causative step rather than a secondary event, and it is very commonly associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, corticobasal degeneration, Alzheimer's and Creutzfeldt-Jacob diseases, and spinocerebelar ataxia type 2

Collective studies confirm that reactive oxygen species contribute to neuronal cell injuries secondary to ischemia and reperfusion (Lehotsky et al., 2004; Burda et al., 2005; Danielisova et al., 2005; Shi and Liu, 2007). Oxidative burst lasting several minutes upon the onset of reperfusion is followed by dysregulation of antioxidant mechanism and moderate but persistently elevated production of oxygen radicals which might initiate cell death signaling pathways after cerebral ischemia and parallels with selective postischemic vulnerability of

One of the main aims of the study of Pavlikova et al. (2009) was to determine whether IRI and IPC would affect the physical and functional properties of hippocampal membrane vesicles including Golgi SP. Neuronal microsomes are vulnerable to physical and functional oxidative damage (Lehotsky et al. 1999, 2002a; Urikova et al. 2006). The nature of the effect of free radicals on SPCA1 protein is not yet known. Authors show here for the first time that SPCA activity is also selectively damaged by free radicals in vitro, the property which is similar to other P-type ATPase such as SERCA and PMCA (Lehotsky et al. 2002b). In the study, authors showed that transient ischemia for 15 min induces considerable LPO and protein oxidation in hippocampal membranes. Protein oxidation pursues disturbances in oxidant/antioxidant balance and depression of enzymatic activities of main antioxidant enzymes detected at later stages after the ischemic insult (Lehotsky et al., 2002a; Urkova et al., 2006). Thus, oxidative alterations detected after IRI may at least partially explain

**secretory pathway Ca2+-ATPases (SPCAs)** play an important role.

(Wootton et al., 2004).

(Gonatas et al., 2006).

**6.1 Effect of oxidative damage on SPCA1** 

the brain (Valko et al., 2007; Shi and Liu, 2007).

functional post-ischemic disturbances of neuronal ion transport mechanisms (Lipton, 1999; Lehotsky et al., 2002a; Obrenovitch, 2008) and inhibition of global proteosynthesis (Burda et al., 2003), which both are implicated in neuronal cell damage and/or recovery from ischemic insult.

IPC caused significant reductions of LPO products and it reduced protein oxidative changes induced by ischemia in the hippocampal membranes in both the ischemic time and in reperfusion period. One of the possible explanations comes from the studies describing upregulation of defense mechanisms (antioxidant enzymes) against oxidative stress due to the preconditioning challenge (Danielisova et al., 2005; Gidday, 2006; Obrenovitch, 2008). In addition, forebrain ischemia causes small but significant drops in **the SPCA-associated Ca2+-ATPase activity** (by about 9%). The activity increases in early reperfusion times. However, it did not reach the control level and reached the highest depression after 24 h reperfusion to 88% of control. In the experiments, the IPC had a partial protective effect on the SPCA-associated Ca2+-ATPase activity. Ischemic insult after IPC pretreatment initiate only non-significant inhibition of Ca2+-ATPase activity compared to preconditioned control. After 1 and 3 h of reperfusion, the activity exceeded the control levels and reached it again after 24 h of reperfusion. However, the changes were not statistically significant at any reperfusion time. As shown in earlier studies, preconditioning upregulates defense mechanisms against oxidative stress (Danielisova et al., 2005; Gidday, 2006; Obrenovitch, 2008), which might partially restore the depression of enzyme activity. Additionally, as shown in the study by Western blot analysis, IPC induced an elevation of SPCA protein level in comparison to corresponding naive ischemic control.

In summary, the experiments conclusively showed that cerebral IRI-induced depression of SPCA activity and lipid and protein oxidation in rat hippocampal membranes. IRI also activates induction of SPCA1 gene expression in later reperfusion periods. IPC partially suppresses oxidative changes in hippocampal membranes and also partially restores the ischemic-induced depression of SPCA activity.

In addition, IPC initiates earlier cellular response to the injury by the significant elevation of mRNA expression (to 142% comparing to 1 h of corresponding reperfusion) and to 154 and 111% comparing to 3 and 24 h of corresponding reperfusion, respectively. Similar patterns were observed on the translational level by Western blot analysis. Results of Pavlikova et al. (2009) indicate the specific SPCA1 expression pattern in injured ischemic hippocampus and might serve to understand the molecular mechanisms involved in the structural integrity and function of the Golgi complex after ischemic challenge. They also suggest for the correlation of SPCA function with the role of SP in response to preischemic challenge.

Collective studies confirm, that reactive oxygen species (ROS) contribute to neuronal cell injuries secondary to ischemia and reperfusion (Lehotsky et al., 2004; Burda et al., 2005; Danielisova et al., 2005; Shi and Liu, 2007) and might initiate cell death signaling pathways after cerebral ischemia and parallels with selective post-ischemic vulnerability of the brain (Valko et al., 2007; Shi and Liu, 2007; Otani, 2008; Dirnagl et al., 2009). As shown by measurement of steady state fluorescence of ANS in hippocampal mitochondria (Racay et al., 2007, 2009a), naive IRI induced significant increase in ANS flurescence (it binds to hydrophobic part of membrane lipids and proteins) of the forebrain in both ischemic and reperfusion periods. These results support data from previous experiments (Lehotsky et al., 2004; Babusikova et al., 2008), which showed that IRI induced structural changes on hippocampal membrane lipids and both, the lipoperoxidation dependent and the direct

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 255

with several CNS disorders, such as stroke (Obeid et al., 2007), epilepsy (Sachdev, 2004; Herrmann et al., 2007), neurodegenerative (Clarke et al., 1998; Mattson et al., 2002) and neuropsychiatric diseases (Diaz-Arrastia, 2000; Bottiglieri, 2005), as well as inborn errors of metabolism (Mudd et al., 2001). In addition, even moderate hyperhomocysteinemia is a factor stimulating the development of dementia and Alzheimer's disease (Seshadri et al.,

Fig. 2. Cross-talk between the fuction of intracellular organelles which follows ischemic insults and results in injured phenopyte in vulnerable neurons. Adapted from Lehotsky et

Ischemic brain stroke in humans represents very complex cerebrovascular disease. A number of conventional risk factors for ischemic stroke are known, such as a previous occurrence of stroke, previous transient ischemic attack (TIA), arterial disease, atrial fibrillation, poor diet and/or obesity and physical inactivity (Prasad, 1999). It has been reported that hyperhomocysteinemia may also be associated with the incidence of ischemic brain stroke (Refsum et al., 1998), mainly due to pleiotropic activity of homocysteine and acceleration of atherosclerotic changes (Refsum et al., 1998; Thambyrajah et al., 2000). In fact, Hcy suppresses NO production by endothelial cells (Upchurch et al., 1997) and platelets (Mutus et al., 2001) and increases generation of reactive oxygen species (ROS) by the release of arachidonic acid from platelets (Signorello et al. 2002). It also inhibits glutathione peroxidase (Upchurch et al., 1997), and thus stimulates proliferation of endothelial cells

In addition, Hcy has been shown to inhibit methyltransferases, to suppress DNA reparation and to facilitate apoptosis when accumulated inside the cells (Duan et al., 2002; Kruman et al., 2002). Autooxidation of Hcy metabolites results in H202 accumulation (Gortz et al., 2004; Boldyrev, 2005) and long term incubation of neurons with Hcy metabolites induces necrotic cell death (Zieminska et al., 2003; Boldyrev et al., 2004). Homocysteine has also been shown to be elevated in other disorders of the CNS, e.g. Alzheimer disease or Parkinson disease

2002).

al. (2009c).

(Toohey, 2007).

(Jeremy et al., 1999; Domagala et al., 1998).

oxidative modifications of membrane proteins. Remarkably, preconditioning (IPC) induces significant decrease of ANS fluorescence, which indicates protective effect of IPC on mitochondrial membranes.

SP are involved in the stress sensing, neuronal aging and transduction of apoptotic signals (Maag et al., 2003; Sepulveda et al., 2008). In order to evaluate whether the severe metabolic stress induced by IRI and/or IPC affects transcription of SPCA1 gene, the mRNA and protein levels of SPCA1 was analyzed (Lehotsky, 1999, 2002a, 2004). As shown by Pavlikova et al. (2009), RT-PCR clearly detected, that hippocampal cells respond to the IRI by induction of mRNA level in reperfusion period with maximum at 3 h reperfusion (to 171% of control). Preconditioning (IPC) initiates earlier tissue response to the injury by the significant elevation of mRNA expression already at 1 h of reperfusion and the level of mRNA expression reached 142% comparing to 1 h ischemia, and to 164% comparing to control.

Neuronal microsomes are vulnerable to physical and functional oxidative damage (Lehotsky et al., 1999, 2002a, 2004; Urikova et al., 2006). The authors showed (Pavlikova et al. (2009) that SPCA activity, similar to other P-type ATPases, is also subject to ischemic damage most likely due to free radicals action (Lehotsky et al., 2002b). In addition, oxidative alterations detected in mitochondria and microsomes after IRI in our experiments, may at least partially explain functional postischemic disturbances of neuronal ion transport mechanisms (Lipton 1999; Lehotsky et al., 2002a; Obrenovitch, 2008) and inhibition of global proteosynthesis (Burda et al., 2003), which are both implicated in neuronal cell damage and/or recovery from ischemic insult, IPC-induced reduction of lipoperoxidation products and protein oxidative changes (Racay et al., 2009; Pavlikova et al., 2009). These may all be probably due to upregulation of defence mechanisms (antioxidant enzymes) against oxidative stress in the preconditioning challenge (Danielisova et al., 2005; Gidday, 2006; Obrenovitch, 2008).

One of the most pronounced morphological features following IRI is the mitochondrial and Golgi swelling and activation, which could be suppressed by neuroprotective treatment (Hicks and Machaner, 2005; Strosznajder et al., 2005; Gonatas et al., 2006). The secretory pathways are apparently involved in sensing stress and transducing signals during the execution phase of apoptosis (Maag et al., 2003; Hicks and Machamer, 2005). Data from Pavlikova (2009) showed a partial recovery of Ca2+-ATPase activity and earlier hippocampal response to later ischemia by the induction of mRNA and protein expression.

**Cross-talk** between the function of intracellular organelles following ischemic insult and reperfusion (Fig.2) and response of the tissue to the preischemic challenge (Fig 3) is depicted bellow .

### **6.2 Effect of hyperhomocysteinemia on SPCA expression**

**Homocysteine (Hcy)** is a sulfur-containing amino acid, which is derived from methionine metabolism. Hyperhomocysteinemia, condition in which Hcy concentration exceeds 16 µmol/l, is the result of perturbed Hcy metabolism and dietary deficiencies in folic acid, vitamin B6, and/or vitamin B12 (Obeid et al., 2007).

**Hyperhomocysteinemia** has been implicated as an independent risk factor for arteriosclerosis and coronary heart disease (Refsum et al., 1998; Thambyrajah et al., 2000). Severe forms of hyperhomocysteinemia results in convulsions and dementia (Watkins et al., 1989; van den Berg et al., 1995) corresponding multiple participation of homocysteine (Hcy) in diverse pathologies that affect the CNS. Likewise, homocysteine has also been associated

oxidative modifications of membrane proteins. Remarkably, preconditioning (IPC) induces significant decrease of ANS fluorescence, which indicates protective effect of IPC on

SP are involved in the stress sensing, neuronal aging and transduction of apoptotic signals (Maag et al., 2003; Sepulveda et al., 2008). In order to evaluate whether the severe metabolic stress induced by IRI and/or IPC affects transcription of SPCA1 gene, the mRNA and protein levels of SPCA1 was analyzed (Lehotsky, 1999, 2002a, 2004). As shown by Pavlikova et al. (2009), RT-PCR clearly detected, that hippocampal cells respond to the IRI by induction of mRNA level in reperfusion period with maximum at 3 h reperfusion (to 171% of control). Preconditioning (IPC) initiates earlier tissue response to the injury by the significant elevation of mRNA expression already at 1 h of reperfusion and the level of mRNA expression reached 142% comparing to 1 h ischemia, and to 164% comparing to control. Neuronal microsomes are vulnerable to physical and functional oxidative damage (Lehotsky et al., 1999, 2002a, 2004; Urikova et al., 2006). The authors showed (Pavlikova et al. (2009) that SPCA activity, similar to other P-type ATPases, is also subject to ischemic damage most likely due to free radicals action (Lehotsky et al., 2002b). In addition, oxidative alterations detected in mitochondria and microsomes after IRI in our experiments, may at least partially explain functional postischemic disturbances of neuronal ion transport mechanisms (Lipton 1999; Lehotsky et al., 2002a; Obrenovitch, 2008) and inhibition of global proteosynthesis (Burda et al., 2003), which are both implicated in neuronal cell damage and/or recovery from ischemic insult, IPC-induced reduction of lipoperoxidation products and protein oxidative changes (Racay et al., 2009; Pavlikova et al., 2009). These may all be probably due to upregulation of defence mechanisms (antioxidant enzymes) against oxidative stress in the preconditioning challenge (Danielisova et al., 2005; Gidday, 2006;

One of the most pronounced morphological features following IRI is the mitochondrial and Golgi swelling and activation, which could be suppressed by neuroprotective treatment (Hicks and Machaner, 2005; Strosznajder et al., 2005; Gonatas et al., 2006). The secretory pathways are apparently involved in sensing stress and transducing signals during the execution phase of apoptosis (Maag et al., 2003; Hicks and Machamer, 2005). Data from Pavlikova (2009) showed a partial recovery of Ca2+-ATPase activity and earlier hippocampal

**Cross-talk** between the function of intracellular organelles following ischemic insult and reperfusion (Fig.2) and response of the tissue to the preischemic challenge (Fig 3) is depicted

**Homocysteine (Hcy)** is a sulfur-containing amino acid, which is derived from methionine metabolism. Hyperhomocysteinemia, condition in which Hcy concentration exceeds 16 µmol/l, is the result of perturbed Hcy metabolism and dietary deficiencies in folic acid,

**Hyperhomocysteinemia** has been implicated as an independent risk factor for arteriosclerosis and coronary heart disease (Refsum et al., 1998; Thambyrajah et al., 2000). Severe forms of hyperhomocysteinemia results in convulsions and dementia (Watkins et al., 1989; van den Berg et al., 1995) corresponding multiple participation of homocysteine (Hcy) in diverse pathologies that affect the CNS. Likewise, homocysteine has also been associated

response to later ischemia by the induction of mRNA and protein expression.

**6.2 Effect of hyperhomocysteinemia on SPCA expression** 

vitamin B6, and/or vitamin B12 (Obeid et al., 2007).

mitochondrial membranes.

Obrenovitch, 2008).

bellow .

with several CNS disorders, such as stroke (Obeid et al., 2007), epilepsy (Sachdev, 2004; Herrmann et al., 2007), neurodegenerative (Clarke et al., 1998; Mattson et al., 2002) and neuropsychiatric diseases (Diaz-Arrastia, 2000; Bottiglieri, 2005), as well as inborn errors of metabolism (Mudd et al., 2001). In addition, even moderate hyperhomocysteinemia is a factor stimulating the development of dementia and Alzheimer's disease (Seshadri et al., 2002).

Fig. 2. Cross-talk between the fuction of intracellular organelles which follows ischemic insults and results in injured phenopyte in vulnerable neurons. Adapted from Lehotsky et al. (2009c).

Ischemic brain stroke in humans represents very complex cerebrovascular disease. A number of conventional risk factors for ischemic stroke are known, such as a previous occurrence of stroke, previous transient ischemic attack (TIA), arterial disease, atrial fibrillation, poor diet and/or obesity and physical inactivity (Prasad, 1999). It has been reported that hyperhomocysteinemia may also be associated with the incidence of ischemic brain stroke (Refsum et al., 1998), mainly due to pleiotropic activity of homocysteine and acceleration of atherosclerotic changes (Refsum et al., 1998; Thambyrajah et al., 2000). In fact, Hcy suppresses NO production by endothelial cells (Upchurch et al., 1997) and platelets (Mutus et al., 2001) and increases generation of reactive oxygen species (ROS) by the release of arachidonic acid from platelets (Signorello et al. 2002). It also inhibits glutathione peroxidase (Upchurch et al., 1997), and thus stimulates proliferation of endothelial cells (Jeremy et al., 1999; Domagala et al., 1998).

In addition, Hcy has been shown to inhibit methyltransferases, to suppress DNA reparation and to facilitate apoptosis when accumulated inside the cells (Duan et al., 2002; Kruman et al., 2002). Autooxidation of Hcy metabolites results in H202 accumulation (Gortz et al., 2004; Boldyrev, 2005) and long term incubation of neurons with Hcy metabolites induces necrotic cell death (Zieminska et al., 2003; Boldyrev et al., 2004). Homocysteine has also been shown to be elevated in other disorders of the CNS, e.g. Alzheimer disease or Parkinson disease (Toohey, 2007).

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 257

attributed to a part of the protective tolerant phenomenon induced by preconditioning

Fig. 4. Comparison of mRNA levels of SPCA1 between naive group (C-nai, Isch-nai), IPC group (C-IPC, Isch-IPC) and hyperhomocysteinemic group (C-Hcy, Isch-Hcy, Isch-IPC-Hcy) in rat cortex. Results are presented as mean ± SEM for *n* = 6. \* *p <* 0.05 compared to C-nai group, + *p <* 0.05 compared to C-IPC groups, † *p <* 0.05 compared to Isch-Hcy group, ‡ *p <*  0.05 compared to C-IPC group, ## *p <* 0.01 compared to Isch-IPC-Hcy group, && *p <* 0.01 compared to Isch-IPC-Hcy group. C-nai, control naive group; Isch-nai, ischemia naive group, C-IPC, control preischemic group; Isch-IPC, preischemic group; C-Hcy, control Hcy group; Isch-Hcy, ischemia Hcy group; Isch-IPC-Hcy, preischemic Hcy group. Adapted from

treatment.

Pavlikova et al. (2011).

A series of papers (Urban et al., 2009; Lehotsky et al., 2009b; Pavlikova et al.,2009) found that ischemia/reperfusion injury (IRI) initiates time dependent differences in endoplasmic reticular gene expression at both the mRNA and protein levels in rat hippocampus and that endoplasmic gene expression is affected by pre-ischemic treatment. More recently, Pavlikova et al. (2011) conducted an investigation into the differences between naive control and hyperhomocysteinemic control animals in each group independently. They showed for the first time that experimental 2 weeks hyperhomocysteinemia significantly decreased the level of SPCA1 mRNA gene expression in cerebral cortex which also led to the nonsignificantly decreased expression levels in hippocampal area. In cortex, ischemic challenge for 15 min. did not change significantly the level of mRNA SPCA1 expression in comparison to controls. Conversely, the gene response to pre-ischemic challenge was clearly shown within the homocysteine group by abrupt stimulation of the mRNA expression level to 249 % of hyperhomocysteinemic ischemic group and to 321% of hyperhomocysteinemic control. Notably, values far exceed those observed in the naive control. However, the effect of IPC challenge was not observed in the naive groups.

Fig. 3. Cross-talk between the fuction of intracellular organelles which follow preischemic maneuver and results in tolerant phenopyte in vulnerable neurons. Adapted from Lehotsky et al. (2009c).

The expression level decreased in the hyperhomocysteinemic control to 259% (p<0.05) of naive control and to 277% of control with IPC. When changes were compared between all ischemic groups, the following were observed: low level of mRNA expression in hyperhomocysteinemic ischemic group (to 201% of naive ischemia and to 185% of ischemic preconditioning. However, there were no significant differences between Hcy-control group and Hcy- ischemic group. Preischemic challenge initiated stimulation of the mRNA expression to 249% of hyperhomocysteinemic ischemic group. This response may be

A series of papers (Urban et al., 2009; Lehotsky et al., 2009b; Pavlikova et al.,2009) found that ischemia/reperfusion injury (IRI) initiates time dependent differences in endoplasmic reticular gene expression at both the mRNA and protein levels in rat hippocampus and that endoplasmic gene expression is affected by pre-ischemic treatment. More recently, Pavlikova et al. (2011) conducted an investigation into the differences between naive control and hyperhomocysteinemic control animals in each group independently. They showed for the first time that experimental 2 weeks hyperhomocysteinemia significantly decreased the level of SPCA1 mRNA gene expression in cerebral cortex which also led to the nonsignificantly decreased expression levels in hippocampal area. In cortex, ischemic challenge for 15 min. did not change significantly the level of mRNA SPCA1 expression in comparison to controls. Conversely, the gene response to pre-ischemic challenge was clearly shown within the homocysteine group by abrupt stimulation of the mRNA expression level to 249 % of hyperhomocysteinemic ischemic group and to 321% of hyperhomocysteinemic control. Notably, values far exceed those observed in the naive control. However, the effect of IPC

Fig. 3. Cross-talk between the fuction of intracellular organelles which follow preischemic maneuver and results in tolerant phenopyte in vulnerable neurons. Adapted from

The expression level decreased in the hyperhomocysteinemic control to 259% (p<0.05) of naive control and to 277% of control with IPC. When changes were compared between all ischemic groups, the following were observed: low level of mRNA expression in hyperhomocysteinemic ischemic group (to 201% of naive ischemia and to 185% of ischemic preconditioning. However, there were no significant differences between Hcy-control group and Hcy- ischemic group. Preischemic challenge initiated stimulation of the mRNA expression to 249% of hyperhomocysteinemic ischemic group. This response may be

challenge was not observed in the naive groups.

Lehotsky et al. (2009c).

attributed to a part of the protective tolerant phenomenon induced by preconditioning treatment.

Fig. 4. Comparison of mRNA levels of SPCA1 between naive group (C-nai, Isch-nai), IPC group (C-IPC, Isch-IPC) and hyperhomocysteinemic group (C-Hcy, Isch-Hcy, Isch-IPC-Hcy) in rat cortex. Results are presented as mean ± SEM for *n* = 6. \* *p <* 0.05 compared to C-nai group, + *p <* 0.05 compared to C-IPC groups, † *p <* 0.05 compared to Isch-Hcy group, ‡ *p <*  0.05 compared to C-IPC group, ## *p <* 0.01 compared to Isch-IPC-Hcy group, && *p <* 0.01 compared to Isch-IPC-Hcy group. C-nai, control naive group; Isch-nai, ischemia naive group, C-IPC, control preischemic group; Isch-IPC, preischemic group; C-Hcy, control Hcy group; Isch-Hcy, ischemia Hcy group; Isch-IPC-Hcy, preischemic Hcy group. Adapted from Pavlikova et al. (2011).

Mechanisms of Ischemic Induced Neuronal Death and Ischemic Tolerance 259

This study was supported by Grants VEGA 0049/09 from the Ministry of Education of the Slovak Republic, UK-55-15/07 from Ministry of Health of Slovak Republic, and APVV VVCE 0064-07 and by project "IDENTIFICATION OF NOVEL MARKERS IN DIAGNOSTIC PANEL OF NEUROLOGICAL DISEASES", code 26220220114, co-financed from EU sources and European Regional Development Fund. The authors are grateful to Dr N. A. Yeboah for

Adachi Y, Yamamoto K, Okada T, Yoshida H, Harada A, Mori K (2008) ATF6 is a

upregulates endothelial nitric oxide synthase in mice. *Stroke* pp. 32:980–986 Aoki M, Tamatani M, Taniguchi M, Yamaguchi A, Bando Y, Kasai K et al (2001)

Babusikova E., Jesenak M., Racay P., Dobrota D., Kaplan P. (2008): Oxidative alterations in

Balduini W, Mazzoni E, Carloni S, De Simoni MG, Perego C, Sironi L (2003) Prophylactic but

Barone FC, White RF, Spera PA, Ellison J, Currie RW, Wang X, Feuerstein GZ. 1998.

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receptors and NAD(P)H mediate Ca2+ signaling required for hypoxic

**8. Acknowledgments** 

**9. References** 

critical reading of the manuscript.

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In results of mRNA SPCA1 expression in hippocampal area no statistically significant changes were found between naive control and IPC control groups. Hyperhomocysteinemia for 14 days supressed mRNA expression, however the changes were not statistically significant. Similarly, as shown in the cortex, the preischemic challenge in hippocampal region initiated stimulation of the mRNA expression by 159% of hyperhomocysteinemic control and by to 131% hyperhomocysteinemic ischemic group. The suggestion was, that this response might also be part of the protective tolerant phenomenon induced by preconditioning treatment.

The previous results showed that IRI insult alters time expression profile of SPCA1 on mRNA and protein level (Pavlikova et al. 2009), and that preischemic challenge (induction of tolerance), not only preserved majority of surviving neuron but also activates partial recovery of the secretory pathways SPCA Ca2+-ATPase activity and earlier hippocampal response to later ischemia by the induction of SPCA1 mRNA and protein expression. We shown here for the first time that chemically induced experimental 2 weeks hyperhomocysteinemia significantly decreased the level of SPCA1 mRNA gene expression in cerebral cortex and also led to the non-significant decreased expression level in hippocampal area. There are no literature data on how the Hcy might affect the expression profile of the Ca2+-transport proteins in neuronal cells. In fact, the general mechanism of transcriptional regulation of SPCA1 gene is not yet fully understood. The transcription factors Sp1 and YY1 were shown to be involved in the gene regulation by the cis-enhancing elements in 5´-untranslated regions (Kawada et al., 2005). Another possibility is the expression of the putative endogenous activator of SPCA or the changes in local membrane environment are suggested as a cause for the increase in SPCA activity (Sepulveda et al. 2008). In fact, hyperhomocysteinemia often results in intracellular Ca2+ mobilization, endoplasmic reticulum (ER) stress, with the subsequent development of apoptotic events, chronic inflammation leading to endothelial dysfunction and remodeling of the extracellular matrix. Homocysteine has also been reported to induce modulation of gene expression through alteration of the methylation status (Dionisio, 2010).

In conclusion, our results indicate that chemically induced hyperhomocysteinemia initiates supression of the SPCA1 gene expression in both brain regions cerebral cortex and hippocampus. Documented response of SPCA gene to preischemic challenge in hyperhomocysteinemic group of animals might suggest for the correlation of SPCA expression with the role of secretory pathways in the proposed phenomenon of ischemic tolerance (Dirnagl et al., 2009; Pignataro et al., 2009). This might also serve to understand the molecular mechanisms involved in the structural integrity and function of the Golgi complex after ischemic challenge.

### **7. Conclusion**

Ischemic induced alterations of mitochondria, endoplasmic reticulum and Golgi apparatus shed more light on understanding the cross-talk between intracellular Ca2+ stores in cerebral ischemia/reperfusion injury. Documented neuroprotective response of intracellular organelles in the phenomenon of ischemic tolerance may also form a basis for future therapeutic interventions to enhance recovery from stroke. Finally, exploration of the protective mechanisms could lead to the recognition of newer strategies and suggestions for development of novel prophylactic/therapeutics for neuronal apoptosis-related diseases.

### **8. Acknowledgments**

258 Advances in the Preclinical Study of Ischemic Stroke

In results of mRNA SPCA1 expression in hippocampal area no statistically significant changes were found between naive control and IPC control groups. Hyperhomocysteinemia for 14 days supressed mRNA expression, however the changes were not statistically significant. Similarly, as shown in the cortex, the preischemic challenge in hippocampal region initiated stimulation of the mRNA expression by 159% of hyperhomocysteinemic control and by to 131% hyperhomocysteinemic ischemic group. The suggestion was, that this response might also be part of the protective tolerant phenomenon induced by

The previous results showed that IRI insult alters time expression profile of SPCA1 on mRNA and protein level (Pavlikova et al. 2009), and that preischemic challenge (induction of tolerance), not only preserved majority of surviving neuron but also activates partial recovery of the secretory pathways SPCA Ca2+-ATPase activity and earlier hippocampal response to later ischemia by the induction of SPCA1 mRNA and protein expression. We shown here for the first time that chemically induced experimental 2 weeks hyperhomocysteinemia significantly decreased the level of SPCA1 mRNA gene expression in cerebral cortex and also led to the non-significant decreased expression level in hippocampal area. There are no literature data on how the Hcy might affect the expression profile of the Ca2+-transport proteins in neuronal cells. In fact, the general mechanism of transcriptional regulation of SPCA1 gene is not yet fully understood. The transcription factors Sp1 and YY1 were shown to be involved in the gene regulation by the cis-enhancing elements in 5´-untranslated regions (Kawada et al., 2005). Another possibility is the expression of the putative endogenous activator of SPCA or the changes in local membrane environment are suggested as a cause for the increase in SPCA activity (Sepulveda et al. 2008). In fact, hyperhomocysteinemia often results in intracellular Ca2+ mobilization, endoplasmic reticulum (ER) stress, with the subsequent development of apoptotic events, chronic inflammation leading to endothelial dysfunction and remodeling of the extracellular matrix. Homocysteine has also been reported to induce modulation of gene expression

In conclusion, our results indicate that chemically induced hyperhomocysteinemia initiates supression of the SPCA1 gene expression in both brain regions cerebral cortex and hippocampus. Documented response of SPCA gene to preischemic challenge in hyperhomocysteinemic group of animals might suggest for the correlation of SPCA expression with the role of secretory pathways in the proposed phenomenon of ischemic tolerance (Dirnagl et al., 2009; Pignataro et al., 2009). This might also serve to understand the molecular mechanisms involved in the structural integrity and function of the Golgi

Ischemic induced alterations of mitochondria, endoplasmic reticulum and Golgi apparatus shed more light on understanding the cross-talk between intracellular Ca2+ stores in cerebral ischemia/reperfusion injury. Documented neuroprotective response of intracellular organelles in the phenomenon of ischemic tolerance may also form a basis for future therapeutic interventions to enhance recovery from stroke. Finally, exploration of the protective mechanisms could lead to the recognition of newer strategies and suggestions for development of novel prophylactic/therapeutics for neuronal apoptosis-related diseases.

through alteration of the methylation status (Dionisio, 2010).

complex after ischemic challenge.

**7. Conclusion** 

preconditioning treatment.

This study was supported by Grants VEGA 0049/09 from the Ministry of Education of the Slovak Republic, UK-55-15/07 from Ministry of Health of Slovak Republic, and APVV VVCE 0064-07 and by project "IDENTIFICATION OF NOVEL MARKERS IN DIAGNOSTIC PANEL OF NEUROLOGICAL DISEASES", code 26220220114, co-financed from EU sources and European Regional Development Fund. The authors are grateful to Dr N. A. Yeboah for critical reading of the manuscript.

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**13** 

*USA* 

**Mitochondrial Ceramide in Stroke** 

*Ralph H. Johnson Veterans Affairs Medical Center and the Departments of Neuroscience and Medicine of Medical University of South Carolina, Charleston, South Carolina,* 

Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation, migration, and apoptosis. Most sphingolipids are ubiquitous, but complex sphingolipids, including sphingomyelin (SM) and glycosphingolipids (GSLs), are more abundant in the brain and are particularly abundant in myelin. Sphingolipids are defined by the presence of a long-chain sphingoid backbone, generally sphingosine. Acylation of the sphingoid base, i.e. addition of a C14–C26 fatty acid to the amino group, yields ceramide, a building block for more complex sphingolipids. Neural cells are particularly enriched in GSLs and SM which is also a major lipid component of myelin. Sphingolipids are abundant in the plasma membranes and have unique molecular structures and conformational properties that cause them to form segregated compositional lipid domains in phospholipid bilayers (Sonnino et al., 2006). Membrane lipid domains, zones of the membrane with reduced fluidity, contain complex lipids of the cell, but are highly enriched in cholesterol and sphingolipids. Importantly, the proteins involved in signal transduction appear to segregate also in the lipid domains where they can exert their functions. Experimental evidence indicating that sphingolipids function through membrane reorganization and formation of lipid domains is summarized in a recent review by Kolesnick and Stancevic (Stancevic and Kolesnick, 2010). In addition to their role as building blocks of cellular membranes, sphingolipids have been reported to be pleotropic modulators of numerous enzymes in intracellular signaling pathways. Basic organization and specific principles of sphingolipid-mediated cell regulation have been reviewed by Hannun and Obeid (Hannun and Obeid, 2008, 2011). After more than a decade of extensive investigations, it has become clear that ceramide is a key sphingolipid messenger regulating a diverse range of cell-stress responses, including apoptosis, cell senescence, and autophagy. Ceramide is tightly regulated in cells, and its participation in cell death signaling pathways is controlled by rapid conversion of ceramide into less deleterious sphingolipids (**Scheme 1**). Thus, ceramide can be metabolized into complex sphingolipids by glucosylceramide synthase or into SM by SM synthase, or into ceramide-1-phosphate by ceramide kinase (Hannun and Obeid, 2002; Ogretmen and Hannun, 2004), or into sphingosine-1-phosphate by ceramidase and sphingosine kinase (Hannun and Obeid, 2008). However, pathological conditions, including cerebral ischemia/reperfusion, could disturb ceramide metabolism resulting in ceramide

**1. Introduction** 

accumulation that ultimately leads to cell death.

Tatyana I. Gudz and Sergei A. Novgorodov


### **Mitochondrial Ceramide in Stroke**

Tatyana I. Gudz and Sergei A. Novgorodov

*Ralph H. Johnson Veterans Affairs Medical Center and the Departments of Neuroscience and Medicine of Medical University of South Carolina, Charleston, South Carolina, USA* 

### **1. Introduction**

268 Advances in the Preclinical Study of Ischemic Stroke

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302–314. doi:10.1006/exnr.1998.7002

Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation, migration, and apoptosis. Most sphingolipids are ubiquitous, but complex sphingolipids, including sphingomyelin (SM) and glycosphingolipids (GSLs), are more abundant in the brain and are particularly abundant in myelin. Sphingolipids are defined by the presence of a long-chain sphingoid backbone, generally sphingosine. Acylation of the sphingoid base, i.e. addition of a C14–C26 fatty acid to the amino group, yields ceramide, a building block for more complex sphingolipids. Neural cells are particularly enriched in GSLs and SM which is also a major lipid component of myelin. Sphingolipids are abundant in the plasma membranes and have unique molecular structures and conformational properties that cause them to form segregated compositional lipid domains in phospholipid bilayers (Sonnino et al., 2006). Membrane lipid domains, zones of the membrane with reduced fluidity, contain complex lipids of the cell, but are highly enriched in cholesterol and sphingolipids. Importantly, the proteins involved in signal transduction appear to segregate also in the lipid domains where they can exert their functions. Experimental evidence indicating that sphingolipids function through membrane reorganization and formation of lipid domains is summarized in a recent review by Kolesnick and Stancevic (Stancevic and Kolesnick, 2010). In addition to their role as building blocks of cellular membranes, sphingolipids have been reported to be pleotropic modulators of numerous enzymes in intracellular signaling pathways. Basic organization and specific principles of sphingolipid-mediated cell regulation have been reviewed by Hannun and Obeid (Hannun and Obeid, 2008, 2011). After more than a decade of extensive investigations, it has become clear that ceramide is a key sphingolipid messenger regulating a diverse range of cell-stress responses, including apoptosis, cell senescence, and autophagy. Ceramide is tightly regulated in cells, and its participation in cell death signaling pathways is controlled by rapid conversion of ceramide into less deleterious sphingolipids (**Scheme 1**). Thus, ceramide can be metabolized into complex sphingolipids by glucosylceramide synthase or into SM by SM synthase, or into ceramide-1-phosphate by ceramide kinase (Hannun and Obeid, 2002; Ogretmen and Hannun, 2004), or into sphingosine-1-phosphate by ceramidase and sphingosine kinase (Hannun and Obeid, 2008). However, pathological conditions, including cerebral ischemia/reperfusion, could disturb ceramide metabolism resulting in ceramide

accumulation that ultimately leads to cell death.

Mitochondrial Ceramide in Stroke 271

**Sphingomyelin**

**SMase**

*De novo* **pathway Recycling or Salvage pathway**

**CH2O H O H**

**C H <sup>2</sup> O H O H N H O**

**GCase**

**NH 3 +**

**Sphingosine Kinase**

**Glucosylceramide**

**Complex sphingolipids**

**Sphingosine**

**Ceramidase**

**CERAMIDE**

**Sphingosine-1-phosphate (S1P)**

**Serine + Palmitoyl CoA**

**SPT 3-ketosphinganine**

**Sphinganine**

**SMase**

**Sphingomyelin**

**Sphingomyelinase pathway**

**C H <sup>2</sup> O H O H N H O**

**Fumonisin B1**

**Myriocin**

**LASS/CerS**

**Fumonisin B1**

Scheme 1. Biosynthesis of ceramide and its conversion into other bioactive sphingolipids. *De novo* ceramide synthesis begins with the conversion of serine and fatty acyl CoA into 3 ketosphinganine by serine palmitoyl transferase (SPT), then 3-ketosphinganine is converted into dihydrosphingosine. Myriocin is a potent inhibitor of SPT activity. (Dihydro) ceramide synthase (LASS/CerS) acylates dihydrosphingosine to form dihydroceramide, which is then reduced to ceramide by dihydroceramide desaturase. Ceramide is also produced by SMases

sphingosine, which is phosphorylated by sphingosine kinase (SK) to generate sphingosine-1-phosphate. Ceramide is phosphorylated by ceramide kinase (CK) yielding ceramide-1 phosphate (C1P). In the salvage or recycling pathway, complex sphingolipids are broken down to ceramide by glucosylceramidase (GCase) and then by ceramidase to sphingosine, which is re-acylated to ceramide by LASS/CerS. Fumonisin B1 inhibits LASS/CerS activity. resulted in increased levels of a specific subset of ceramide species. It has been demonstrated that CerS1 exhibits high specificity for C**18:0**-CoA generating C18:0-ceramide (Mizutani et al., 2005). CerS2, CerS4, and CerS3 appear to have broader specificity (Laviad et al., 2008; Mizutani et al., 2006). CerS2 or CerS4 mainly synthesizes C**20:0**-, C**22:0**-, C**24:1**-, C**24:0**-, C**26:1** and C**26:0** ceramide, but is unable to synthesize C**16:0**- or C**18:0**-ceramide (Laviad et al., 2008; Mizutani et al., 2005). CerS3 generates C**18:0**-, C**20:0**-, C**22:0**- and C**24:0**-ceramide (Mizutani et al., 2006). It has been shown that CerS5 generates C**14:0**-, C**16:0**-, C**18:0**-, and C**18:1**-ceramide (Lahiri and Futerman, 2005; Mizutani

through SM degradation in SMase pathway. Ceramidase converts ceramide into

et al., 2005); and CerS6 produces C**14:0**-, C**16:0**-, and C**18:0**-ceramide (Mizutani et al., 2005).

The availability of certain fatty acyl-CoA species and the characteristic distribution pattern of CerS family members in tissues seem to regulate the tissue-specificity of the ceramide species. Northern blot and real-time RT-PCR analysis revealed broad expression of CerS5, CerS4, and CerS6 genes in mammalian tissues, but CerS1 expression was limited to the brain and skeletal muscle (Laviad et al., 2008; Mizutani et al., 2006). Interestingly, CerS2 mRNA

**Ceramidase**

**Dihydroceramide**

**(Dihydro) ceramide synthase (LASS/CerS)**

**C1P**

**CK**

**Fatty acid acyl CoA**

**3-ketosphingane reductase**

**CERAMIDE Dihydroceramide desaturase**

**SM synthase**

### **2. Pathways of ceramide generation**

Ceramide is a family comprised of about 50 distinct molecular species characterized by various acyl chains, their desaturation, and hydroxylation. Ceramide is an Nacylsphingosine consisting of a fatty acid bound to the amino group of the sphingoid base, sphingosine. Ceramides can contain monounsaturated or saturated fatty acids of various lengths from 2 to 28 carbon atoms, and the fatty acid chain length profoundly alters ceramide's biophysical properties. Short-chain ceramides with fatty acyl chains of fewer than 12 carbons can be easily dispersed in water and serve as detergents (Sot et al., 2005b). In contrast, most ceramides found in mammalian cellular membranes contain long fatty acyl chains of 16-28 carbon atoms rendering them hydrophobic lipids lacking detergent properties. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or non-existent. *In situ* enzymatic generation, or external addition, of long-chain ceramides in membranes has at least three important effects: (i) the lipid monolayer has an increased tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure (Graham and Kozlov, 2010), (ii) the bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) a transbilayer (flip-flop) lipid motion is promoted (Goni et al., 2005).

As a result, ceramide metabolism is restricted to cellular membranes and is highly compartmentalized. Hydrophobic ceramides are generated by membrane-associated enzymes, and exert their effects either in close proximity to the generation site or require specific transport mechanisms to reach their targets in another intracellular compartment (Futerman and Riezman, 2005). Long-chain ceramides appear to be able to flip-flop across the membrane (Lopez-Montero et al., 2005); however, spontaneous inter-bilayer transfer is extremely slow (Contreras et al., 2010). Therefore, the transfer of ceramide between intracellular compartments is facilitated by vesicular transport pathways (Perry and Ridgway, 2005). Alternatively, ceramide is transported by a non-vesicular pathway involving a transfer protein, CERT, from its generation site in the endoplasmic reticulum (ER) to the Golgi where it is required for SM synthesis (Hanada et al., 2003). In addition to *de novo* biosynthesis, ceramide is generated by sphingomyelinases (SMases) from SM in two major pathways: the neutral SMase (nSMase)-dependent pathway and aSMase (aSMase) dependent pathway or salvage (recycling) pathway (**Scheme 1**).

### **2.1** *De novo* **ceramide biosynthesis**

Remarkable progress has been made toward identifying enzymes involved in ceramide biosynthesis (Futerman and Riezman, 2005). (Dihydro) ceramide synthase (EC 2.3.1.24) is a key enzyme in *de novo* ceramide synthasis, and it utilizes fatty acid acyl CoA for N-acylation of sphinganine (dihydrosphingosine) yielding dihydroceramide that is converted to ceramide by desaturase (**Scheme 1**). In yeast, longevity assurance gene 1 (LAG1) was identified as a component of ceramide synthase. Deletion of LAG1 in haploid cells resulted in a pronounced increase (~50%) in mean and maximum life spans (D'Mello N et al., 1994).

Mammalian homologs of LAG1, which belong to the LASS (longevity assurance gene homolog) family, were cloned and characterized (Futerman and Riezman, 2005). Each of the 6 known LASS (also known as CerS) genes appears to regulate synthesis of a specific subset of ceramides, and displays a unique substrate specificity profile for chain-length and/or saturation in fatty acid acyl CoA. Over-expression of any CerS protein in mammalian cells

Ceramide is a family comprised of about 50 distinct molecular species characterized by various acyl chains, their desaturation, and hydroxylation. Ceramide is an Nacylsphingosine consisting of a fatty acid bound to the amino group of the sphingoid base, sphingosine. Ceramides can contain monounsaturated or saturated fatty acids of various lengths from 2 to 28 carbon atoms, and the fatty acid chain length profoundly alters ceramide's biophysical properties. Short-chain ceramides with fatty acyl chains of fewer than 12 carbons can be easily dispersed in water and serve as detergents (Sot et al., 2005b). In contrast, most ceramides found in mammalian cellular membranes contain long fatty acyl chains of 16-28 carbon atoms rendering them hydrophobic lipids lacking detergent properties. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or non-existent. *In situ* enzymatic generation, or external addition, of long-chain ceramides in membranes has at least three important effects: (i) the lipid monolayer has an increased tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure (Graham and Kozlov, 2010), (ii) the bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) a transbilayer (flip-flop)

As a result, ceramide metabolism is restricted to cellular membranes and is highly compartmentalized. Hydrophobic ceramides are generated by membrane-associated enzymes, and exert their effects either in close proximity to the generation site or require specific transport mechanisms to reach their targets in another intracellular compartment (Futerman and Riezman, 2005). Long-chain ceramides appear to be able to flip-flop across the membrane (Lopez-Montero et al., 2005); however, spontaneous inter-bilayer transfer is extremely slow (Contreras et al., 2010). Therefore, the transfer of ceramide between intracellular compartments is facilitated by vesicular transport pathways (Perry and Ridgway, 2005). Alternatively, ceramide is transported by a non-vesicular pathway involving a transfer protein, CERT, from its generation site in the endoplasmic reticulum (ER) to the Golgi where it is required for SM synthesis (Hanada et al., 2003). In addition to *de novo* biosynthesis, ceramide is generated by sphingomyelinases (SMases) from SM in two major pathways: the neutral SMase (nSMase)-dependent pathway and aSMase (aSMase)-

Remarkable progress has been made toward identifying enzymes involved in ceramide biosynthesis (Futerman and Riezman, 2005). (Dihydro) ceramide synthase (EC 2.3.1.24) is a key enzyme in *de novo* ceramide synthasis, and it utilizes fatty acid acyl CoA for N-acylation of sphinganine (dihydrosphingosine) yielding dihydroceramide that is converted to ceramide by desaturase (**Scheme 1**). In yeast, longevity assurance gene 1 (LAG1) was identified as a component of ceramide synthase. Deletion of LAG1 in haploid cells resulted in a pronounced increase (~50%) in mean and maximum life spans (D'Mello N et al., 1994). Mammalian homologs of LAG1, which belong to the LASS (longevity assurance gene homolog) family, were cloned and characterized (Futerman and Riezman, 2005). Each of the 6 known LASS (also known as CerS) genes appears to regulate synthesis of a specific subset of ceramides, and displays a unique substrate specificity profile for chain-length and/or saturation in fatty acid acyl CoA. Over-expression of any CerS protein in mammalian cells

**2. Pathways of ceramide generation** 

lipid motion is promoted (Goni et al., 2005).

**2.1** *De novo* **ceramide biosynthesis** 

dependent pathway or salvage (recycling) pathway (**Scheme 1**).

Scheme 1. Biosynthesis of ceramide and its conversion into other bioactive sphingolipids. *De novo* ceramide synthesis begins with the conversion of serine and fatty acyl CoA into 3 ketosphinganine by serine palmitoyl transferase (SPT), then 3-ketosphinganine is converted into dihydrosphingosine. Myriocin is a potent inhibitor of SPT activity. (Dihydro) ceramide synthase (LASS/CerS) acylates dihydrosphingosine to form dihydroceramide, which is then reduced to ceramide by dihydroceramide desaturase. Ceramide is also produced by SMases through SM degradation in SMase pathway. Ceramidase converts ceramide into sphingosine, which is phosphorylated by sphingosine kinase (SK) to generate sphingosine-1-phosphate. Ceramide is phosphorylated by ceramide kinase (CK) yielding ceramide-1 phosphate (C1P). In the salvage or recycling pathway, complex sphingolipids are broken down to ceramide by glucosylceramidase (GCase) and then by ceramidase to sphingosine, which is re-acylated to ceramide by LASS/CerS. Fumonisin B1 inhibits LASS/CerS activity.

resulted in increased levels of a specific subset of ceramide species. It has been demonstrated that CerS1 exhibits high specificity for C**18:0**-CoA generating C18:0-ceramide (Mizutani et al., 2005). CerS2, CerS4, and CerS3 appear to have broader specificity (Laviad et al., 2008; Mizutani et al., 2006). CerS2 or CerS4 mainly synthesizes C**20:0**-, C**22:0**-, C**24:1**-, C**24:0**-, C**26:1** and C**26:0** ceramide, but is unable to synthesize C**16:0**- or C**18:0**-ceramide (Laviad et al., 2008; Mizutani et al., 2005). CerS3 generates C**18:0**-, C**20:0**-, C**22:0**- and C**24:0**-ceramide (Mizutani et al., 2006). It has been shown that CerS5 generates C**14:0**-, C**16:0**-, C**18:0**-, and C**18:1**-ceramide (Lahiri and Futerman, 2005; Mizutani et al., 2005); and CerS6 produces C**14:0**-, C**16:0**-, and C**18:0**-ceramide (Mizutani et al., 2005).

The availability of certain fatty acyl-CoA species and the characteristic distribution pattern of CerS family members in tissues seem to regulate the tissue-specificity of the ceramide species. Northern blot and real-time RT-PCR analysis revealed broad expression of CerS5, CerS4, and CerS6 genes in mammalian tissues, but CerS1 expression was limited to the brain and skeletal muscle (Laviad et al., 2008; Mizutani et al., 2006). Interestingly, CerS2 mRNA

Mitochondrial Ceramide in Stroke 273

(Clarke et al., 2011). nSMase1 is localized to the ER and nucleus (Tomiuk et al., 2000). nSMase2 had a dynamic intracellular localization (Clarke et al., 2006), having been found in the Golgi of sub-confluent cells, at the plasma membrane at the regions of cell-cell contact (Marchesini et al., 2004) and in recycling compartments (Milhas et al., 2010). Furthermore, it appears that oxidative stress could induce nSMase2 trafficking to the plasma membrane, whereas antioxidant (glutathione) directed its translocation to the perinuclear region (Levy et al., 2006). MA-nSMase is found within the mitochondria and associated membranes (Wu et al., 2010). The physiological role of neutral SMase isoforms may be dictated by their immediate environment in the specific intracellular compartment. **Alkaline SMase** lacks homology to neutral or aSMase and its mRNA is abundant in the intestine where the

Ceramide is also produced during the recycling of sphingosine in the process termed the "salvage pathway" (Kitatani et al., 2008). In this process, complex sphingolipids are broken down to ceramide and then to sphingosine, which is then used by ceramide synthase to yield ceramide. SM is converted to ceramide by aSMase. Ceramide accumulation via the salvage pathway requires ceramide synthase which is important in *de novo* synthesis of ceramide. Complex sphingolipids undergo constitutive degradation in the late endosomes and the lysosomes yielding ceramide which does not leave the lysosomes (Chatelut et al., 1998) unless converted into sphingosine by acid ceramidase. Free sphingosine could be

released from the lysosomes and re-acylated by ceramide synthase to form ceramide.

Mitochondria arise as important intracellular compartment for ceramide metabolism, and they have been shown to contain a variety of sphingolipids, including SM and ceramide (Ardail et al., 2001; Tserng and Griffin, 2003). Mounting evidence suggests a local action of ceramide on mitochondria in intact cells. Thus, selective hydrolysis of a mitochondrial pool of SM by overexpressed sphingomyelinase (bSMase) targeted to mitochondria resulted in apoptosis. In contrast, generation of ceramide in the plasma membrane, ER, or Golgi apparatus by bSMase targeted to these compartments had no effect on cell viability (Birbes et al., 2001). Recently, mitochondrial ceramide engagement in apoptosis has been shown using loss-of-function mutants of ceramide synthase in the germ cell line of C. *elegans* (Deng et al., 2008). In this study, an ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase, and restored upon microinjection of long-chain ceramide. Radiation-induced increase in ceramide localized to mitochondria was required for activation of CED-3 caspase and apoptosis. These studies underscore the physiological significance of the mitochondrial ceramide and SM pools (Andreyev et al., 2010; Ardail et al., 2001; Dai et al., 2004; Monette et al., 2010; Tserng and Griffin, 2003; Yabu

Although several enzyme activities involved in ceramide metabolism have been demonstrated in mitochondria, the nature of enzymes generating ceramide in this organelle is still a matter of debate (Laviad et al., 2008). Mitochondria evolve as a specialized compartment of sphingolipid metabolism with their own subset of ceramide synthesizing and degrading enzymes. Three possibilities may account for ceramide generation in

enzyme plays a major role in digestion of dietary SM (Nilsson and Duan, 2006).

**2.3 Recycling or salvage pathway** 

**3. Ceramide generation in mitochondria** 

et al., 2009).

mitochondria.

was more abundant than other CerS family members and had the broadest tissue distribution (Laviad et al., 2008). Except for a weak display in skin, CerS3 mRNA expression is limited almost solely to testis, implying that CerS3 plays an important role in this gland (Mizutani et al., 2006).

CerS are integral membrane proteins, but the exact number of transmembrane domains and their topology has not been resolved experimentally. All of the CerS genes have a highly conserved stretch of 52 amino acids known as the Lag1p motif which is essential for enzyme activity (Spassieva et al., 2006). Some of the CerS proteins are post-translationally modified, and, for instance, CerS6 is expressed as a native and an N-glycosylated form. The Nglycosylation site is conserved in CerS6, CerS2, and CerS5, but this post-translational modification is not required for ceramide synthase activity (Mizutani et al., 2005). Intriguingly, CerS1 phosphorylation appears to regulate the protein turnover (Sridevi et al., 2009)*.* All CerS except CerS1 contain a homeobox domain, suggesting involvement in developmental regulation (Venkataraman and Futerman, 2002). *De novo* synthesis of ceramide is required for cell survival *in vivo*, and is widespread among cell types and tissues. Regulation of ceramide synthesis is only beginning to be understood. Regulation at the transcriptional level has been observed with a number of agents, including endotoxin and cytokines, UVB irradiation, and retinoic acid (Merrill, 2002).

*De novo* ceramide biosynthesis occurs in the endoplasmic reticulum (ER) where all the participating enzymes have been found (Hirschberg et al., 1993; Mandon et al., 1992; Michel and van Echten-Deckert, 1997). Ceramide is synthesized at the cytosolic side of the ER (Mandon et al., 1992; Merrill, 2002), serving as a precursor for the biosynthesis of glycosphingolipids and SM in the Golgi (Futerman et al., 1990; Kolter et al., 2002).

### **2.2 Sphingomyelin hydrolysis**

SM hydrolysis by one of several SMases is another source of cellular ceramide. Three groups of SMases, acid, neutral, and alkaline, are distinguished by their primary structure, catalytic pH optimum, and localization.

### **2.2.1 Acid SMase**

A well-characterized enzyme, acid SMase (aSMase) contributes to the catabolism of SM and ceramide formation in lysosomes (Tani et al., 2005; Tani et al., 2007). aSMase could relocate from intracellular compartments to the plasma membrane where it plays an important role in SM hydrolysis and ceramide generation within lipid rafts (Bollinger et al., 2005). aSMase is a soluble enzyme with no transmembrane domains, and the mechanism of aSMase association with the membrane, at which its substrate, SM, resides, remains unclear. aSMase is also secreted through the Golgi secretory pathway, and it is constitutively present in plasma (Spence et al., 1989) where it is involved in hydrolysis of lipoprotein-bound SM, the second most abundant lipid in human plasma. Intriguingly, aSMase hydrolyzes SM bound to oxidized LDL more effectively than SM bound to intact LDL (Schissel et al., 1998). The accelerated hydrolysis of SM could enhance LDL aggregation leading to macrophage foam cell formation, suggesting a role for secretory aSMase in the pathogenesis of arteriosclerosis (Tabas, 1999).

### **2.2.2 Neutral SMase**

Three mammalian closely related isoforms of neutral SMase (nSMase) have been recently cloned, including nSMase1, nSMase2 and mitochondria-associated nSMase (MA-nSMase)

was more abundant than other CerS family members and had the broadest tissue distribution (Laviad et al., 2008). Except for a weak display in skin, CerS3 mRNA expression is limited almost solely to testis, implying that CerS3 plays an important role in this gland

CerS are integral membrane proteins, but the exact number of transmembrane domains and their topology has not been resolved experimentally. All of the CerS genes have a highly conserved stretch of 52 amino acids known as the Lag1p motif which is essential for enzyme activity (Spassieva et al., 2006). Some of the CerS proteins are post-translationally modified, and, for instance, CerS6 is expressed as a native and an N-glycosylated form. The Nglycosylation site is conserved in CerS6, CerS2, and CerS5, but this post-translational modification is not required for ceramide synthase activity (Mizutani et al., 2005). Intriguingly, CerS1 phosphorylation appears to regulate the protein turnover (Sridevi et al., 2009)*.* All CerS except CerS1 contain a homeobox domain, suggesting involvement in developmental regulation (Venkataraman and Futerman, 2002). *De novo* synthesis of ceramide is required for cell survival *in vivo*, and is widespread among cell types and tissues. Regulation of ceramide synthesis is only beginning to be understood. Regulation at the transcriptional level has been observed with a number of agents, including endotoxin

*De novo* ceramide biosynthesis occurs in the endoplasmic reticulum (ER) where all the participating enzymes have been found (Hirschberg et al., 1993; Mandon et al., 1992; Michel and van Echten-Deckert, 1997). Ceramide is synthesized at the cytosolic side of the ER (Mandon et al., 1992; Merrill, 2002), serving as a precursor for the biosynthesis of

SM hydrolysis by one of several SMases is another source of cellular ceramide. Three groups of SMases, acid, neutral, and alkaline, are distinguished by their primary structure, catalytic

A well-characterized enzyme, acid SMase (aSMase) contributes to the catabolism of SM and ceramide formation in lysosomes (Tani et al., 2005; Tani et al., 2007). aSMase could relocate from intracellular compartments to the plasma membrane where it plays an important role in SM hydrolysis and ceramide generation within lipid rafts (Bollinger et al., 2005). aSMase is a soluble enzyme with no transmembrane domains, and the mechanism of aSMase association with the membrane, at which its substrate, SM, resides, remains unclear. aSMase is also secreted through the Golgi secretory pathway, and it is constitutively present in plasma (Spence et al., 1989) where it is involved in hydrolysis of lipoprotein-bound SM, the second most abundant lipid in human plasma. Intriguingly, aSMase hydrolyzes SM bound to oxidized LDL more effectively than SM bound to intact LDL (Schissel et al., 1998). The accelerated hydrolysis of SM could enhance LDL aggregation leading to macrophage foam cell formation, suggesting a role for secretory aSMase in the pathogenesis of arteriosclerosis

Three mammalian closely related isoforms of neutral SMase (nSMase) have been recently cloned, including nSMase1, nSMase2 and mitochondria-associated nSMase (MA-nSMase)

glycosphingolipids and SM in the Golgi (Futerman et al., 1990; Kolter et al., 2002).

and cytokines, UVB irradiation, and retinoic acid (Merrill, 2002).

(Mizutani et al., 2006).

**2.2 Sphingomyelin hydrolysis** 

pH optimum, and localization.

**2.2.1 Acid SMase** 

(Tabas, 1999).

**2.2.2 Neutral SMase** 

(Clarke et al., 2011). nSMase1 is localized to the ER and nucleus (Tomiuk et al., 2000). nSMase2 had a dynamic intracellular localization (Clarke et al., 2006), having been found in the Golgi of sub-confluent cells, at the plasma membrane at the regions of cell-cell contact (Marchesini et al., 2004) and in recycling compartments (Milhas et al., 2010). Furthermore, it appears that oxidative stress could induce nSMase2 trafficking to the plasma membrane, whereas antioxidant (glutathione) directed its translocation to the perinuclear region (Levy et al., 2006). MA-nSMase is found within the mitochondria and associated membranes (Wu et al., 2010). The physiological role of neutral SMase isoforms may be dictated by their immediate environment in the specific intracellular compartment. **Alkaline SMase** lacks homology to neutral or aSMase and its mRNA is abundant in the intestine where the enzyme plays a major role in digestion of dietary SM (Nilsson and Duan, 2006).

### **2.3 Recycling or salvage pathway**

Ceramide is also produced during the recycling of sphingosine in the process termed the "salvage pathway" (Kitatani et al., 2008). In this process, complex sphingolipids are broken down to ceramide and then to sphingosine, which is then used by ceramide synthase to yield ceramide. SM is converted to ceramide by aSMase. Ceramide accumulation via the salvage pathway requires ceramide synthase which is important in *de novo* synthesis of ceramide. Complex sphingolipids undergo constitutive degradation in the late endosomes and the lysosomes yielding ceramide which does not leave the lysosomes (Chatelut et al., 1998) unless converted into sphingosine by acid ceramidase. Free sphingosine could be released from the lysosomes and re-acylated by ceramide synthase to form ceramide.

### **3. Ceramide generation in mitochondria**

Mitochondria arise as important intracellular compartment for ceramide metabolism, and they have been shown to contain a variety of sphingolipids, including SM and ceramide (Ardail et al., 2001; Tserng and Griffin, 2003). Mounting evidence suggests a local action of ceramide on mitochondria in intact cells. Thus, selective hydrolysis of a mitochondrial pool of SM by overexpressed sphingomyelinase (bSMase) targeted to mitochondria resulted in apoptosis. In contrast, generation of ceramide in the plasma membrane, ER, or Golgi apparatus by bSMase targeted to these compartments had no effect on cell viability (Birbes et al., 2001). Recently, mitochondrial ceramide engagement in apoptosis has been shown using loss-of-function mutants of ceramide synthase in the germ cell line of C. *elegans* (Deng et al., 2008). In this study, an ionizing radiation-induced apoptosis of germ cells was obliterated upon inactivation of ceramide synthase, and restored upon microinjection of long-chain ceramide. Radiation-induced increase in ceramide localized to mitochondria was required for activation of CED-3 caspase and apoptosis. These studies underscore the physiological significance of the mitochondrial ceramide and SM pools (Andreyev et al., 2010; Ardail et al., 2001; Dai et al., 2004; Monette et al., 2010; Tserng and Griffin, 2003; Yabu et al., 2009).

Although several enzyme activities involved in ceramide metabolism have been demonstrated in mitochondria, the nature of enzymes generating ceramide in this organelle is still a matter of debate (Laviad et al., 2008). Mitochondria evolve as a specialized compartment of sphingolipid metabolism with their own subset of ceramide synthesizing and degrading enzymes. Three possibilities may account for ceramide generation in mitochondria.

Mitochondrial Ceramide in Stroke 275

mitochondria, and that ceramide formation may occur from sphingosine and palmitoyl-CoA from coupled activities of a mitochondrial thioesterase and nCDase catalyzing the reverse reaction (**Scheme 2**). Another possibility is that ceramide could be also transported from the

**FFA**

**CoA Sph**

**NCD MTE**

**Mitochondrial membrane**

Scheme 2. Ceramide formation from Acyl-CoA and sphingosine (Sph) mediated by coupled

Ceramide accumulation has been demonstrated in various *in vivo* models of IR and it has been implicated as an important mediator of apoptosis in the injured tissue, but mechanisms of ceramide generation are not well-defined and the downstream targets of ceramide remain unresolved. The IR-induced accumulation of ceramide appears to be a general phenomenon for heart, kidney, liver and brain. The identification and characterization of key proteins of ceramide synthesis are expected to expand our understanding of molecular mechanisms

Research progress has been hampered by a lack of appropriate techniques that would allow simultaneous analysis of multiple sphingolipid species. Thus, the most common method for quantification of ceramide, the diglyceride (DG) kinase assay (Bielawska et al., 2001) has significant disadvantages including a limited separation of ceramide from dihydroceramide and the inability to determine the individual molecular species of ceramide. Recent advances in the development of new mass spectroscopy-based methods for quantitative analysis of sphingolipid molecular species may allow further dissection of ceramide specific

Increasing evidence suggests that the fatty acid chain of ceramide is an important determinant of the biological effect mediated by the individual ceramide species. Most of the experimental evidence indicating the important roles of ceramides containing distinct fatty acids is summarized in an excellent review by Futerman and his colleagues (Pewzner-Jung et al., 2006), and new studies further support the notion of distinct roles of ceramide species in cell metabolism(Hannun and Obeid, 2011). It has been demonstrated that generation of C18:0-ceramide, and not C16:0-ceramide repressed human telomerase reverse transcriptase promoter in lung carcinoma cells (Wooten-Blanks et al., 2007). Activation of aSMase in the salvage pathway brought about a selective accumulation of C16:0-ceramide (Chudakova et al., 2008; Kitatani et al., 2006) due to the involvement of ceramide synthase CerS5 localized in mitochondria-associated membranes (Kitatani et al., 2006). In another

**Acyl-CoA**

ER to mitochondria through the contact sites between them (Stiban et al., 2008).

**ceramide**

activities of mitochondrial thioesterase (MTE) and nCDase (NCD).

**4. Ceramide accumulation in Ischemia/Reperfusion (IR)** 

behind ceramide's involvement in IR-induced tissue damage.

pathways (Pettus et al., 2004; Sullards, 2000).

First, experimental evidence suggests the presence of ceramide synthase activity in mitochondria. Thus, ceramide synthase activity was first detected (Morell and Radin, 1970; Ullman and Radin, 1972) and partially purified from a bovine brain mitochondria-enriched fraction (Shimeno et al., 1995) which was not characterized in terms of marker enzyme activities. Mitochondrial enzymes had almost 2-fold higher specific ceramide synthase activity than the ceramide synthase from the ER. The mitochondrial enzyme had a pH optimum around 7.5 and maximal catalytic efficiency with C16:0- or C18:0-acyl CoA. The addition of liposomes to the mitochondrial enzyme increased ceramide synthase activity (approx. 7.8-fold) (Shimeno et al., 1995). Purification of ceramide synthase from bovine liver mitochondria yielded two major protein bands, 62 and 72 kDa on a SDS-gel (Shimeno et al., 1998). This enzyme had an apparent Km of 146 µM and Vmax of 11.1 nmol/min/mg protein with C18:0-acyl CoA, and corresponding values of Vmax 144 µM and 8.5 nmol/min/mg protein towards sphinganine.

Detailed analysis of ceramide synthase activity in highly purified mitochondria by Bionda *et al*. essentially confirmed previous findings (Bionda et al., 2004). Ceramide synthase activity was demonstrated in rat liver mitochondria and in the sub-compartment of the ER closely associated with mitochondria. Further sub-mitochondrial investigation of ceramide synthase activity revealed that both outer and inner mitochondrial membranes can synthesize ceramide (Bionda et al., 2004). Recent reports describing several ceramide synthase isoforms, including CerS1, CerS2, CerS4 and CerS6, in purified mouse brain mitochondria (Novgorodov et al., 2011a; Yu et al., 2007) support the notion that several ceramide synthesizing enzymes could be localized in mitochondria (Futerman, 2006). No such association was found in HeLa cells (Mesicek et al., 2010), suggesting that this might be a cell type/tissue specific event. The intramitochondrial localization of CerS was examined in purified brain mitochondria by immunoprecipitation (Novgorodov et al., 2011a). These studies reveal a selective CerS6 association with adenine nucleotide translocase, the inner membrane component of the mitochondrial permeability transition pore (MPTP). In contrast, CerS2 associated with the outer membrane resident protein Tom20, a receptor of the protein import complex. The data suggest CerS6/ceramide could regulate MPTP activity and mitochondrial Ca2+ homeostasis whereas CerS2/ceramide could modulate the mitochondrial protein import machinery.

Secondly, recent studies identified two novel SMases, which hydrolyze SM to ceramide, and phosphocholine in mitochondria from zebrafish (Yabu et al., 2009) and mouse tissues (Wu et al., 2010). Notably, in yeast, the mammalian nSMase ortholog Isc1p associates with mitochondria in the post-diauxic phase of yeast growth and regulates mitochondrial sphingolipid metabolism (Kitagaki et al., 2007; Vaena de Avalos et al., 2004).

Thirdly, the additional source of ceramide in mitochondria is a reverse reaction of a neutral ceramidase (nCDase), e.g., formation of ceramide as a result of condensation of palmitate and sphingosine (El Bawab et al., 2001). On the basis of molecular cloning and confocal microscopy data, this activity was ascribed to mitochondria (El Bawab et al., 2000), and it was demonstrated in purified mitochondria (Bionda et al., 2004). Recent studies describe the molecular mechanism of ceramide generation from palmitate and sphingosine in purified liver mitochondria that requires concerted action of two enzymes nCDase and thioesterase (which hydrolyzes palmitoyl-CoA to CoA and fatty acid)(Novgorodov et al., 2011b). Thus, mitochondria from nCDase-deficient mice have significantly decreased formation of ceramide from sphingosine and palmitoyl-CoA (or palmitate) compared to mitochondria from wild type mice, indicating that nCDase participates in ceramide formation in liver

First, experimental evidence suggests the presence of ceramide synthase activity in mitochondria. Thus, ceramide synthase activity was first detected (Morell and Radin, 1970; Ullman and Radin, 1972) and partially purified from a bovine brain mitochondria-enriched fraction (Shimeno et al., 1995) which was not characterized in terms of marker enzyme activities. Mitochondrial enzymes had almost 2-fold higher specific ceramide synthase activity than the ceramide synthase from the ER. The mitochondrial enzyme had a pH optimum around 7.5 and maximal catalytic efficiency with C16:0- or C18:0-acyl CoA. The addition of liposomes to the mitochondrial enzyme increased ceramide synthase activity (approx. 7.8-fold) (Shimeno et al., 1995). Purification of ceramide synthase from bovine liver mitochondria yielded two major protein bands, 62 and 72 kDa on a SDS-gel (Shimeno et al., 1998). This enzyme had an apparent Km of 146 µM and Vmax of 11.1 nmol/min/mg protein with C18:0-acyl CoA, and corresponding values of Vmax 144 µM and 8.5 nmol/min/mg

Detailed analysis of ceramide synthase activity in highly purified mitochondria by Bionda *et al*. essentially confirmed previous findings (Bionda et al., 2004). Ceramide synthase activity was demonstrated in rat liver mitochondria and in the sub-compartment of the ER closely associated with mitochondria. Further sub-mitochondrial investigation of ceramide synthase activity revealed that both outer and inner mitochondrial membranes can synthesize ceramide (Bionda et al., 2004). Recent reports describing several ceramide synthase isoforms, including CerS1, CerS2, CerS4 and CerS6, in purified mouse brain mitochondria (Novgorodov et al., 2011a; Yu et al., 2007) support the notion that several ceramide synthesizing enzymes could be localized in mitochondria (Futerman, 2006). No such association was found in HeLa cells (Mesicek et al., 2010), suggesting that this might be a cell type/tissue specific event. The intramitochondrial localization of CerS was examined in purified brain mitochondria by immunoprecipitation (Novgorodov et al., 2011a). These studies reveal a selective CerS6 association with adenine nucleotide translocase, the inner membrane component of the mitochondrial permeability transition pore (MPTP). In contrast, CerS2 associated with the outer membrane resident protein Tom20, a receptor of the protein import complex. The data suggest CerS6/ceramide could regulate MPTP activity and mitochondrial Ca2+ homeostasis whereas CerS2/ceramide could modulate the mitochondrial protein import machinery. Secondly, recent studies identified two novel SMases, which hydrolyze SM to ceramide, and phosphocholine in mitochondria from zebrafish (Yabu et al., 2009) and mouse tissues (Wu et al., 2010). Notably, in yeast, the mammalian nSMase ortholog Isc1p associates with mitochondria in the post-diauxic phase of yeast growth and regulates mitochondrial

sphingolipid metabolism (Kitagaki et al., 2007; Vaena de Avalos et al., 2004).

Thirdly, the additional source of ceramide in mitochondria is a reverse reaction of a neutral ceramidase (nCDase), e.g., formation of ceramide as a result of condensation of palmitate and sphingosine (El Bawab et al., 2001). On the basis of molecular cloning and confocal microscopy data, this activity was ascribed to mitochondria (El Bawab et al., 2000), and it was demonstrated in purified mitochondria (Bionda et al., 2004). Recent studies describe the molecular mechanism of ceramide generation from palmitate and sphingosine in purified liver mitochondria that requires concerted action of two enzymes nCDase and thioesterase (which hydrolyzes palmitoyl-CoA to CoA and fatty acid)(Novgorodov et al., 2011b). Thus, mitochondria from nCDase-deficient mice have significantly decreased formation of ceramide from sphingosine and palmitoyl-CoA (or palmitate) compared to mitochondria from wild type mice, indicating that nCDase participates in ceramide formation in liver

protein towards sphinganine.

mitochondria, and that ceramide formation may occur from sphingosine and palmitoyl-CoA from coupled activities of a mitochondrial thioesterase and nCDase catalyzing the reverse reaction (**Scheme 2**). Another possibility is that ceramide could be also transported from the ER to mitochondria through the contact sites between them (Stiban et al., 2008).

Scheme 2. Ceramide formation from Acyl-CoA and sphingosine (Sph) mediated by coupled activities of mitochondrial thioesterase (MTE) and nCDase (NCD).

### **4. Ceramide accumulation in Ischemia/Reperfusion (IR)**

Ceramide accumulation has been demonstrated in various *in vivo* models of IR and it has been implicated as an important mediator of apoptosis in the injured tissue, but mechanisms of ceramide generation are not well-defined and the downstream targets of ceramide remain unresolved. The IR-induced accumulation of ceramide appears to be a general phenomenon for heart, kidney, liver and brain. The identification and characterization of key proteins of ceramide synthesis are expected to expand our understanding of molecular mechanisms behind ceramide's involvement in IR-induced tissue damage.

Research progress has been hampered by a lack of appropriate techniques that would allow simultaneous analysis of multiple sphingolipid species. Thus, the most common method for quantification of ceramide, the diglyceride (DG) kinase assay (Bielawska et al., 2001) has significant disadvantages including a limited separation of ceramide from dihydroceramide and the inability to determine the individual molecular species of ceramide. Recent advances in the development of new mass spectroscopy-based methods for quantitative analysis of sphingolipid molecular species may allow further dissection of ceramide specific pathways (Pettus et al., 2004; Sullards, 2000).

Increasing evidence suggests that the fatty acid chain of ceramide is an important determinant of the biological effect mediated by the individual ceramide species. Most of the experimental evidence indicating the important roles of ceramides containing distinct fatty acids is summarized in an excellent review by Futerman and his colleagues (Pewzner-Jung et al., 2006), and new studies further support the notion of distinct roles of ceramide species in cell metabolism(Hannun and Obeid, 2011). It has been demonstrated that generation of C18:0-ceramide, and not C16:0-ceramide repressed human telomerase reverse transcriptase promoter in lung carcinoma cells (Wooten-Blanks et al., 2007). Activation of aSMase in the salvage pathway brought about a selective accumulation of C16:0-ceramide (Chudakova et al., 2008; Kitatani et al., 2006) due to the involvement of ceramide synthase CerS5 localized in mitochondria-associated membranes (Kitatani et al., 2006). In another

Mitochondrial Ceramide in Stroke 277

Recently, increases of specific ceramide species in the rat heart were investigated after 30 min global ischemia/30 min reperfusion. IR increased accumulation of only 7 out of 14 ceramide species identified in the heart (Beresewicz et al., 2002). Of note, the relative magnitude of IR-induced myocardial accumulation of ceramide species was not proportional to their basal tissue concentrations. For instance, although C16:0-ceramide and C18:0-ceramide are the most abundant in rat heart (40% and 23% of total, respectively), IR increased their content by 48–54%. However, C18:2-ceramide, which contributes only 3.2% of total myocardial ceramides, was increased by 281%. These findings suggest the role of specific ceramide species signaling in the mechanism of post-ischemic myocardial injury. *In vitro*, hypoxia/reoxygenation activated nSMase and ceramide accumulation in cardiomyocytes implicating the production of free radicals (Hernandez et al., 2000). nSMase activation could be abrogated by inhibition of a factor associated with nSMase activation (FAN) which is an adaptor protein connecting neutral SMase to the TNF receptor signaling

Ceramide was elevated in injured liver tissue after cold ischemia and warm reperfusion during liver transplantation (Bradham et al., 1997). A critical role of aSMase and ceramide accumulation was demonstrated in another study of hepatic IR injury (Llacuna et al., 2006). Hepatic ceramide transiently increased after the reperfusion phase due to activation of aSMase followed by acid ceramidase stimulation. Knocking down aSMase by *in vivo* administration of siRNA decreased ceramide generation during IR, and attenuated hepatocellular necrosis, cytochrome c release, and caspase-3 activation. The study draws attention to an important role of ceramide in IR-induced liver damage and suggests that

In the whole kidney IR model, ceramide content was increased about 1.8-fold in the injured tissue during the reperfusion phase (Zager et al., 1997) which was not accompanied by SM hydrolysis. In fact, there was no SM content change in post-IR tissue. Analysis of SMase activity revealed that ischemia induced declines (50%) in both acid and neutral SMase activity, and these persisted throughout the 24-h reperfusion period (Zager et al., 1998). C16:0-, C22:0-, and C24:0-ceramides comprised 20%, 10%, and 70% of the total ceramide content in kidney tissue, respectively (Kalhorn and Zager, 1999). IR dramatically increased C16:0 ceramide (4-fold), and all other ceramides increased modestly. Interestingly, IR induced a striking shift towards unsaturated (vs. saturated) fatty acyl within C22:0- and C24:0- (but not C16:0-) ceramide pools. The data imply that IR-induced inhibition of sphingomyelin hydrolysis results in accumulation of ceramide, the substrate in sphingomyelin synthetic

Ischemic stroke occurs when cerebral or precerebral arteries are occluded or significantly stenosed by emboli or by local atherosclerotic disease. Within minutes of interrupted blood flow, mitochondrial energy production is shut down due to lack of oxygen resulting in membrane depolarization and excessive release of neurotransmitters, specifically, glutamate. Extracellular glutamate accumulation over-stimulates glutamate receptors,

modulation of aSMase could be of therapeutic relevance in liver transplantation.

pathway (O'Brien et al., 2003).

**4.2 Liver ischemia** 

**4.3 Kidney ischemia** 

pathway.

**4.4 Cerebral ischemia** 

study, the effects of chronic hypoxia on selected ceramide species were examined in cardiac tissue in a neonatal mouse model (Noureddine et al., 2008). The study revealed the differential involvement of the right ventricle with regard to levels of C16:0-ceramide and its precursor, dihydro-C16:0-ceramide. The decrease in C16:0-ceramide observed in both hypoxic and control right ventricles over time occurred along with a significant increase in dihydro-C16:0-ceramide in hypoxic but not control tissues suggesting a role for dihydro-C16:0 ceramide in the adaptive tissue response to hypoxia. Although ceramide species could have different effects on biophysical properties of the membrane lipid bilayer (Sot et al., 2005a), it remains unclear how ceramides containing different fatty acids exert their effects upon cell physiology.

### **4.1 Cardiac ischemia**

In several studies, elevated ceramide has been reported in myocardium after ischemia and IR. In the rat heart left coronary artery occlusion model, ischemia with subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells indicated by DNA laddering and measurement of soluble chromatin degradation products (Bielawska et al., 1997). The content of ceramide in ischemic myocardium was elevated to 155% baseline levels after 30 min ischemia, and was further increased to 250% after 3 h reperfusion. In the rabbit heart left coronary artery occlusion model, ceramide content was increased during the first minute of ischemia, peaking at 5 min with mean ceramide ~127% of baseline. However, this peak was transient because ceramide content returned to near-baseline values as soon as 10 min into the sustained ischemia (Argaud et al., 2004). In another study with the rat heart left coronary artery occlusion model, ceramide content in reperfused myocardium was found to increase up to 50%. This increase was not associated with enhanced neutral or aSMase activity, but rather with reduced activity of ceramidase, a ceramide-metabolizing enzyme (**Scheme 1**) (Zhang et al., 2001). In a global rat heart ischemia model, ceramide content was elevated about 2-fold after 30 min ischemia/30 min reperfusion which was attributed to SM hydrolysis. Thus, there was about 50% less SM in reperfused myocardium after IR (Cordis et al., 1998). This finding was confirmed in recent studies by the same group who reported an increased accumulation of ceramide in ischemic myocardium after 30 min ischemia/2 h reperfusion (Cui et al., 2004; Der et al., 2006). An inhibitor of aSMase activity desipramine prevented ceramide accumulation and provided cardioprotection. Intriguingly, a significant amount of ceramide accumulated in the caveolin-1-rich membrane microdomains after IR was abolished by pre-treatment with desipramine (Der et al., 2006). The ceramide–caveolin-1 interaction is believed to occur within lipid raft microdomains in membranes leading to rafts stabilization (Xu et al., 2001) and alteration of receptor tyrosine kinase signal transduction (Zundel et al., 2000).

In a very interesting study, analysis of cardiac tissues from mice subjected to IR revealed significant elevation of ceramide and inhibition of sphingosine kinase 1 activity (**Scheme 1**) that could ultimately result in decreased sphingosine-1-phosphate (Pchejetski et al., 2007). Furthermore, sphingosine kinase 1 inhibition, ceramide accumulation, cardiomyocyte apoptosis, and infarct size were significantly decreased in mitochondrial monoamine oxidase (MAO-A)-deficient mice after IR. MAO-A appears to play an important role in reactive oxygen species (ROS)-dependent cardiomyocytes apoptosis and postischemic cardiac damage (Bianchi et al., 2005). The data imply that the upregulation of ceramide/sphingosine-1-phosphate ratio is a critical event in MAO-A-dependent cardiac cell apoptosis in IR.

study, the effects of chronic hypoxia on selected ceramide species were examined in cardiac tissue in a neonatal mouse model (Noureddine et al., 2008). The study revealed the differential involvement of the right ventricle with regard to levels of C16:0-ceramide and its precursor, dihydro-C16:0-ceramide. The decrease in C16:0-ceramide observed in both hypoxic and control right ventricles over time occurred along with a significant increase in dihydro-C16:0-ceramide in hypoxic but not control tissues suggesting a role for dihydro-C16:0 ceramide in the adaptive tissue response to hypoxia. Although ceramide species could have different effects on biophysical properties of the membrane lipid bilayer (Sot et al., 2005a), it remains unclear how ceramides containing different fatty acids exert their effects upon cell

In several studies, elevated ceramide has been reported in myocardium after ischemia and IR. In the rat heart left coronary artery occlusion model, ischemia with subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells indicated by DNA laddering and measurement of soluble chromatin degradation products (Bielawska et al., 1997). The content of ceramide in ischemic myocardium was elevated to 155% baseline levels after 30 min ischemia, and was further increased to 250% after 3 h reperfusion. In the rabbit heart left coronary artery occlusion model, ceramide content was increased during the first minute of ischemia, peaking at 5 min with mean ceramide ~127% of baseline. However, this peak was transient because ceramide content returned to near-baseline values as soon as 10 min into the sustained ischemia (Argaud et al., 2004). In another study with the rat heart left coronary artery occlusion model, ceramide content in reperfused myocardium was found to increase up to 50%. This increase was not associated with enhanced neutral or aSMase activity, but rather with reduced activity of ceramidase, a ceramide-metabolizing enzyme (**Scheme 1**) (Zhang et al., 2001). In a global rat heart ischemia model, ceramide content was elevated about 2-fold after 30 min ischemia/30 min reperfusion which was attributed to SM hydrolysis. Thus, there was about 50% less SM in reperfused myocardium after IR (Cordis et al., 1998). This finding was confirmed in recent studies by the same group who reported an increased accumulation of ceramide in ischemic myocardium after 30 min ischemia/2 h reperfusion (Cui et al., 2004; Der et al., 2006). An inhibitor of aSMase activity desipramine prevented ceramide accumulation and provided cardioprotection. Intriguingly, a significant amount of ceramide accumulated in the caveolin-1-rich membrane microdomains after IR was abolished by pre-treatment with desipramine (Der et al., 2006). The ceramide–caveolin-1 interaction is believed to occur within lipid raft microdomains in membranes leading to rafts stabilization (Xu et al., 2001) and alteration of receptor tyrosine

In a very interesting study, analysis of cardiac tissues from mice subjected to IR revealed significant elevation of ceramide and inhibition of sphingosine kinase 1 activity (**Scheme 1**) that could ultimately result in decreased sphingosine-1-phosphate (Pchejetski et al., 2007). Furthermore, sphingosine kinase 1 inhibition, ceramide accumulation, cardiomyocyte apoptosis, and infarct size were significantly decreased in mitochondrial monoamine oxidase (MAO-A)-deficient mice after IR. MAO-A appears to play an important role in reactive oxygen species (ROS)-dependent cardiomyocytes apoptosis and postischemic cardiac damage (Bianchi et al., 2005). The data imply that the upregulation of ceramide/sphingosine-1-phosphate ratio is a critical event in MAO-A-dependent cardiac

physiology.

**4.1 Cardiac ischemia** 

kinase signal transduction (Zundel et al., 2000).

cell apoptosis in IR.

Recently, increases of specific ceramide species in the rat heart were investigated after 30 min global ischemia/30 min reperfusion. IR increased accumulation of only 7 out of 14 ceramide species identified in the heart (Beresewicz et al., 2002). Of note, the relative magnitude of IR-induced myocardial accumulation of ceramide species was not proportional to their basal tissue concentrations. For instance, although C16:0-ceramide and C18:0-ceramide are the most abundant in rat heart (40% and 23% of total, respectively), IR increased their content by 48–54%. However, C18:2-ceramide, which contributes only 3.2% of total myocardial ceramides, was increased by 281%. These findings suggest the role of specific ceramide species signaling in the mechanism of post-ischemic myocardial injury. *In vitro*, hypoxia/reoxygenation activated nSMase and ceramide accumulation in cardiomyocytes implicating the production of free radicals (Hernandez et al., 2000). nSMase activation could be abrogated by inhibition of a factor associated with nSMase activation (FAN) which is an adaptor protein connecting neutral SMase to the TNF receptor signaling pathway (O'Brien et al., 2003).

### **4.2 Liver ischemia**

Ceramide was elevated in injured liver tissue after cold ischemia and warm reperfusion during liver transplantation (Bradham et al., 1997). A critical role of aSMase and ceramide accumulation was demonstrated in another study of hepatic IR injury (Llacuna et al., 2006). Hepatic ceramide transiently increased after the reperfusion phase due to activation of aSMase followed by acid ceramidase stimulation. Knocking down aSMase by *in vivo* administration of siRNA decreased ceramide generation during IR, and attenuated hepatocellular necrosis, cytochrome c release, and caspase-3 activation. The study draws attention to an important role of ceramide in IR-induced liver damage and suggests that modulation of aSMase could be of therapeutic relevance in liver transplantation.

### **4.3 Kidney ischemia**

In the whole kidney IR model, ceramide content was increased about 1.8-fold in the injured tissue during the reperfusion phase (Zager et al., 1997) which was not accompanied by SM hydrolysis. In fact, there was no SM content change in post-IR tissue. Analysis of SMase activity revealed that ischemia induced declines (50%) in both acid and neutral SMase activity, and these persisted throughout the 24-h reperfusion period (Zager et al., 1998). C16:0-, C22:0-, and C24:0-ceramides comprised 20%, 10%, and 70% of the total ceramide content in kidney tissue, respectively (Kalhorn and Zager, 1999). IR dramatically increased C16:0 ceramide (4-fold), and all other ceramides increased modestly. Interestingly, IR induced a striking shift towards unsaturated (vs. saturated) fatty acyl within C22:0- and C24:0- (but not C16:0-) ceramide pools. The data imply that IR-induced inhibition of sphingomyelin hydrolysis results in accumulation of ceramide, the substrate in sphingomyelin synthetic pathway.

### **4.4 Cerebral ischemia**

Ischemic stroke occurs when cerebral or precerebral arteries are occluded or significantly stenosed by emboli or by local atherosclerotic disease. Within minutes of interrupted blood flow, mitochondrial energy production is shut down due to lack of oxygen resulting in membrane depolarization and excessive release of neurotransmitters, specifically, glutamate. Extracellular glutamate accumulation over-stimulates glutamate receptors,

Mitochondrial Ceramide in Stroke 279

limited permeability state of MPTP opening (Novgorodov and Gudz, 1996) that only depolarizes mitochondria without causing swelling (Brustovetsky and Dubinsky, 2000b). This depolarization dramatically reduces the driving force for Ca2+ influx via mitochondrial Ca2+ uniporter , thus limiting the mitochondrial ability to sequester Ca2+(Brustovetsky and Dubinsky, 2000a). The lower CerS6 expression and C16:0-ceramide content were associated with reduced mitochondrial CLC in adult brain mitochondria, whereas exogenous C16:0 ceramide restored CLC to that of young brain mitochondria. This is in line with the finding that long-chain ceramides, including C16:0-ceramide, are potent inhibitors of MPTP activity (Novgorodov et al., 2008). This suggests that CerS6-generated ceramide could prevent

MPTP opening, leading to increased Ca2+ accumulation in the mitochondrial matrix.

an important future target for neuroprotection.

**5. Ceramide and mitochondrial injury in stroke**

**5.1 Respiratory chain** 

mimic the properties of naturally occurring long-chain ceramides.

The role of CerS6 in cell survival was examined in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development or following cerebral IR. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of *de novo* ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect. Importantly, blocking mitochondrial Ca2+ uptake or decreasing Ca2+-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. The data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca2+ homeostasis and calpain, which could be important in cell death after cerebral IR (**Scheme 3**). These studies illuminate a novel determinant in cerebral IR, mitochondrial ceramide synthase CerS6 which could be

*In vitro, de novo* synthesized ceramide increased after brief exposure of cultured brain cells to hypoxia, oxygen/glucose deprivation, or TNF (Ginis et al., 1999; Liu et al., 2000). In neuronal precursor cells, hypoxia/reoxygenation triggered a robust elevation in C14:0- and C16:0-ceramides, and a small increase in C18:0-, C18:1- and C20:0-ceramides, and no increase in C24:0- and C24:1-ceramides (Jin et al., 2008). The elevations in ceramides were primarily due to the actions of aSMase and ceramide synthase CerS5, demonstrating the involvement of the salvage pathway. Interestingly, C2-ceramide infusion protected the brain against IR injury (Chen et al., 2001; Furuya et al., 2001). However, this effect could be also attributed to the intracellular/extracellular conversion of ceramide into sphingosine-1-phosphate, which is known to protect cells from apoptosis (Hait et al., 2006; Taha et al., 2006; Tani et al., 2007).

Although IR–induced mitochondrial injury has been extensively studied and mitochondrial functions affected by IR are characterized (Sims and Anderson, 2002), crucial information is needed regarding the cause of mitochondrial dysfunction. Our studies suggest that exogenously added ceramide could provoke mitochondrial dysfunctions similar to that occurring in cerebral IR (Yu et al., 2007). Of note, some data on mitochondrial effects of ceramide have been obtained using synthetic short-chain analogs, which may not fully

The restriction on mitochondrial respiratory chain function has been shown in various rodent models of stroke (Sims and Anderson, 2002). An impairment of Complex III has been

promoting cytosolic Ca2+ overload, the generation of ROS, and mitochondrial dysfunction leading to cell death.

A few studies reported ceramide accumulation during cerebral ischemia and IR (Kubota et al., 1989; Nakane et al., 2000), and it appears that the mechanism of ceramide accumulation depends on the severity of the insult to the brain. Thus, severe and lethal cerebral IR resulted in ceramide accumulation via activation of aSMase and SM hydrolysis (Kubota et al., 1989; Kubota et al., 1996; Nakane et al., 2000) or inhibition of ceramide utilization by glucosylceramide synthase (Takahashi et al., 2004). Consistent with these data, the extent of brain tissue damage was decreased in mice lacking aSMase (Yu et al., 2000). In a recent study, severe cerebral IR induced SM hydrolysis and increased ceramide and sphingosine in the ischemic brain (Chudakova et al., 2008). Similarly, chronic cerebral ischemia caused ceramide accumulation due to activation of SM degradation accompanied by reduced ceramide utilization via glucosylceramide synthase (Ohtani et al., 2004). The data highlight IR-induced deregulation of complex sphingolipids metabolism.

In mild IR, ceramide accumulation resulted from *de novo* ceramide biosynthesis rather from hydrolysis of SM (Yu et al., 2007). There is apparent tissue specificity in the expression of individual ceramide species that might reflect the tissue specificity of the ceramide synthases. In brain, C18:0-, C18:1- and C24:1-ceramide are the major species expressed (39.5%, 34%, and 12.5% of total ceramide, respectively) whereas C16:0- ceramide contributes only 4% of total ceramide. All ceramide species were elevated in the ischemic brain about 1.5–2-fold. The enhanced accumulation of sphingolipids seems to occur during the reperfusion phase; there were no changes in sphingolipid content after ischemia without reperfusion. This finding is in line with data which show that both ischemia and the restoration of blood flow to ischemic tissue (reperfusion) causes cellular damage by different molecular mechanisms (Chan, 2004; Gustafsson and Gottlieb, 2008).

Investigation of intracellular sites of ceramide accumulation after mild IR revealed the elevation of ceramide species both in purified mitochondria and in the ER (Yu et al., 2007). In mitochondria, only C18:0-, C18:1- and C16:0-ceramides were increased, but all ceramide species increased in the ER suggesting activation of different ceramide synthases in these intracellular compartments. Indeed, several ceramide synthases were identified in mitochondria and the ER, including CerS1, CerS2, and CerS6, but CerS5 was localized only in the ER in the brain. Activity measurements indicated activation of CerS6 in ischemic mitochondria apparently via post-translational mechanisms; IR did not affect the CerS6 protein expression (Yu et al., 2007).

It appears that CerS6 is developmentally regulated and primarily generates C16:0-ceramide in brain mitochondria (Novgorodov et al., 2011a). An investigation into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation during brain development is associated with dramatically decreased mitochondrial Ca2+-loading capacity (CLC) which could be rescued by addition of ceramide. Ceramide-mediated blockade of MPTP opening seems to be the underlying mechanism of the increased CLC in brain mitochondria isolated from young animals. In fact, mitochondria maintain low cytosolic Ca2+ levels by sequestering Ca2+ inside the mitochondrial matrix complexed with phosphate. Energized mitochondria take up Ca2+ via the mitochondrial calcium uniporter which has been recently described as a highly selective, inwardly rectifying channel (Kirichok et al., 2004). Excessive accumulation of Ca2+ in the mitochondrial matrix could trigger opening of MPTP at a high conductance state, which would be accompanied by dissipation of the transmembrane potential and mitochondrial swelling. In brain mitochondria, Ca2+ may also activate a

promoting cytosolic Ca2+ overload, the generation of ROS, and mitochondrial dysfunction

A few studies reported ceramide accumulation during cerebral ischemia and IR (Kubota et al., 1989; Nakane et al., 2000), and it appears that the mechanism of ceramide accumulation depends on the severity of the insult to the brain. Thus, severe and lethal cerebral IR resulted in ceramide accumulation via activation of aSMase and SM hydrolysis (Kubota et al., 1989; Kubota et al., 1996; Nakane et al., 2000) or inhibition of ceramide utilization by glucosylceramide synthase (Takahashi et al., 2004). Consistent with these data, the extent of brain tissue damage was decreased in mice lacking aSMase (Yu et al., 2000). In a recent study, severe cerebral IR induced SM hydrolysis and increased ceramide and sphingosine in the ischemic brain (Chudakova et al., 2008). Similarly, chronic cerebral ischemia caused ceramide accumulation due to activation of SM degradation accompanied by reduced ceramide utilization via glucosylceramide synthase (Ohtani et al., 2004). The data highlight

In mild IR, ceramide accumulation resulted from *de novo* ceramide biosynthesis rather from hydrolysis of SM (Yu et al., 2007). There is apparent tissue specificity in the expression of individual ceramide species that might reflect the tissue specificity of the ceramide synthases. In brain, C18:0-, C18:1- and C24:1-ceramide are the major species expressed (39.5%, 34%, and 12.5% of total ceramide, respectively) whereas C16:0- ceramide contributes only 4% of total ceramide. All ceramide species were elevated in the ischemic brain about 1.5–2-fold. The enhanced accumulation of sphingolipids seems to occur during the reperfusion phase; there were no changes in sphingolipid content after ischemia without reperfusion. This finding is in line with data which show that both ischemia and the restoration of blood flow to ischemic tissue (reperfusion) causes cellular damage by different molecular mechanisms

Investigation of intracellular sites of ceramide accumulation after mild IR revealed the elevation of ceramide species both in purified mitochondria and in the ER (Yu et al., 2007). In mitochondria, only C18:0-, C18:1- and C16:0-ceramides were increased, but all ceramide species increased in the ER suggesting activation of different ceramide synthases in these intracellular compartments. Indeed, several ceramide synthases were identified in mitochondria and the ER, including CerS1, CerS2, and CerS6, but CerS5 was localized only in the ER in the brain. Activity measurements indicated activation of CerS6 in ischemic mitochondria apparently via post-translational mechanisms; IR did not affect the CerS6

It appears that CerS6 is developmentally regulated and primarily generates C16:0-ceramide in brain mitochondria (Novgorodov et al., 2011a). An investigation into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation during brain development is associated with dramatically decreased mitochondrial Ca2+-loading capacity (CLC) which could be rescued by addition of ceramide. Ceramide-mediated blockade of MPTP opening seems to be the underlying mechanism of the increased CLC in brain mitochondria isolated from young animals. In fact, mitochondria maintain low cytosolic Ca2+ levels by sequestering Ca2+ inside the mitochondrial matrix complexed with phosphate. Energized mitochondria take up Ca2+ via the mitochondrial calcium uniporter which has been recently described as a highly selective, inwardly rectifying channel (Kirichok et al., 2004). Excessive accumulation of Ca2+ in the mitochondrial matrix could trigger opening of MPTP at a high conductance state, which would be accompanied by dissipation of the transmembrane potential and mitochondrial swelling. In brain mitochondria, Ca2+ may also activate a

IR-induced deregulation of complex sphingolipids metabolism.

(Chan, 2004; Gustafsson and Gottlieb, 2008).

protein expression (Yu et al., 2007).

leading to cell death.

limited permeability state of MPTP opening (Novgorodov and Gudz, 1996) that only depolarizes mitochondria without causing swelling (Brustovetsky and Dubinsky, 2000b). This depolarization dramatically reduces the driving force for Ca2+ influx via mitochondrial Ca2+ uniporter , thus limiting the mitochondrial ability to sequester Ca2+(Brustovetsky and Dubinsky, 2000a). The lower CerS6 expression and C16:0-ceramide content were associated with reduced mitochondrial CLC in adult brain mitochondria, whereas exogenous C16:0 ceramide restored CLC to that of young brain mitochondria. This is in line with the finding that long-chain ceramides, including C16:0-ceramide, are potent inhibitors of MPTP activity (Novgorodov et al., 2008). This suggests that CerS6-generated ceramide could prevent MPTP opening, leading to increased Ca2+ accumulation in the mitochondrial matrix.

The role of CerS6 in cell survival was examined in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development or following cerebral IR. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of *de novo* ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect. Importantly, blocking mitochondrial Ca2+ uptake or decreasing Ca2+-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. The data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca2+ homeostasis and calpain, which could be important in cell death after cerebral IR (**Scheme 3**). These studies illuminate a novel determinant in cerebral IR, mitochondrial ceramide synthase CerS6 which could be an important future target for neuroprotection.

*In vitro, de novo* synthesized ceramide increased after brief exposure of cultured brain cells to hypoxia, oxygen/glucose deprivation, or TNF (Ginis et al., 1999; Liu et al., 2000). In neuronal precursor cells, hypoxia/reoxygenation triggered a robust elevation in C14:0- and C16:0-ceramides, and a small increase in C18:0-, C18:1- and C20:0-ceramides, and no increase in C24:0- and C24:1-ceramides (Jin et al., 2008). The elevations in ceramides were primarily due to the actions of aSMase and ceramide synthase CerS5, demonstrating the involvement of the salvage pathway. Interestingly, C2-ceramide infusion protected the brain against IR injury (Chen et al., 2001; Furuya et al., 2001). However, this effect could be also attributed to the intracellular/extracellular conversion of ceramide into sphingosine-1-phosphate, which is known to protect cells from apoptosis (Hait et al., 2006; Taha et al., 2006; Tani et al., 2007).

### **5. Ceramide and mitochondrial injury in stroke**

Although IR–induced mitochondrial injury has been extensively studied and mitochondrial functions affected by IR are characterized (Sims and Anderson, 2002), crucial information is needed regarding the cause of mitochondrial dysfunction. Our studies suggest that exogenously added ceramide could provoke mitochondrial dysfunctions similar to that occurring in cerebral IR (Yu et al., 2007). Of note, some data on mitochondrial effects of ceramide have been obtained using synthetic short-chain analogs, which may not fully mimic the properties of naturally occurring long-chain ceramides.

### **5.1 Respiratory chain**

The restriction on mitochondrial respiratory chain function has been shown in various rodent models of stroke (Sims and Anderson, 2002). An impairment of Complex III has been

Mitochondrial Ceramide in Stroke 281

chain ceramide could increase the generation of ROS in isolated mitochondria (Garcia-Ruiz et

**Cytochrome c**

**Ascorbate**

**Complex IV O2**

**Complex III**

**ROS**

Scheme 4**.** Mitochondrial respiratory chain complexes. Mitochondrial respiratory chain consists of four multi-protein complexes Complex I-IV. The respiratory chain function is determined using substrates such as glutamate, succinate or ascorbate, which are oxidized via different complexes of respiratory chain. An inhibition of the electron transport through

Excessive accumulation of Ca2+ in mitochondrial matrix could trigger opening of the MPTP at a high conductance state that is accompanied with dissipation of transmembrane potential and swelling of mitochondria. In brain mitochondria, Ca2+ may also activate a limited permeability state of MPTP opening that only depolarizes mitochondria without swelling (Novgorodov and Gudz, 1996). This depolarization dramatically reduces the driving force for Ca2+ influx via the uniporter channel in the inner membrane, thus limiting

MPTP opening at a high conductance state appears to be a crucial event leading to cell death by necrosis (Fiskum, 2000; Galluzzi et al., 2009; Kroemer et al., 1998). Severe insult causes widespread opening of the MPTP in mitochondria. Cell death proceeds through necrosis when the MPTP remains open, causing the inhibition of ATP production. If the initial insult is not too severe and MPTP does not open, cellular ATP can be maintained to support the energy demand of apoptosis. This provides an explanation for the coexistence of apoptotic and necrotic cell death in IR-injured tissue. It has been emphasized that MPTP regulates necrotic, but not apoptotic cell death in cardiac and cerebral IR (Nakagawa et al., 2005; Schinzel et al., 2005). Mice deficient in cyclophilin D (CyD), the main regulatory component of MPTP, developed up to 37% smaller brain infarcts after IR. CyD-deficient cells died normally in response to various apoptotic stimuli, but were resistant to necrotic cell death induced by ROS and Ca2+ overload. MPTP opening at a high conductance state has been proposed to occur in cerebral IR, but the evidence is largely indirect (Sims and Anderson, 2002; Stavrovskaya and Kristal, 2005). The opening of the MPTP at a high conductance state has been implicated as another mechanism of cytochrome c release from mitochondria due to short-chain ceramide

(Arora et al., 1997; Novgorodov et al., 2005; Pastorino et al., 1999a; Szalai et al., 1999).

**Complex I**

**ROS**

**Complex II**

the Complex I or III could result in generation of ROS.

the mitochondrial ability to sequester Ca2+.

**5.3 Mitochondrial Permeability Transition Pore (MPTP)** 

**Succinate**

**Glutamate**

al., 1997).

Scheme 3. Hypothetical role of CerS6/ceramide in mitochondria after cerebral IR. IR triggers glutamate-induced cytosolic Ca2+ influx into the mitochondria and an activation of mitochondrial CerS6 that elevates ceramide. Ceramide blocks the MPTP opening at a low conductance state, leading to increased Ca2+ in the mitochondrial matrix. This MPTP inactivation would allow mitochondria to support adequate ATP production for formation of the apoptosome, and might be responsible for the initial raise in ATP production (and hence Δψ) during apoptosis (Atlante et al., 2005), an observation that corresponds well with the reported transient mitochondrial hyper-polarization in the apoptosis induced by IL-3 withdrawal (Vander Heiden et al., 1997). Rising mitochondrial Ca2+ activates calpain 10, which could cleave protein components of the MPTP (Arrington et al., 2006) resulting in the MPTP opening at a high conductance state, swelling, and rupture of the outer mitochondrial membrane leading to necrotic cell death.

implicated (**Scheme 4**), but the mechanisms remain unresolved, and a Complex I defect has not been ruled out. We and others have reported that short-chain ceramide could directly suppress respiratory chain Complex III activity (Di Paola et al., 2000; Gudz et al., 1997). Also, ceramide seems to participate in displacement of cytochrome c from its binding site on Complex III (Yuan et al., 2003) corresponds to an apparent mitochondrial Complex III defect in IR.

### **5.2 Reactive Oxygen Species (ROS)**

Free radical formation occurs during cerebral IR. In fact, mitochondria are the major site of production of ROS, and are the likely source for the generation of peroxynitrite, formed from nitric oxide and superoxide during IR (Chan, 2001). Studies in isolated mitochondria indicated that Complex I and III are potential sites of superoxide formation (Votyakova and Reynolds, 2001). Complex III deficiency observed in cerebral IR strongly implicates Complex III as the major and relevant site of ROS generation; however, Complex I remains to be ruled out. Short-

**Ca2+**

**Calpain**

**Ca2+**

**Loss of ∆Ψ<sup>m</sup>**

**Necrosis**

**CerS6**

**MPTP open**

**ceramide**

**ceramide**

**MPTP closed**

glutamate-induced cytosolic Ca2+ influx into the mitochondria and an activation of mitochondrial CerS6 that elevates ceramide. Ceramide blocks the MPTP opening at a low conductance state, leading to increased Ca2+ in the mitochondrial matrix. This MPTP inactivation would allow mitochondria to support adequate ATP production for formation of the apoptosome, and might be responsible for the initial raise in ATP production (and hence Δψ) during apoptosis (Atlante et al., 2005), an observation that corresponds well with the reported transient mitochondrial hyper-polarization in the apoptosis induced by IL-3 withdrawal (Vander Heiden et al., 1997). Rising mitochondrial Ca2+ activates calpain 10, which could cleave protein components of the MPTP (Arrington et al., 2006) resulting in the MPTP opening at a high conductance state, swelling, and rupture of the outer mitochondrial

Scheme 3. Hypothetical role of CerS6/ceramide in mitochondria after cerebral IR. IR triggers

implicated (**Scheme 4**), but the mechanisms remain unresolved, and a Complex I defect has not been ruled out. We and others have reported that short-chain ceramide could directly suppress respiratory chain Complex III activity (Di Paola et al., 2000; Gudz et al., 1997). Also, ceramide seems to participate in displacement of cytochrome c from its binding site on Complex III (Yuan et al., 2003) corresponds to an apparent mitochondrial Complex III defect

Free radical formation occurs during cerebral IR. In fact, mitochondria are the major site of production of ROS, and are the likely source for the generation of peroxynitrite, formed from nitric oxide and superoxide during IR (Chan, 2001). Studies in isolated mitochondria indicated that Complex I and III are potential sites of superoxide formation (Votyakova and Reynolds, 2001). Complex III deficiency observed in cerebral IR strongly implicates Complex III as the major and relevant site of ROS generation; however, Complex I remains to be ruled out. Short-

**CerS6**

**Ca2+**

**Activated Bax**

**Ca2+**

**Pro-apoptotic proteins release**

**Apoptosis**

membrane leading to necrotic cell death.

**5.2 Reactive Oxygen Species (ROS)** 

in IR.

**Glutamate**

chain ceramide could increase the generation of ROS in isolated mitochondria (Garcia-Ruiz et al., 1997).

### **Succinate**

Scheme 4**.** Mitochondrial respiratory chain complexes. Mitochondrial respiratory chain consists of four multi-protein complexes Complex I-IV. The respiratory chain function is determined using substrates such as glutamate, succinate or ascorbate, which are oxidized via different complexes of respiratory chain. An inhibition of the electron transport through the Complex I or III could result in generation of ROS.

### **5.3 Mitochondrial Permeability Transition Pore (MPTP)**

Excessive accumulation of Ca2+ in mitochondrial matrix could trigger opening of the MPTP at a high conductance state that is accompanied with dissipation of transmembrane potential and swelling of mitochondria. In brain mitochondria, Ca2+ may also activate a limited permeability state of MPTP opening that only depolarizes mitochondria without swelling (Novgorodov and Gudz, 1996). This depolarization dramatically reduces the driving force for Ca2+ influx via the uniporter channel in the inner membrane, thus limiting the mitochondrial ability to sequester Ca2+.

MPTP opening at a high conductance state appears to be a crucial event leading to cell death by necrosis (Fiskum, 2000; Galluzzi et al., 2009; Kroemer et al., 1998). Severe insult causes widespread opening of the MPTP in mitochondria. Cell death proceeds through necrosis when the MPTP remains open, causing the inhibition of ATP production. If the initial insult is not too severe and MPTP does not open, cellular ATP can be maintained to support the energy demand of apoptosis. This provides an explanation for the coexistence of apoptotic and necrotic cell death in IR-injured tissue. It has been emphasized that MPTP regulates necrotic, but not apoptotic cell death in cardiac and cerebral IR (Nakagawa et al., 2005; Schinzel et al., 2005). Mice deficient in cyclophilin D (CyD), the main regulatory component of MPTP, developed up to 37% smaller brain infarcts after IR. CyD-deficient cells died normally in response to various apoptotic stimuli, but were resistant to necrotic cell death induced by ROS and Ca2+ overload. MPTP opening at a high conductance state has been proposed to occur in cerebral IR, but the evidence is largely indirect (Sims and Anderson, 2002; Stavrovskaya and Kristal, 2005). The opening of the MPTP at a high conductance state has been implicated as another mechanism of cytochrome c release from mitochondria due to short-chain ceramide (Arora et al., 1997; Novgorodov et al., 2005; Pastorino et al., 1999a; Szalai et al., 1999).

Mitochondrial Ceramide in Stroke 283

apoptotic members of the Bcl-2 family of proteins that, in turn, alter the outer mitochondrial membrane permeability for cytochrome c and other pro-apoptotic molecules. Protein targets for ceramide in the cytoplasm include protein phosphatases PP1A and PP2A, protein kinases PKC ζ, raf-1, and kinase-suppressor Ras (Snook et al., 2006). In the lysosomal compartment, ceramide activates aspartate protease cathepsin D (Bidere et al., 2003; Heinrich et al., 2004; Heinrich et al., 1999; Pettus et al., 2002). Among these targets, cathepsin D, PP2A, and PP1A could propagate a pro-apoptotic ceramide signal to the level of the mitochondria (Pettus et al., 2002). Interaction of ceramide with cathepsin D results in cleavage of Bid to active tBid with subsequent activation of caspase-9 and caspase-3 (Heinrich et al., 2004). Activation by ceramides of serine/threonine protein phosphatase PP2A is involved in regulation of the apoptotic/anti-apoptotic activity of Bcl-2 family proteins by changing their phosphorylation status. Ceramide-activated PP2A increases the pro-apoptotic potential of Bcl-2 family proteins by dephosphorylation of Bax (activation) (Xin and Deng, 2006), or Bcl-2 (inactivation) (Ruvolo et al., 1999). An additional substrate for PP2A is serine/threonine kinase Akt/PKB (Pettus et al., 2002). Ceramide-dependent activation of PP2A leads to inactivation of Akt (Garcia et al., 2003; Millward et al., 1999; Pettus et al., 2002) that, in turn, results in dephosphorylation and activation of pro-apoptotic Bad, an Akt substrate (Datta et al., 1997). At the same time PP2A can directly dephosphorylate Bad, thus increasing its pro-apoptotic activity (Chiang et al., 2003). PP1A also can exert its effect on mitochondria by Bad dephosphorylation (Garcia et al., 2003). Interestingly, ceramide by itself can trigger transition of Bax into the active conformation, insertion in to the outer mitochondrial membrane with the subsequent release of cytochrome c and Smac in a cell-free system (Kashkar et al., 2005). Potentiation of Bax binding by ceramides to the outer mitochondrial membrane was shown by Birbes and colleagues (Birbes et al., 2005) and in energized mitochondria ceramide-induced Baxdependent MPTP opening (Pastorino et al., 1999b). Critical involvement of ceramide in triggering Bax translocation to the mitochondria was demonstrated during hypoxia/reoxygenation in neuronal cells (Jin et al., 2008). Attenuation of Bax translocation by knockdown of ceramide synthase CerS5 or aSMase suggests contribution of the activated salvage pathway in ceramide upregulation; however, the mechanisms by which ceramide

Less-defined, indirect mechanisms include interaction of ceramide with protein kinases PKC δ, p38 and JNK. Short-chain ceramides induce translocation of PKC δ from the cytoplasm to the mitochondria in LNCaP cells (Sumitomo et al., 2002). The translocation of PKC δ was accompanied by cytochrome c release. Mitochondrial translocation of PKC δ and activation of kinase activity was also evident when endogenous ceramides were raised by activation of de novo and neutral SMase-dependent pathways of ceramide production. Endogenous ceramide-induced PKC δ translocation similarly promoted release of cytochrome c and caspase-9 activation. A report by Huwiler et al. (Huwiler et al., 1998) indicates that ceramide can directly target PKC δ. Thus, increased ceramide during I/R can potentially contribute to mitochondrial translocation/activation of PKC δ, which enhances cytochrome c release in heart I/R (Murriel et al., 2004). Although potential mitochondrial PKC δ targets for which phosphorylation results in cytochrome c release remain illusive, PKC δ-dependant accumulation and dephosphorylation of Bad may contribute to the initiation of apoptotic

The member of the mitogen-activated protein kinase (MAPK) superfamily p38 MAPK was implicated in ceramide-induced apoptosis in cardiomyocytes (Kong et al., 2005). Short-chain

exerts its effect remain unknown.

program (Murriel et al., 2004).

### **5.4 Release of pro-apoptotic proteins from mitochondria**

Apoptosis is mediated by two major pathways: the extrinsic and the intrinsic (or mitochondrial) pathway. The release of mitochondrial cytochrome c and/or Smac, which antagonizes apoptotic protein inhibitor, into the cytosol initiates the activation of caspase-9 leading to the proteolytic activation of executioner caspase-3 and -7. Cytochrome c release from mitochondria is well documented in different models of ischemia (Galluzzi et al., 2009). Recent studies have showed caspase-independent apoptosis involving the release of mitochondrial proteins, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) and their translocation to the nucleus in brain IR (Galluzzi et al., 2009; Joza et al., 2009). Emerging evidence indicates an important role of Bax in cytochrome c and Smac (but not AIF and EndoG) release from brain mitochondria (Brustovetsky et al., 2003). Importantly, mitochondrial calpain has been implicated in AIF release from mitochondria (Kar et al.). The release of mitochondrial proteins implies that the outer and /or the inner mitochondrial membrane is compromised in IR, but the precise mechanisms of the protein release remain unclear. Short-chain ceramide accelerated the release of cytochrome c and AIF from heart mitochondria (Di Paola et al., 2004), and natural C16-ceramide has been shown to form large channels in the outer mitochondrial membrane permeable to cytochrome c (Siskind and Colombini, 2000).

### **6. Ceramide and mitochondria in cell death**

Irrespective of the type of IR, IR-related physiological events have a common final consequence: alteration of mitochondrial function and release of mitochondrial proteins, leading to cell death. Cells with hallmarks of necrosis or apoptosis have been detected in animal models of IR (Li et al., 1995). The mitochondrial changes appear to be one essential step in tissue damage in IR, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function (Nakai et al., 1997). Multiple studies show intimate connections between ceramide signaling and functioning of mitochondria (Mimeault, 2002; Morales et al., 2007), which play central role in integration of cellular signals to determine the outcome among apoptosis, necrosis, or proliferation (Brenner and Kroemer, 2000; Ferri and Kroemer, 2001; Kroemer et al., 2007).

 Several lines of evidence have implicated changes in mitochondrial function as an intermediate step in transduction of ceramide signals that culminate in apoptotic or necrotic cell death (Morales et al., 2007; Taha et al., 2006). First, ceramide-induced apoptosis is accompanied by release of pro-apoptotic proteins from mitochondria (Birbes et al., 2001; Hearps et al., 2002; Zhang et al., 2008), increased generation of mitochondrial ROS (Won and Singh, 2006), and discharge of mitochondrial transmembrane potential, ∆ψ (Gendron et al., 2001; Hearps et al., 2002; Lin et al., 2004; Zamzami et al., 1995). Second, interventions that specifically prevent mitochondrial dysfunction suppress ceramide-induced apoptosis: inhibitors of the MPTP bongkrekic acid (Gendron et al., 2001; Stoica et al., 2003) and cyclosporin A (Pacher and Hajnoczky, 2001; Pastorino et al., 1996; Stoica et al., 2003); and over-expression of Bcl-2 (Geley et al., 1997; Gendron et al., 2001; Scaffidi et al., 1999; Zamzami et al., 1995; Zhang et al., 1996). Third, TNF-α-, ischemia/reperfusion-, etoposide-, or UV-induced apoptosis is associated with simultaneous increase in mitochondrial ceramide (Birbes et al., 2005; Dai et al., 2004; Garcia-Ruiz et al., 1997; Yu et al., 2007).

Depending on cell type and stimuli, ceramide can alter mitochondrial function indirectly or directly (**Scheme 5**). Indirectly, ceramide modifies activity of pro-apoptotic and anti-

Apoptosis is mediated by two major pathways: the extrinsic and the intrinsic (or mitochondrial) pathway. The release of mitochondrial cytochrome c and/or Smac, which antagonizes apoptotic protein inhibitor, into the cytosol initiates the activation of caspase-9 leading to the proteolytic activation of executioner caspase-3 and -7. Cytochrome c release from mitochondria is well documented in different models of ischemia (Galluzzi et al., 2009). Recent studies have showed caspase-independent apoptosis involving the release of mitochondrial proteins, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) and their translocation to the nucleus in brain IR (Galluzzi et al., 2009; Joza et al., 2009). Emerging evidence indicates an important role of Bax in cytochrome c and Smac (but not AIF and EndoG) release from brain mitochondria (Brustovetsky et al., 2003). Importantly, mitochondrial calpain has been implicated in AIF release from mitochondria (Kar et al.). The release of mitochondrial proteins implies that the outer and /or the inner mitochondrial membrane is compromised in IR, but the precise mechanisms of the protein release remain unclear. Short-chain ceramide accelerated the release of cytochrome c and AIF from heart mitochondria (Di Paola et al., 2004), and natural C16-ceramide has been shown to form large channels in the outer mitochondrial membrane permeable to cytochrome c (Siskind and

Irrespective of the type of IR, IR-related physiological events have a common final consequence: alteration of mitochondrial function and release of mitochondrial proteins, leading to cell death. Cells with hallmarks of necrosis or apoptosis have been detected in animal models of IR (Li et al., 1995). The mitochondrial changes appear to be one essential step in tissue damage in IR, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function (Nakai et al., 1997). Multiple studies show intimate connections between ceramide signaling and functioning of mitochondria (Mimeault, 2002; Morales et al., 2007), which play central role in integration of cellular signals to determine the outcome among apoptosis, necrosis, or proliferation (Brenner and

 Several lines of evidence have implicated changes in mitochondrial function as an intermediate step in transduction of ceramide signals that culminate in apoptotic or necrotic cell death (Morales et al., 2007; Taha et al., 2006). First, ceramide-induced apoptosis is accompanied by release of pro-apoptotic proteins from mitochondria (Birbes et al., 2001; Hearps et al., 2002; Zhang et al., 2008), increased generation of mitochondrial ROS (Won and Singh, 2006), and discharge of mitochondrial transmembrane potential, ∆ψ (Gendron et al., 2001; Hearps et al., 2002; Lin et al., 2004; Zamzami et al., 1995). Second, interventions that specifically prevent mitochondrial dysfunction suppress ceramide-induced apoptosis: inhibitors of the MPTP bongkrekic acid (Gendron et al., 2001; Stoica et al., 2003) and cyclosporin A (Pacher and Hajnoczky, 2001; Pastorino et al., 1996; Stoica et al., 2003); and over-expression of Bcl-2 (Geley et al., 1997; Gendron et al., 2001; Scaffidi et al., 1999; Zamzami et al., 1995; Zhang et al., 1996). Third, TNF-α-, ischemia/reperfusion-, etoposide-, or UV-induced apoptosis is associated with simultaneous increase in mitochondrial

ceramide (Birbes et al., 2005; Dai et al., 2004; Garcia-Ruiz et al., 1997; Yu et al., 2007).

Depending on cell type and stimuli, ceramide can alter mitochondrial function indirectly or directly (**Scheme 5**). Indirectly, ceramide modifies activity of pro-apoptotic and anti-

**5.4 Release of pro-apoptotic proteins from mitochondria**

**6. Ceramide and mitochondria in cell death** 

Kroemer, 2000; Ferri and Kroemer, 2001; Kroemer et al., 2007).

Colombini, 2000).

apoptotic members of the Bcl-2 family of proteins that, in turn, alter the outer mitochondrial membrane permeability for cytochrome c and other pro-apoptotic molecules. Protein targets for ceramide in the cytoplasm include protein phosphatases PP1A and PP2A, protein kinases PKC ζ, raf-1, and kinase-suppressor Ras (Snook et al., 2006). In the lysosomal compartment, ceramide activates aspartate protease cathepsin D (Bidere et al., 2003; Heinrich et al., 2004; Heinrich et al., 1999; Pettus et al., 2002). Among these targets, cathepsin D, PP2A, and PP1A could propagate a pro-apoptotic ceramide signal to the level of the mitochondria (Pettus et al., 2002). Interaction of ceramide with cathepsin D results in cleavage of Bid to active tBid with subsequent activation of caspase-9 and caspase-3 (Heinrich et al., 2004). Activation by ceramides of serine/threonine protein phosphatase PP2A is involved in regulation of the apoptotic/anti-apoptotic activity of Bcl-2 family proteins by changing their phosphorylation status. Ceramide-activated PP2A increases the pro-apoptotic potential of Bcl-2 family proteins by dephosphorylation of Bax (activation) (Xin and Deng, 2006), or Bcl-2 (inactivation) (Ruvolo et al., 1999). An additional substrate for PP2A is serine/threonine kinase Akt/PKB (Pettus et al., 2002). Ceramide-dependent activation of PP2A leads to inactivation of Akt (Garcia et al., 2003; Millward et al., 1999; Pettus et al., 2002) that, in turn, results in dephosphorylation and activation of pro-apoptotic Bad, an Akt substrate (Datta et al., 1997). At the same time PP2A can directly dephosphorylate Bad, thus increasing its pro-apoptotic activity (Chiang et al., 2003). PP1A also can exert its effect on mitochondria by Bad dephosphorylation (Garcia et al., 2003). Interestingly, ceramide by itself can trigger transition of Bax into the active conformation, insertion in to the outer mitochondrial membrane with the subsequent release of cytochrome c and Smac in a cell-free system (Kashkar et al., 2005). Potentiation of Bax binding by ceramides to the outer mitochondrial membrane was shown by Birbes and colleagues (Birbes et al., 2005) and in energized mitochondria ceramide-induced Baxdependent MPTP opening (Pastorino et al., 1999b). Critical involvement of ceramide in triggering Bax translocation to the mitochondria was demonstrated during hypoxia/reoxygenation in neuronal cells (Jin et al., 2008). Attenuation of Bax translocation by knockdown of ceramide synthase CerS5 or aSMase suggests contribution of the activated salvage pathway in ceramide upregulation; however, the mechanisms by which ceramide exerts its effect remain unknown.

Less-defined, indirect mechanisms include interaction of ceramide with protein kinases PKC δ, p38 and JNK. Short-chain ceramides induce translocation of PKC δ from the cytoplasm to the mitochondria in LNCaP cells (Sumitomo et al., 2002). The translocation of PKC δ was accompanied by cytochrome c release. Mitochondrial translocation of PKC δ and activation of kinase activity was also evident when endogenous ceramides were raised by activation of de novo and neutral SMase-dependent pathways of ceramide production. Endogenous ceramide-induced PKC δ translocation similarly promoted release of cytochrome c and caspase-9 activation. A report by Huwiler et al. (Huwiler et al., 1998) indicates that ceramide can directly target PKC δ. Thus, increased ceramide during I/R can potentially contribute to mitochondrial translocation/activation of PKC δ, which enhances cytochrome c release in heart I/R (Murriel et al., 2004). Although potential mitochondrial PKC δ targets for which phosphorylation results in cytochrome c release remain illusive, PKC δ-dependant accumulation and dephosphorylation of Bad may contribute to the initiation of apoptotic program (Murriel et al., 2004).

The member of the mitogen-activated protein kinase (MAPK) superfamily p38 MAPK was implicated in ceramide-induced apoptosis in cardiomyocytes (Kong et al., 2005). Short-chain

Mitochondrial Ceramide in Stroke 285

Kurinna et al., 2004); however, direct interaction of JNK with the mitochondrial pool of BclxL was suggested (Kharbanda et al., 2000). Alternatively, activated JNK can induce mitochondrial dysfunction by phosphorylation of a pro-apoptotic member of the Bcl-2 family protein, Bim (Kurinna et al., 2004; Lei and Davis, 2003; Llacuna et al., 2006). Translocation of activated Bim to mitochondria initiates Bax-dependent cytochrome c release and apoptosis (Lei and Davis, 2003). Several other members of Bcl-2 family proteins have been proposed to mediate pro-apoptotic JNK signaling(Weston and Davis, 2007). Overall, the increased ratio of pro-apoptotic/anti-apoptotic proteins bound to mitochondria is generally considered to trigger permeabilization of the outer mitochondrial membrane for cytochrome c and other mitochondrial inter-membrane resident proteins, initiators of apoptosis (Armstrong, 2006; Kroemer et al., 2007). The increases in cell ceramide species contents are expected to contribute to the induction of apoptosis by this mechanism. Among non-protein indirect pathways, those associated with Ca2+ signaling attract special attention because of the well-known ability of these organelles both to respond to Ca2+ and to shape and propagate the Ca2+ signal within the cell (Giorgi et al., 2008; Szabadkai and Duchen, 2008). In this pathway, the Ca2+ pool of the ER is a target for ceramide (Pinton et al., 2001; Scorrano et al., 2003). Ca2+ released by ceramide from the ER is readily accumulated in

mitochondria that, in turn, results in MPTP opening, and cytochrome c release.

mitochondrial respiratory chain (Therade-Matharan et al., 2005).

Evidence is also accruing to implicate a direct action of ceramide on mitochondria. In this context, modulation by ceramide of mitochondrial functions at the level of isolated organelles has provided further evidence in support of this mechanism. It has been reported that ceramides directly suppress respiratory chain activity at the level of respiratory chain Complex III and/or Complex I (Di Paola et al., 2000; Garcia-Ruiz et al., 1997; Gudz et al., 1997; Yu et al., 2007). Suppression of the respiratory chain by ceramides results in increased production of ROS (Andrieu-Abadie et al., 2001; Di Paola et al., 2000; Garcia-Ruiz et al., 1997; Quillet-Mary et al., 1997), well-known inducers of an apoptotic cell response (Andrieu-Abadie et al., 2001; Ott et al., 2007). Increased ROS production by endothelial cells after hypoxia/reoxygenation was linked to the ceramide-induced suppression of the

Moreover, current research is focused on the ability of ceramides to release cytochrome c or other pro-apoptotic proteins from the mitochondrial inter-membrane space. Within the model of Colombini and co-workers, pro-apoptotic protein release is due to formation of large pores in the outer mitochondrial membrane by ceramide itself, whereas the inner membrane is viewed as being ceramide-insensitive. This model is supported by extensive experimental material using isolated mitochondria (Di Paola et al., 2004; Ghafourifar et al., 1999; Siskind et al., 2002, 2006) and artificial membranes (liposomes and black lipid membranes) (Montes et al., 2002; Siskind et al., 2002). Importantly, it was recently shown that anti-apoptotic Bcl-2 can disassemble ceramide channels in the outer mitochondrial membrane and black lipid membranes (Siskind et al., 2008), thus providing the mechanistic explanation for the original observation of Ghafourifar *et al*. (Ghafourifar et al., 1999) that Bcl-2 suppresses ceramide-induced cytochrome c release from isolated mitochondria. However, the formation of ceramide channels seems to be highly dependent on the conditions employed, and has been questioned in a number of publications (Kristal and Brown, 1999; Novgorodov et al., 2005; Szalai et al., 1999; Yuan H, 2003). Besides, a few reports suggest that the permeabilization of the inner mitochondrial membrane via the opening of the MPTP could be a primary event in initiation of cytochrome c release in the presence of ceramides (Pastorino et al., 1999b; Szalai et al., 1999). The switch between

Scheme 5. Ceramide modulates mitochondrial functions through direct and indirect mechanisms. **A**. Indirect modulation of mitochondrial functions by ceramide occurs through the change in the ratio of pro-apoptotic/anti-apoptotic proteins of Bcl-2 family at the outer mitochondrial membrane. **B**. Direct modulation of mitochondrial functions by ceramide include a) formation of ceramide channels permeable for cytochrome c in the outer mitochondrial membrane; b) potentiation of mitochondrial permeability transition pore opening (MPTP) in the inner membrane in the presence of Ca2+ or Bax (ceramide-induced Ca2+ release from the endoplasmic reticulum (ER) can contribute to the processes; c) potentiation of Bax insertion (activation) in to the outer membrane, d) inhibition of the respiratory chain (RC) with a subsequent increase in ROS formation.

ceramide treatment induced phosphorylation/activation of p38 MAPK which was accompanied by release of cytochrome c from the mitochondria and by the discharge of mitochondrial membrane potential. P38 MAPK inhibitor, SB 202190, abrogated the effect of ceramide both on p38 MAPK phosphorylation and on mitochondrial dysfunction. An interesting aspect of the study was the phosphorylation of the mitochondria-associated p38 MAPK pool under the influence of ceramide. This observation might indicate local signaling in mitochondria-mediated cell death. Although mitochondria-related targets for ceramideactivated p38 MAPK are not well defined, a recent report by Capano and Crompton (Capano and Crompton, 2006) demonstrates that activation of p38 MAPK during simulated ischemia in cardiomyocytes is a key regulatory point of Bax translocation from the cytosol to the mitochondria. The evidence of p38 MAPK-dependent phosphorylation of BimEL in apoptotic cell response has been provided (Cai et al., 2006) . Another member of the MAPK superfamily, JNK, was shown to be readily activated by both endogenous ceramide generation in liver I/R (Llacuna et al., 2006) and by addition of exogenous ceramide (Kurinna et al., 2004). Activated JNK translocates to the mitochondria and initiates cytochrome c release and cell death by yet unidentified mechanisms (Eminel et al., 2004;

**Dephosphorylation**

**PP2A (-)**

**apoptosis**

mechanisms. **A**. Indirect modulation of mitochondrial functions by ceramide occurs through the change in the ratio of pro-apoptotic/anti-apoptotic proteins of Bcl-2 family at the outer mitochondrial membrane. **B**. Direct modulation of mitochondrial functions by ceramide include a) formation of ceramide channels permeable for cytochrome c in the outer mitochondrial membrane; b) potentiation of mitochondrial permeability transition pore opening (MPTP) in the inner membrane in the presence of Ca2+ or Bax (ceramide-induced Ca2+ release from the endoplasmic reticulum (ER) can contribute to the processes; c) potentiation of Bax insertion (activation) in to the outer membrane, d) inhibition of the

ceramide treatment induced phosphorylation/activation of p38 MAPK which was accompanied by release of cytochrome c from the mitochondria and by the discharge of mitochondrial membrane potential. P38 MAPK inhibitor, SB 202190, abrogated the effect of ceramide both on p38 MAPK phosphorylation and on mitochondrial dysfunction. An interesting aspect of the study was the phosphorylation of the mitochondria-associated p38 MAPK pool under the influence of ceramide. This observation might indicate local signaling in mitochondria-mediated cell death. Although mitochondria-related targets for ceramideactivated p38 MAPK are not well defined, a recent report by Capano and Crompton (Capano and Crompton, 2006) demonstrates that activation of p38 MAPK during simulated ischemia in cardiomyocytes is a key regulatory point of Bax translocation from the cytosol to the mitochondria. The evidence of p38 MAPK-dependent phosphorylation of BimEL in apoptotic cell response has been provided (Cai et al., 2006) . Another member of the MAPK superfamily, JNK, was shown to be readily activated by both endogenous ceramide generation in liver I/R (Llacuna et al., 2006) and by addition of exogenous ceramide (Kurinna et al., 2004). Activated JNK translocates to the mitochondria and initiates cytochrome c release and cell death by yet unidentified mechanisms (Eminel et al., 2004;

Scheme 5. Ceramide modulates mitochondrial functions through direct and indirect

Bax insertion **ROS**

**SH H2O** RC **H+**

Ca**2+**

**(+)**

**ER**

**Ca2+**

MPTP opening; outer membrane rupture

MPTP

**(+)**

**(-)**

Bax

**(+)**

**Ca ceramide 2+**

**Ceramide**

Ceramide channels

**(+)**

Bax channels

respiratory chain (RC) with a subsequent increase in ROS formation.

**Bcl-2 (-) Bax (+) Bid Bad**

**phorylation**

**cleavage Dephos-**

**tBid,Bax,Bad/Bcl-2**

**tBid**

**Cytochrome c release**

**Cathepsin D PP2A Akt/PKB**

**(+) (+) (+)**

**PP1**

**(+)**

**A B**

**CERAMIDE**

Kurinna et al., 2004); however, direct interaction of JNK with the mitochondrial pool of BclxL was suggested (Kharbanda et al., 2000). Alternatively, activated JNK can induce mitochondrial dysfunction by phosphorylation of a pro-apoptotic member of the Bcl-2 family protein, Bim (Kurinna et al., 2004; Lei and Davis, 2003; Llacuna et al., 2006). Translocation of activated Bim to mitochondria initiates Bax-dependent cytochrome c release and apoptosis (Lei and Davis, 2003). Several other members of Bcl-2 family proteins have been proposed to mediate pro-apoptotic JNK signaling(Weston and Davis, 2007). Overall, the increased ratio of pro-apoptotic/anti-apoptotic proteins bound to mitochondria is generally considered to trigger permeabilization of the outer mitochondrial membrane for cytochrome c and other mitochondrial inter-membrane resident proteins, initiators of apoptosis (Armstrong, 2006; Kroemer et al., 2007). The increases in cell ceramide species contents are expected to contribute to the induction of apoptosis by this mechanism. Among non-protein indirect pathways, those associated with Ca2+ signaling attract special attention because of the well-known ability of these organelles both to respond to Ca2+ and to shape and propagate the Ca2+ signal within the cell (Giorgi et al., 2008; Szabadkai and Duchen, 2008). In this pathway, the Ca2+ pool of the ER is a target for ceramide (Pinton et al., 2001; Scorrano et al., 2003). Ca2+ released by ceramide from the ER is readily accumulated in mitochondria that, in turn, results in MPTP opening, and cytochrome c release.

Evidence is also accruing to implicate a direct action of ceramide on mitochondria. In this context, modulation by ceramide of mitochondrial functions at the level of isolated organelles has provided further evidence in support of this mechanism. It has been reported that ceramides directly suppress respiratory chain activity at the level of respiratory chain Complex III and/or Complex I (Di Paola et al., 2000; Garcia-Ruiz et al., 1997; Gudz et al., 1997; Yu et al., 2007). Suppression of the respiratory chain by ceramides results in increased production of ROS (Andrieu-Abadie et al., 2001; Di Paola et al., 2000; Garcia-Ruiz et al., 1997; Quillet-Mary et al., 1997), well-known inducers of an apoptotic cell response (Andrieu-Abadie et al., 2001; Ott et al., 2007). Increased ROS production by endothelial cells after hypoxia/reoxygenation was linked to the ceramide-induced suppression of the mitochondrial respiratory chain (Therade-Matharan et al., 2005).

Moreover, current research is focused on the ability of ceramides to release cytochrome c or other pro-apoptotic proteins from the mitochondrial inter-membrane space. Within the model of Colombini and co-workers, pro-apoptotic protein release is due to formation of large pores in the outer mitochondrial membrane by ceramide itself, whereas the inner membrane is viewed as being ceramide-insensitive. This model is supported by extensive experimental material using isolated mitochondria (Di Paola et al., 2004; Ghafourifar et al., 1999; Siskind et al., 2002, 2006) and artificial membranes (liposomes and black lipid membranes) (Montes et al., 2002; Siskind et al., 2002). Importantly, it was recently shown that anti-apoptotic Bcl-2 can disassemble ceramide channels in the outer mitochondrial membrane and black lipid membranes (Siskind et al., 2008), thus providing the mechanistic explanation for the original observation of Ghafourifar *et al*. (Ghafourifar et al., 1999) that Bcl-2 suppresses ceramide-induced cytochrome c release from isolated mitochondria. However, the formation of ceramide channels seems to be highly dependent on the conditions employed, and has been questioned in a number of publications (Kristal and Brown, 1999; Novgorodov et al., 2005; Szalai et al., 1999; Yuan H, 2003). Besides, a few reports suggest that the permeabilization of the inner mitochondrial membrane via the opening of the MPTP could be a primary event in initiation of cytochrome c release in the presence of ceramides (Pastorino et al., 1999b; Szalai et al., 1999). The switch between

Mitochondrial Ceramide in Stroke 287

Thus, the proposed mechanisms by which ceramides may affect mitochondria vary, and the combination of direct and indirect mechanisms involved in propagation of ceramide signals

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction and the release of mitochondrial proteins leading to cell death. It has become increasingly clear that ceramide, a membrane sphingolipid and a key mediator of cell-stress responses, could play a critical role in cerebral IR - induced mitochondrial injury. Mitochondria are being appreciated as vital intracellular compartments for ceramide metabolism in cerebral IR. Emerging data suggest that the subcellular location of ceramide generation plays a fundamental role in dictating its downstream targets and cell responses to stress stimuli. Continued research efforts are required to better understand the pathophysiological mechanisms of cerebral IR injury, to identify and test new protective agents. Further studies of the molecular basis of the role of ceramide in the ischemic brain are warranted. Because many assumptions regarding ceramide functions in IR-induced tissue injury were based on *in vitro* studies employing artificial ceramides, we must critically evaluate the mitochondrial dysfunctions in IR-injured brain and define a possible role of long-chain ceramides as causes of the mitochondrial impairment. This will allow the discovery of novel and groundbreaking therapeutic approaches to mitigate diseases that may result from elevations

We thank Dr. Jennifer G. Schnellmann for help with preparation of the manuscript. This work is supported by the NIH/NCCR COBRE in Lipidomics and Pathobiology P20 RR 17677 (TIG), VA Merit Awards from BLRD and RRD Service (TIG), and SAN was supported

Andreyev, A.Y., Fahy, E., Guan, Z., Kelly, S., Li, X., McDonald, J.G., Milne, S., Myers, D.,

Andrieu-Abadie, N., Gouaze, V., Salvayre, R., and Levade, T. (2001). Ceramide in apoptosis signaling: relationship with oxidative stress. Free Radic Biol Med *31*, 717-728. Ardail, D., Popa, I., Alcantara, K., Pons, A., Zanetta, J.P., Louisot, P., Thomas, L., and

Argaud, L., Prigent, A.F., Chalabreysse, L., Loufouat, J., Lagarde, M., and Ovize, M. (2004).

Armstrong, J.S. (2006). Mitochondrial membrane permeabilization: the sine qua non for cell

macrophages. J Lipid Res *51*, 2785-2797.

Park, H., Ryan, A.*, et al.* (2010). Subcellular organelle lipidomics in TLR-4-activated

Portoukalian, J. (2001). Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria. FEBS

Ceramide in the antiapoptotic effect of ischemic preconditioning. American journal

to mitochondria depends on cell type and the nature of the stimuli employed.

**7. Conclusion** 

in ceramide and its metabolites.

letters *488*, 160-164.

of physiology *286*, H246-251.

death. Bioessays *28*, 253-260.

**8. Acknowledgements** 

by NIH grant AG16583.

**9. References** 

selective permeabilization of the outer membrane vs. permeabilization of the inner membrane in the presence of ceramide appears to depend on the composition of incubation medium and the nature of ceramide employed (Di Paola et al., 2004).

Additional direct effects of ceramide on mitochondria include modulation of the ionic permeability of the lipid component of the inner membrane (Di Paola et al., 2000) and displacement of cytochrome c from the inner membrane as a result of the direct interaction with the proteins in the respiratory chain Complex III (Ghafourifar et al., 1999; Yuan H, 2003). Emerging evidence suggests involvement of ceramides in reorganization of the mitochondrial network. Both exogenous and endogenously generated ceramides induce mitochondrial fission (Parra et al., 2008; Zeidan et al., 2008), which may contribute to apoptotic cell death (Suen et al., 2008). What particular effectors of mitochondrial fission (DRP1, Fis1, or Bax) or fusion (OPA-1, Mitofusins) machineries are the targets of ceramide in this process remains to be determined. However, in cardiomyocytes exposed to exogenous C2-ceramide, an increased expression of mitochondrial resident Fis1 and enhanced recruitment of cytosolic DRP1 to mitochondrial fission foci may contribute to disintegration of the mitochondrial network (Parra et al., 2008).

It should be appreciated that the ceramide/mitochondria interaction in the control of apoptosis should be considered in conjunction with the effects of its pro-apoptotic metabolites such as ganglioside GD3 and sphingosine. GD3 shares with ceramide the same properties with respect to its effects on isolated mitochondria and mitochondria *in situ*. In cells, it disrupts mitochondrial membrane potential in a Bcl-2-sensitive manner (Rippo et al., 2000) and induces ROS production (Colell et al., 2001). At the level of isolated mitochondria, it inhibits the mitochondrial respiratory chain at the level of Complex III (Scorrano et al., 1999), increases ROS production (Garcia-Ruiz et al., 2000), opens the MPTP with the subsequent release of cytochrome c (Inoki et al., 2000; Kristal and Brown, 1999; Scorrano et al., 1999), and potentiates interaction of Bax with mitochondria (Pastorino et al., 1999b). Interestingly, while the effect of CD3 on ROS production is relatively nonspecific (lactosylceramide, GM1, GD1a and glucosylceramide produce a similar response (Garcia-Ruiz et al., 1997)), the effect of GD3 in the induction of apoptosis and MPTP opening shows considerable specificity. GM3, GM1 GD1, GD1a, GT1 has no or a slight inhibitory effect (Kristal and Brown, 1999; Pastorino et al., 1999b; Scorrano et al., 1999). In some instances, the effect of ceramide on mitochondria in cells can be explained by its conversion to GD3 (De Maria et al., 1997; Rippo et al., 2000).

Another pro-apoptotic ceramide derivative, sphingosine, releases cytochrome c from mitochondria that could be inhibited by over-expression of anti-apoptotic Bcl-xL (Cuvillier et al., 2000). In contrast to ceramides and GD3, sphingosine suppresses the MPTP in isolated mitochondria, and thus MPTP-dependent cytochrome c release (Broekemeier and Pfeiffer, 1995; Scorrano et al., 2001). It also inhibits ceramide channel formation in the outer mitochondrial membrane (Elrick et al., 2006). This indicates that indirect pathways of cytochrome c release, for example, by recruitment of Bax to mitochondria (Phillips et al., 2007), are predominant in the action of sphingosine on mitochondria. At the same time, similar to ceramide, sphingosine suppresses respiratory chain activity (Hassoun et al., 2006) and increases ROS production by mitochondria, although at higher concentrations (Garcia-Ruiz et al., 1997). Interaction of ceramide, sphingosine, and ganglioside pathways in the control of mitochondrial functions in the time-course of apoptosis remains to be established. Thus, the proposed mechanisms by which ceramides may affect mitochondria vary, and the combination of direct and indirect mechanisms involved in propagation of ceramide signals to mitochondria depends on cell type and the nature of the stimuli employed.

### **7. Conclusion**

286 Advances in the Preclinical Study of Ischemic Stroke

selective permeabilization of the outer membrane vs. permeabilization of the inner membrane in the presence of ceramide appears to depend on the composition of incubation

Additional direct effects of ceramide on mitochondria include modulation of the ionic permeability of the lipid component of the inner membrane (Di Paola et al., 2000) and displacement of cytochrome c from the inner membrane as a result of the direct interaction with the proteins in the respiratory chain Complex III (Ghafourifar et al., 1999; Yuan H, 2003). Emerging evidence suggests involvement of ceramides in reorganization of the mitochondrial network. Both exogenous and endogenously generated ceramides induce mitochondrial fission (Parra et al., 2008; Zeidan et al., 2008), which may contribute to apoptotic cell death (Suen et al., 2008). What particular effectors of mitochondrial fission (DRP1, Fis1, or Bax) or fusion (OPA-1, Mitofusins) machineries are the targets of ceramide in this process remains to be determined. However, in cardiomyocytes exposed to exogenous C2-ceramide, an increased expression of mitochondrial resident Fis1 and enhanced recruitment of cytosolic DRP1 to mitochondrial fission foci may contribute to disintegration

It should be appreciated that the ceramide/mitochondria interaction in the control of apoptosis should be considered in conjunction with the effects of its pro-apoptotic metabolites such as ganglioside GD3 and sphingosine. GD3 shares with ceramide the same properties with respect to its effects on isolated mitochondria and mitochondria *in situ*. In cells, it disrupts mitochondrial membrane potential in a Bcl-2-sensitive manner (Rippo et al., 2000) and induces ROS production (Colell et al., 2001). At the level of isolated mitochondria, it inhibits the mitochondrial respiratory chain at the level of Complex III (Scorrano et al., 1999), increases ROS production (Garcia-Ruiz et al., 2000), opens the MPTP with the subsequent release of cytochrome c (Inoki et al., 2000; Kristal and Brown, 1999; Scorrano et al., 1999), and potentiates interaction of Bax with mitochondria (Pastorino et al., 1999b). Interestingly, while the effect of CD3 on ROS production is relatively nonspecific (lactosylceramide, GM1, GD1a and glucosylceramide produce a similar response (Garcia-Ruiz et al., 1997)), the effect of GD3 in the induction of apoptosis and MPTP opening shows considerable specificity. GM3, GM1 GD1, GD1a, GT1 has no or a slight inhibitory effect (Kristal and Brown, 1999; Pastorino et al., 1999b; Scorrano et al., 1999). In some instances, the effect of ceramide on mitochondria in cells can be explained by its conversion to GD3 (De

Another pro-apoptotic ceramide derivative, sphingosine, releases cytochrome c from mitochondria that could be inhibited by over-expression of anti-apoptotic Bcl-xL (Cuvillier et al., 2000). In contrast to ceramides and GD3, sphingosine suppresses the MPTP in isolated mitochondria, and thus MPTP-dependent cytochrome c release (Broekemeier and Pfeiffer, 1995; Scorrano et al., 2001). It also inhibits ceramide channel formation in the outer mitochondrial membrane (Elrick et al., 2006). This indicates that indirect pathways of cytochrome c release, for example, by recruitment of Bax to mitochondria (Phillips et al., 2007), are predominant in the action of sphingosine on mitochondria. At the same time, similar to ceramide, sphingosine suppresses respiratory chain activity (Hassoun et al., 2006) and increases ROS production by mitochondria, although at higher concentrations (Garcia-Ruiz et al., 1997). Interaction of ceramide, sphingosine, and ganglioside pathways in the control of mitochondrial functions in the time-course of apoptosis remains to be established.

medium and the nature of ceramide employed (Di Paola et al., 2004).

of the mitochondrial network (Parra et al., 2008).

Maria et al., 1997; Rippo et al., 2000).

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction and the release of mitochondrial proteins leading to cell death. It has become increasingly clear that ceramide, a membrane sphingolipid and a key mediator of cell-stress responses, could play a critical role in cerebral IR - induced mitochondrial injury. Mitochondria are being appreciated as vital intracellular compartments for ceramide metabolism in cerebral IR. Emerging data suggest that the subcellular location of ceramide generation plays a fundamental role in dictating its downstream targets and cell responses to stress stimuli. Continued research efforts are required to better understand the pathophysiological mechanisms of cerebral IR injury, to identify and test new protective agents. Further studies of the molecular basis of the role of ceramide in the ischemic brain are warranted. Because many assumptions regarding ceramide functions in IR-induced tissue injury were based on *in vitro* studies employing artificial ceramides, we must critically evaluate the mitochondrial dysfunctions in IR-injured brain and define a possible role of long-chain ceramides as causes of the mitochondrial impairment. This will allow the discovery of novel and groundbreaking therapeutic approaches to mitigate diseases that may result from elevations in ceramide and its metabolites.

### **8. Acknowledgements**

We thank Dr. Jennifer G. Schnellmann for help with preparation of the manuscript. This work is supported by the NIH/NCCR COBRE in Lipidomics and Pathobiology P20 RR 17677 (TIG), VA Merit Awards from BLRD and RRD Service (TIG), and SAN was supported by NIH grant AG16583.

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**Part 3** 

**Novel Approaches to Neuroprotection** 

