**8. Neuronal injury and nerve cell death: basic sciences**

Neuronal death is normal during development of the nervous system, but it is abnormal in brain and spinal cord disease and injury. The available evidence indicates that the survival of neurons and their death are highly regulated and finely orchestrated dynamic events that depend on a number of internal and external factors. Two types of cell death are recognized: cell necrosis resulting from injury and causes inflammation and apoptosis, observed normally in development and now identified as programmed cell death. Apoptosis and necrosis are types of cell death. They are generally considered to be distinct forms of cell death, but there is mounting evidence supporting an apoptosisnecrosis cell death continuum (Portera-Calliau, et al, 1997). In this continuum, neuronal death can result from varying contributions of coexisting apoptotic and necrotic mechanisms, resulting in some of the distinctions between apoptosis and necrosis becoming blurred. Today it is believed that apoptosis may contribute to the neuronal degeneration in neurological injuries such as cerebral ischemia and trauma (Kerr et al 1972; MacManus and Linnik, 1997; Martin, 2001).

Necrosis can result from acute oxidative stress characterized by passive cell swelling, rapid energy losss, and generalized disruption of internal homeostasis with lysis of the nucleus, intra-nuclear organelles and plasma membranes leading to the release of intracellular components that induce a local inflammatory response that in turn, result in edema and injury to neighboring cells. Morphologically, cell death is characterized by swelling of organelles and rupture. Necrotic cell death is characterized by inflammation and widespread damage.

Apoptosis is a process of cell suicide, the mechanisms of which are encoded in the chromosomes of all nucleated cells. Although the capacity to carry out apoptosis appears to be inherent in all cells, the susceptibility to apoptosis varies markedly and is influenced by external and cell-autonomous events. Apoptosis is regulated by complex molecular signaling systems resulting in an orderly, energy-dependent enzymatic breakdown into characteristic molecular fragments, DNA, lipids and other macromolecules. Apoptosis can be induced by cell surface receptor engagement, growth factor withdrawal and DNA damage. In contrast to those observed in cell necrosis, the morphological changes that occur during developmental cell death include cell shrinkage, membrane blebbing, chromatin condensation and DNA fragmentation. Earlier studies showed that one of the biochemical hallmarks of apoptosis is DNA cleavage at internucleosomal linker regions, resulting in ladder formation of DNA of 180-200 bp or multiples thereof. However, this ladder-type DNA fragmentation is also found in some cells dying of necrosis, indicating that DNA fragmentation cannot be the sole criterion, but simultaneous morphological assessment must also be done for identifying apoptosis.

#### **8.1 Mechanisms of apoptotic cell death**

Several families of proteins and specific biochemical signal-transduction pathways regulate cell death. Cell death signaling can involve plasma membrane death receptors, mitochondrial death proteins, proteases, kinases and transcription factors. Predominant factors in cell death and cell survival include fas receptor, Bcl-2 and Bax (and their homologues), cytochrome c, caspases, p53 and extra cellular signal-regulated protein kinases. Some forms of cell death require gene activation, RNA synthesis and protein synthesis, whereas other forms are transcriptionally-translationally independent and are

Neurologic Injury Following Hypothermic Circulatory Arrest 253

**Motor Cortex** 3.28+0.32\* 1.79+0.38+ 0.50+0.22 **Sensory Cortex** 3.88+0.13\*\* 1.60+0.31+++ 0.14+0.14

**Hippocampus** 2.67+0.36\* 1.39+0.24++ 0.17+0.17 **Cerebellum** 2.13+0.48 1.82+0.23++ 0.71+0.18

**Medulla** 2.00+0.41 2.08+0.23+++ 0.57+0.20 **Thalamus** 2.33+0.67 1.54+0.31+++ 0.00+0.00

All values are expressed as mean + SE. \*p<0.006 and \*\*p<0.0001 compared to values from animals treated with 10oC HCA. +p<0.05; ++p<0.005; +++p<0.002 compared to normal

Age and temperature appear to influence neuronal injury, by making certain nerve cell populations more vulnerable to injury. In particular, the hippocampus, cerebellum, striatum, thalamus, amygdala and neocortex have been reported vulnerable in adult normothermic ischemia. In contrast, newborns were more vulnerable to injury in the neocortex and striatum. In the present model of hypothermic ischemia in juvenile pigs, the neocortex and hippocampus demonstrated the greatest vulnerability to insult during HCA. The apparent higher level of TUNEL positive cells in the primary sensory cortex (post-

Although these previous studies clearly support that some of the cell death observed in HCA is via an apoptotic pathway, the experimental conditions used may underestimate the contribution of apoptosis to the cerebral sequelae after HCA (Kurth et al, 1999; Hagl et al, 2001). In this regard, some authors have expressed concern regarding the temporal pattern of brain damage and apoptosis after HCA. Thus, although recently improved methods of perfusion-fixation and more sophisticated analysis, have clearly shown the HCA initiates a series of events that ultimately leads to cell death via a typical apoptotic pattern, the time course of these events remains unclear. Most of these previous studies use the classic 90-min HCA, 20 oC model, which results in more severe cerebral injury than that usually observed clinically, where HCA is carried out for shorter intervals. The results from earlier studies also demonstrated that serious cell injury exists as early as 6 h after HCA, and that this

The importance of understanding the time course of events is underscored by an earlier study of long-term survivors of the 90-min, 20 oC protocol. Although treatment with CsA was reported to improve behavioral recovery after 7 days, at the 7 days time point after HCA there was no difference between CsA treated animals and controls for apoptosis measures. The authors concluded that they had missed the peak of apoptosis, and that an effective reduction in nerve cell injury would be found most likely with CsA treatment had

We found no morphological evidence of apoptosis or necrosis, but significantly greater levels of TUNEL positive cells in the brain regions assessed, compared to normal control animals. We hypothesize, that these findings indicate an early point of activation of the apoptotic

Table 2. TUNEL Scores in Brain Regions of Animals Treated with HCA

 **10 C o Control C**

**Brain Region 18 <sup>o</sup>**

control levels.

central gyrus) is not clear.

process continues for at least 72h.

they examined brain tissues at an earlier time point.

driven by posttranslational mechanisms such as protein phosphorylation and protein translocation.

The precise signaling cascade starting from the detection of the signal at the cell surface to the events that occur in the nucleus in apoptosis is not well established, with several grey zones in most suggested pathways. However, many events that occur at the cell surface and intracellularly during apoptosis in the nervous system have been reported. Following an appropriate stimulus, the first stage or "decision phase" of apoptosis is the genetic control point of cell death. This is followed by the second state or "execution phase", which is responsible for the morphological changes of apoptosis. The decision phase or genetic control appears to be mediated by two genes Bcl-2 and p53, while the execution phase appears to result from the activation of caspases. It has become apparent that the Bcl-2 family of proteins constitutes a critical intracellular checkpoint within a distal common pathway of programmed cell death (Almeida et al, 2000).

#### **8.2 Selective vulnerability of neural populations to neural insult after HCA**

After assessing acute neuronal injury in various regions of the brain after HCA in a porcine animal model, we found that neurons in the sensory and motor neocortex, as well as those in the hippocampus, were vulnerable to cell injury acutely after 75 min of HCA at 18oC, as determined by a positive TUNEL reaction for DNA fragmentation (Ananiadou et al, 2005). TUNEL positive cells are identified by a red-stained, condensed nucleus with apoptotic bodies, along with a diminutive or absent cytoplasm. (Figure 1) Although nerve cell populations in the cerebellum, thalamus and ventral medulla were also found vulnerable to cell injury, the percentage of TUNEL positive cells in these areas was significantly less than that observed in the primary motor and sensory gray matter, and in the hippocampus. (Table 2)

Fig. 1. Typical Presentation of TUNEL Positive Cells.

Photomicrograph showing apoptosis in the brain following HCA in an acute porcine model. Cluster of TUNEL (+) apoptotic neurons (nucleus is red stained) are interspersed among normal neurons in the anteroventral medulla. (magnification x400)

driven by posttranslational mechanisms such as protein phosphorylation and protein

The precise signaling cascade starting from the detection of the signal at the cell surface to the events that occur in the nucleus in apoptosis is not well established, with several grey zones in most suggested pathways. However, many events that occur at the cell surface and intracellularly during apoptosis in the nervous system have been reported. Following an appropriate stimulus, the first stage or "decision phase" of apoptosis is the genetic control point of cell death. This is followed by the second state or "execution phase", which is responsible for the morphological changes of apoptosis. The decision phase or genetic control appears to be mediated by two genes Bcl-2 and p53, while the execution phase appears to result from the activation of caspases. It has become apparent that the Bcl-2 family of proteins constitutes a critical intracellular checkpoint within a distal common

pathway of programmed cell death (Almeida et al, 2000).

Fig. 1. Typical Presentation of TUNEL Positive Cells.

normal neurons in the anteroventral medulla. (magnification x400)

**8.2 Selective vulnerability of neural populations to neural insult after HCA** 

After assessing acute neuronal injury in various regions of the brain after HCA in a porcine animal model, we found that neurons in the sensory and motor neocortex, as well as those in the hippocampus, were vulnerable to cell injury acutely after 75 min of HCA at 18oC, as determined by a positive TUNEL reaction for DNA fragmentation (Ananiadou et al, 2005). TUNEL positive cells are identified by a red-stained, condensed nucleus with apoptotic bodies, along with a diminutive or absent cytoplasm. (Figure 1) Although nerve cell populations in the cerebellum, thalamus and ventral medulla were also found vulnerable to cell injury, the percentage of TUNEL positive cells in these areas was significantly less than that observed in the primary motor and sensory gray matter, and in the hippocampus.

Photomicrograph showing apoptosis in the brain following HCA in an acute porcine model. Cluster of TUNEL (+) apoptotic neurons (nucleus is red stained) are interspersed among

translocation.

(Table 2)


Table 2. TUNEL Scores in Brain Regions of Animals Treated with HCA All values are expressed as mean + SE. \*p<0.006 and \*\*p<0.0001 compared to values from animals treated with 10oC HCA. +p<0.05; ++p<0.005; +++p<0.002 compared to normal control levels.

Age and temperature appear to influence neuronal injury, by making certain nerve cell populations more vulnerable to injury. In particular, the hippocampus, cerebellum, striatum, thalamus, amygdala and neocortex have been reported vulnerable in adult normothermic ischemia. In contrast, newborns were more vulnerable to injury in the neocortex and striatum. In the present model of hypothermic ischemia in juvenile pigs, the neocortex and hippocampus demonstrated the greatest vulnerability to insult during HCA. The apparent higher level of TUNEL positive cells in the primary sensory cortex (postcentral gyrus) is not clear.

Although these previous studies clearly support that some of the cell death observed in HCA is via an apoptotic pathway, the experimental conditions used may underestimate the contribution of apoptosis to the cerebral sequelae after HCA (Kurth et al, 1999; Hagl et al, 2001). In this regard, some authors have expressed concern regarding the temporal pattern of brain damage and apoptosis after HCA. Thus, although recently improved methods of perfusion-fixation and more sophisticated analysis, have clearly shown the HCA initiates a series of events that ultimately leads to cell death via a typical apoptotic pattern, the time course of these events remains unclear. Most of these previous studies use the classic 90-min HCA, 20 oC model, which results in more severe cerebral injury than that usually observed clinically, where HCA is carried out for shorter intervals. The results from earlier studies also demonstrated that serious cell injury exists as early as 6 h after HCA, and that this process continues for at least 72h.

The importance of understanding the time course of events is underscored by an earlier study of long-term survivors of the 90-min, 20 oC protocol. Although treatment with CsA was reported to improve behavioral recovery after 7 days, at the 7 days time point after HCA there was no difference between CsA treated animals and controls for apoptosis measures. The authors concluded that they had missed the peak of apoptosis, and that an effective reduction in nerve cell injury would be found most likely with CsA treatment had they examined brain tissues at an earlier time point.

We found no morphological evidence of apoptosis or necrosis, but significantly greater levels of TUNEL positive cells in the brain regions assessed, compared to normal control animals. We hypothesize, that these findings indicate an early point of activation of the apoptotic

Neurologic Injury Following Hypothermic Circulatory Arrest 255

ischemia because it has a high metabolic rate and small reserve of high-energy carbohydrates and phosphates. Several studies have indicated that cerebral metabolism is reduced effectively at profound levels of hypothermia, suggesting that protection of the brain should be greater when HCA is performed at even lower temperatures, such as that

We have found that profound hypothermia at 10°C during HCA resulted in a significant reduction in neurological injury in selectively vulnerable brain regions. TUNEL (+) staining was significantly less at 10°C in the motor and sensory cortex and the hippocampus compared to 18°C HCA, indicating that there was increased cerebral protection (Ananiadou et al, 2008). These findings are compatible with previous reports that profound hypothermia results in a superior neurological outcome compared to conventional HCA methods. Although this study does not elucidate the mechanisms, it does affirm that profound

Apoptosis is controlled genetically, and two genes, Bcl-2 and p53 are now believed to be important. It is now established that proteins encoded by the Bcl-2 gene family are major regulatory components of the apoptotic pathway (Kroemer, 1997). Within the apoptotic cascade, several proteins that facilitate neuronal survival compete with molecules that contribute to cell death. Ultimately, the final balance between cell survival–promoting proteins versus cell death–promoting proteins determines the fate of the cell. The Bcl-2

This hypothesis is supported by our findings in Bcl-2 expression. The Bcl-2 family of proteins is important for the regulation of apoptosis during the "decision phase." An increase of Bcl-2 has been suggested as an internal protective mechanism against apoptotic cell death, where Bcl-2 is persistently expressed in neurons that survive in ischemia. In the present study, brain regions that were selectively vulnerable to neurologic injury, particularly the neocortex and hippocampus, showed higher levels of Bcl-2 expression after HCA at 18°C compared with other brain regions (thalamus, cerebellum, and medulla). Moreover, profound hypothermia at 10°C resulted in a significant decrease in TUNEL staining in these brain regions. Although a concomitant increase in Bcl-2 expression was observed in the neocortex, it remains unclear whether profound hypothermia deters from neuronal injury by activation of anti-apoptotic protein Bcl-2 expression (Ananiadou et al,

**Sensory Cortex 18oC 10oC Control** 

**TUNEL** 3.88+0.13\*\* 1.60+0.31\*\*\* 0.14+0.14 **Bcl-2** 0.83 + 0.31 1.8 + 0.31\* 1.8 + 0.63

Table 3. TUNEL Scores and Bcl-2 Immunoreactivity in the Sensory Neocortex of Animals

Treated with HCA at 18oC or 10oC Compared to Controls

\*p<0.05 compared to values from animals treated with 18oC HCA. \*\*p<0.0001 compared to values from animals treated with 10oC HCA.

All values are expressed as mean + SE.

\*\*\*p<0.002 compared to normal control levels

**8.4 The decision phase for apoptotic nerve cell death: evidence for Bcl-2** 

family of proteins plays an important role in this cell survival-cell death decision.

used in our studies (Strauch et al, 2005).

hypothermia exerts a neuroprotective effect.

2007). (Table 3)

pathway. This is consistent with the rapid cell death observed in normal cell suicide programs that can kill a cell within 2 to 3 h. At an earlier time point, such as that in this study, we would not anticipate completion of the apoptotic mechanism, resulting in cell death with its classic morphological characteristics, but rather the initiation of the cellular response cascade.

Certain cell populations appeart to be more vulnerable to injury. In particular, the hippocampus, cerebelleum, striatum, thalamus, amygdala and neocortex are more vulnerable in adult normothermic ischemia. In contrast, newborns are more vulnerable to injury in the neocortex and striatum. Our studies show that hypothermia does not provide equal protection to all regions of the brain. In the juvenile pig model, the neocortex and hippocampus demonstrated the greatest vulnerability to insult during HCA. The apparent higher level of TUNEL positive cells in the primary sensory cortex (post-central gyrus) is not clear, and demands further investigation. (Figure 2)

Fig. 2. Positive TUNEL Reaction in Vulnerable Neural Regions Regional pattern of neuronal death after deep hypothermic circulatory arrest in juvenile pigs. Each point represents the mean score from six experimental animals for each brain region. The brackets indicate the S.D. Among the HCA treated animals, significantly higher concentrations of TUNEL (+) cells were observed in the sensory cortex, motor cortex and hippocampus, compared to the cerebellum, thalamus and medulla (P< 0.05 by ANOVA followed by Fisher PSLD). Although not statistically significantly greater than the motor neocortex and hippocampus, the postcentral gyrus had greater TUNEL scores compared to the medulla and thalamus (P<0.01). [\*P<0.05 vs sensory and motor cortex, and hippocampus; \*\* P<0.001 vs sensory cortex}

#### **8.3 Profound hpothermia rduces aoptotic nurologic ijury ater HCA**

The use of HCA in aortic repair and congenital heart surgery is based on the idea of reducing the metabolic rate and thus, allowing a more prolonged interval without perfusion that can be safely tolerated by the brain. The brain, in general, is very sensitive to hypoxia-

pathway. This is consistent with the rapid cell death observed in normal cell suicide programs that can kill a cell within 2 to 3 h. At an earlier time point, such as that in this study, we would not anticipate completion of the apoptotic mechanism, resulting in cell death with its classic

Certain cell populations appeart to be more vulnerable to injury. In particular, the hippocampus, cerebelleum, striatum, thalamus, amygdala and neocortex are more vulnerable in adult normothermic ischemia. In contrast, newborns are more vulnerable to injury in the neocortex and striatum. Our studies show that hypothermia does not provide equal protection to all regions of the brain. In the juvenile pig model, the neocortex and hippocampus demonstrated the greatest vulnerability to insult during HCA. The apparent higher level of TUNEL positive cells in the primary sensory cortex (post-central gyrus) is not

morphological characteristics, but rather the initiation of the cellular response cascade.

clear, and demands further investigation. (Figure 2)

Fig. 2. Positive TUNEL Reaction in Vulnerable Neural Regions

hippocampus; \*\* P<0.001 vs sensory cortex}

Regional pattern of neuronal death after deep hypothermic circulatory arrest in juvenile pigs. Each point represents the mean score from six experimental animals for each brain region. The brackets indicate the S.D. Among the HCA treated animals, significantly higher concentrations of TUNEL (+) cells were observed in the sensory cortex, motor cortex and hippocampus, compared to the cerebellum, thalamus and medulla (P< 0.05 by ANOVA followed by Fisher PSLD). Although not statistically significantly greater than the motor neocortex and hippocampus, the postcentral gyrus had greater TUNEL scores compared to

The use of HCA in aortic repair and congenital heart surgery is based on the idea of reducing the metabolic rate and thus, allowing a more prolonged interval without perfusion that can be safely tolerated by the brain. The brain, in general, is very sensitive to hypoxia-

the medulla and thalamus (P<0.01). [\*P<0.05 vs sensory and motor cortex, and

**8.3 Profound hpothermia rduces aoptotic nurologic ijury ater HCA** 

ischemia because it has a high metabolic rate and small reserve of high-energy carbohydrates and phosphates. Several studies have indicated that cerebral metabolism is reduced effectively at profound levels of hypothermia, suggesting that protection of the brain should be greater when HCA is performed at even lower temperatures, such as that used in our studies (Strauch et al, 2005).

We have found that profound hypothermia at 10°C during HCA resulted in a significant reduction in neurological injury in selectively vulnerable brain regions. TUNEL (+) staining was significantly less at 10°C in the motor and sensory cortex and the hippocampus compared to 18°C HCA, indicating that there was increased cerebral protection (Ananiadou et al, 2008). These findings are compatible with previous reports that profound hypothermia results in a superior neurological outcome compared to conventional HCA methods. Although this study does not elucidate the mechanisms, it does affirm that profound hypothermia exerts a neuroprotective effect.
