**Part 3**

**Novel Approaches to Neuroprotection** 

302 Advances in the Preclinical Study of Ischemic Stroke

Zager, R.A., Conrad, S., Lochhead, K., Sweeney, E.A., Igarashi, Y., and Burkhart, K.M.

Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S.A.,

Zeidan, Y.H., Wu, B.X., Jenkins, R.W., Obeid, L.M., and Hannun, Y.A. (2008). A novel role

Zhang, D.X., Fryer, R.M., Hsu, A.K., Zou, A.P., Gross, G.J., Campbell, W.B., and Li, P.L.

Zhang, J., Alter, N., Reed, J.C., Borner, C., Obeid, L.M., and Hannun, Y.A. (1996). Bcl-2

National Academy of Sciences of the United States of America *93*, 5325-5328. Zhang, X., Li, B., Zhang, Y., and Liu, J. (2008). Ceramide induces release of mitochondrial

Zundel, W., Swiersz, L.M., and Giaccia, A. (2000). Caveolin 1-mediated regulation of

renal failure. Kidney international *52*, 60-70.

cells. Science in China *51*, 66-71.

in early programmed cell death. J Exp Med *182*, 367-377.

UV light-induced mitochondrial injury. Faseb J *22*, 183-193.

myocardium of rats. Basic research in cardiology *96*, 267-274.

ceramide. Molecular and cellular biology *20*, 1507-1514.

(1998). Altered sphingomyelinase and ceramide expression in the setting of ischemic and nephrotoxic acute renal failure. Kidney international *53*, 573-582. Zager, R.A., Iwata, M., Conrad, D.S., Burkhart, K.M., and Igarashi, Y. (1997). Altered

ceramide and sphingosine expression during the induction phase of ischemic acute

Petit, P.X., Mignotte, B., and Kroemer, G. (1995). Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species

for protein kinase Cdelta-mediated phosphorylation of acid sphingomyelinase in

(2001). Production and metabolism of ceramide in normal and ischemic-reperfused

interrupts the ceramide-mediated pathway of cell death. Proceedings of the

proapoptotic proteins in caspase-dependent and -independent manner in HT-29

receptor tyrosine kinase-associated phosphatidylinositol 3-kinase activity by

**14** 

*Morelia, Michoacán* 

*Mexico* 

**Neuroprotection in Animal Models** 

María Esther Olvera-Cortés3 and Gabriela Moralí4

*2Laboratorio de Psicobiología, División de Neurociencias,* 

*Instituto Mexicano del Seguro Social, Guadalajara, Jalisco* 

*Instituto Mexicano del Seguro Social, Morelia, Michoacán* 

*Centro de Investigación, Biomédica de Occidente* 

*4Unidad de Investigación Médica en Farmacología, UMAE Hospital de Especialidades, CMN S XXI, Instituto Mexicano del Seguro Social, México, D.F.* 

*3Laboratorio de Neurofisiología Experimental, Centro de Investigación Biomédica de Michoacán,* 

*1Laboratorio de Neurociencias. Facultad de Ciencias Médicas y Biológicas "Dr. Ignacio Chávez", Universidad Michoacana de San Nicolás de Hidalgo,* 

Miguel Cervantes1, Ignacio González-Burgos2, Graciela Letechipía-Vallejo1,

The present chapter deals with some of the main lines of experimental research on global cerebral ischemia, through which a substantial knowledge has been generated, that has contributed in an important measure both to the understanding of the mechanisms of cerebral damage induced by ischemia, and of the subsequent post-ischemic neuroregenerative and cerebral plastic processes taking place in the remaining or newly differentiated neurons. Thus, data obtained from experimental designs in animal models of global cerebral ischemia, on key molecular and cellular events triggered by this condition, have provided a substantial background from which neuroprotection can be rationally approached, in order to develop strategies aimed to antagonize, to interrupt, or to slow the sequence of injurious biochemical and molecular events that would result in irreversible ischemic injury; as well as to promote brain repair and plasticity processes which can favor

Transient global cerebral ischemia, which can mainly occur during cardiac arrest and cardiopulmonary resuscitation, but also during asphyxiation, hypotensive shock, or extracorporeal circulation, is a pathophysiological condition that is associated with great morbidity and requires intensive medical treatment (Madl & Holzer, 2004). In certain clinical situations (surgical repair of the thoracic aorta, complex congenital heart lesions, and also during implantable cardiac defibrillator testing in patients with drug-resistant

functional preservation or recovery after global cerebral ischemia.

**1. Introduction** 

**of Global Cerebral Ischemia** 

### **Neuroprotection in Animal Models of Global Cerebral Ischemia**

Miguel Cervantes1, Ignacio González-Burgos2, Graciela Letechipía-Vallejo1, María Esther Olvera-Cortés3 and Gabriela Moralí4 *1Laboratorio de Neurociencias. Facultad de Ciencias Médicas y Biológicas "Dr. Ignacio Chávez", Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán 2Laboratorio de Psicobiología, División de Neurociencias, Centro de Investigación, Biomédica de Occidente Instituto Mexicano del Seguro Social, Guadalajara, Jalisco 3Laboratorio de Neurofisiología Experimental, Centro de Investigación Biomédica de Michoacán, Instituto Mexicano del Seguro Social, Morelia, Michoacán 4Unidad de Investigación Médica en Farmacología, UMAE Hospital de Especialidades, CMN S XXI, Instituto Mexicano del Seguro Social, México, D.F. Mexico* 

### **1. Introduction**

The present chapter deals with some of the main lines of experimental research on global cerebral ischemia, through which a substantial knowledge has been generated, that has contributed in an important measure both to the understanding of the mechanisms of cerebral damage induced by ischemia, and of the subsequent post-ischemic neuroregenerative and cerebral plastic processes taking place in the remaining or newly differentiated neurons. Thus, data obtained from experimental designs in animal models of global cerebral ischemia, on key molecular and cellular events triggered by this condition, have provided a substantial background from which neuroprotection can be rationally approached, in order to develop strategies aimed to antagonize, to interrupt, or to slow the sequence of injurious biochemical and molecular events that would result in irreversible ischemic injury; as well as to promote brain repair and plasticity processes which can favor functional preservation or recovery after global cerebral ischemia.

Transient global cerebral ischemia, which can mainly occur during cardiac arrest and cardiopulmonary resuscitation, but also during asphyxiation, hypotensive shock, or extracorporeal circulation, is a pathophysiological condition that is associated with great morbidity and requires intensive medical treatment (Madl & Holzer, 2004). In certain clinical situations (surgical repair of the thoracic aorta, complex congenital heart lesions, and also during implantable cardiac defibrillator testing in patients with drug-resistant

Neuroprotection in Animal Models of Global Cerebral Ischemia 307

Animal models of global cerebral ischemia allow studying, at different levels of biological organization of the central nervous system, the development and temporal course of those processes that may result in irreversible ischemic neuronal damage, as well as in the subsequent cell repair and plasticity underlying either permanent cerebral functional impairment or recovery as a result of intrinsic brain mechanisms or neuroprotective procedures. Thus, animal-related factors (species, strain, age, sex, co-morbidities), animalmodel-related factors (choice of ischemic model, anesthetic procedures, duration of ischemia, reperfusion, survival, possibility of monitoring of physiological parameters), selective vulnerability of specific neuron types in several brain structures, outcome assessment (histopathological, biochemical, functional, parameters of brain injury in specific cerebral structures), short- or long-term experimental design, pharmacological characteristics of the presumptive neuroprotective agent itself, timing and dose-response of neuroprotective drug administration with reference to starting and ending of the ischemic

Models of cerebral ischemia have been also developed in *in vitro* models, in particular brain tissue slices and neuronal cultures, allowing to study in detail the cellular phenomena leading either to neuronal damage or to neural recovery and plasticity after ischemia (Benítez-King, 2006; Goldberg & Choi, 1993; Kasai et al., 2003; Whittingham et al., 1984). Several conditions have to be fulfilled by animal models of global cerebral ischemia in order to become appropriate counterparts of these pathophysiological conditions in human beings, as well as to yield reliable and valid results in supporting clinical therapeutic approaches. Thus**,** it could be expected that in animal models of global cerebral ischemia the ischemic episode can be induced in a constant and reproducible manner: low variation for the extent, temporal course, and magnitude of the resulting ischemic brain injury under specific experimental conditions, including duration of the ischemic episode; easy control of possible deviations of important physiological variables, feasible neurological, neuropathological, and functional evaluations; lack of influence of anesthetic drugs and surgical procedures on the mechanisms of brain injury, brain recovery and/or neuroprotection; short-, intermediate- and long-term follow up of the outcome; and economical, easily available experimental animals of those species better accepted by public animal welfare concerns to be used in experimental protocols of cerebral ischemia and

Models of global cerebral ischemia have been performed in both large (monkeys, sheep, dogs, pigs, cats, rabbits) and small animals (gerbils, rats, mice). Among these, both advantages and disadvantages can be recognized according to several practical aspects: main objectives of the model; monitoring procedures to be used; nature, number and timing of simultaneous parameters to be recorded in order to evaluate the ischemic brain injury and recovery; degree of similarity of structural and functional characteristics of brains of experimental animals to those of the human brain; and updated ethical outlines for the use

Since the whole brain is exposed to transient ischemia and reperfusion as a result of cardiac arrest and the subsequent cardiorespiratory resuscitation to allow survival in human beings, animal models of global cerebral ischemia have been designed attempting to totally or

episode, may account for the relevance of results from these investigations.

**2. Animal models of global cerebral ischemia** 

neuroprotection.

**2.1 Main animal models of global cerebral ischemia** 

of experimental animals in research protocols.

ventricular fibrillation) the possible occurrence of transient global cerebral ischemia, and some neuroprotective procedures, can be anticipated (Hogue et al., 2008); however, this is not the case of cardiac arrest.

Cardiopulmonary arrest remains as one among the most frequent causes of death and disability around the world. Despite quick emergency responses and better techniques of defibrillation, the chances of survival following cardiac arrest are still poor, between 20-50% of patients in whom cardiopulmonary resuscitation is attempted. A complex pathophysiological condition is elicited by cardiac arrest, since it results in whole-body ischemia which compromises systemic circulatory homeostasis and cerebral, pulmonary, renal, and cardiac functions. In the course of cardiac arrest, global cerebral blood flow is severely impaired with the consequent risk of ischemic damage of brain cells, which magnitude seems to be associated with the cumulative time staying in cardiac arrest. Thus, most deaths (60%) during the post-resuscitation period have been attributed to extensive brain injury and neuronal damage that develops as a consequence of alteration of cell processes triggered by cerebral ischemia and reperfusion, during and after cardiac arrest. In addition, it is known that transient interruption or reduction of blood flow in the whole brain, are main causes of permanent brain damage and functional disruptions in human beings, and near around a half of surviving patients show permanent impairment of cognitive functions, such as learning and memory, attention, and executive functioning, and only a small proportion (less than 10%) of those survivors are able to reassume their former usual life styles (Geocardin et al., 2008; Grubb et al., 2000; Krause et al, 1986; Schneider et al., 2009). Thus, development of effective cytoprotective therapies that may be common to the organs more sensitive to cardiac arrest, such as heart or brain, could result in improvement of survival and better outcome following this whole ischemic episode (Karanjia & Geocardin, 2011).

Experimental protocols aimed to gain relevant information regarding those pathophysiological phenomena leading to cerebral damage elicited by ischemia have included, since long time, the use of animal models of cerebral ischemia, in order to support better diagnostic, prophylactic and clinical-therapeutic procedures for ischemic cerebrovascular diseases in human beings (Ginsberg & Busto, 1989; Gupta & Briyal, 2004; Hartman et al., 2005, Hossmann, 2008; Traystman, 2003). Thus, biochemical, electrophysiological, histological, and behavioral parameters of ischemic brain damage have been included in experimental designs to evaluate the efficacy and safety of pharmacological and non pharmacological neuroprotective procedures against brain injury resulting from the significant reduction of blood supply to the whole brain, in several animal models of global cerebral ischemia.

Even though a great number of pharmacological agents have proven to exert effective neuroprotective actions against cellular events leading to ischemic brain injury in experimental models of global cerebral ischemia, unfortunately they have not had enough clinical relevance to date. On the other hand, after evaluation of its effectiveness as a neuroprotective strategy in animal models of global cerebral ischemia, hypothermia has been tested in clinical trials in patients having suffered cardiac arrest, the most frequent cause of global cerebral ischemia in human beings (Castren et al., 2009; Geocardin et al., 2008; Greer, 2006; Inamasu et al., 2010; Knapp et al., 2011; Seder & Jarrah, 2008,). It seems that new and better strategies to translate preclinical data supporting the potential clinical usefulness of neuroprotective drugs to clinical trials, must be developed.

ventricular fibrillation) the possible occurrence of transient global cerebral ischemia, and some neuroprotective procedures, can be anticipated (Hogue et al., 2008); however, this is

Cardiopulmonary arrest remains as one among the most frequent causes of death and disability around the world. Despite quick emergency responses and better techniques of defibrillation, the chances of survival following cardiac arrest are still poor, between 20-50% of patients in whom cardiopulmonary resuscitation is attempted. A complex pathophysiological condition is elicited by cardiac arrest, since it results in whole-body ischemia which compromises systemic circulatory homeostasis and cerebral, pulmonary, renal, and cardiac functions. In the course of cardiac arrest, global cerebral blood flow is severely impaired with the consequent risk of ischemic damage of brain cells, which magnitude seems to be associated with the cumulative time staying in cardiac arrest. Thus, most deaths (60%) during the post-resuscitation period have been attributed to extensive brain injury and neuronal damage that develops as a consequence of alteration of cell processes triggered by cerebral ischemia and reperfusion, during and after cardiac arrest. In addition, it is known that transient interruption or reduction of blood flow in the whole brain, are main causes of permanent brain damage and functional disruptions in human beings, and near around a half of surviving patients show permanent impairment of cognitive functions, such as learning and memory, attention, and executive functioning, and only a small proportion (less than 10%) of those survivors are able to reassume their former usual life styles (Geocardin et al., 2008; Grubb et al., 2000; Krause et al, 1986; Schneider et al., 2009). Thus, development of effective cytoprotective therapies that may be common to the organs more sensitive to cardiac arrest, such as heart or brain, could result in improvement of survival and better outcome following this whole ischemic episode (Karanjia &

Experimental protocols aimed to gain relevant information regarding those pathophysiological phenomena leading to cerebral damage elicited by ischemia have included, since long time, the use of animal models of cerebral ischemia, in order to support better diagnostic, prophylactic and clinical-therapeutic procedures for ischemic cerebrovascular diseases in human beings (Ginsberg & Busto, 1989; Gupta & Briyal, 2004; Hartman et al., 2005, Hossmann, 2008; Traystman, 2003). Thus, biochemical, electrophysiological, histological, and behavioral parameters of ischemic brain damage have been included in experimental designs to evaluate the efficacy and safety of pharmacological and non pharmacological neuroprotective procedures against brain injury resulting from the significant reduction of blood supply to the whole brain, in several

Even though a great number of pharmacological agents have proven to exert effective neuroprotective actions against cellular events leading to ischemic brain injury in experimental models of global cerebral ischemia, unfortunately they have not had enough clinical relevance to date. On the other hand, after evaluation of its effectiveness as a neuroprotective strategy in animal models of global cerebral ischemia, hypothermia has been tested in clinical trials in patients having suffered cardiac arrest, the most frequent cause of global cerebral ischemia in human beings (Castren et al., 2009; Geocardin et al., 2008; Greer, 2006; Inamasu et al., 2010; Knapp et al., 2011; Seder & Jarrah, 2008,). It seems that new and better strategies to translate preclinical data supporting the potential clinical

usefulness of neuroprotective drugs to clinical trials, must be developed.

not the case of cardiac arrest.

Geocardin, 2011).

animal models of global cerebral ischemia.

### **2. Animal models of global cerebral ischemia**

Animal models of global cerebral ischemia allow studying, at different levels of biological organization of the central nervous system, the development and temporal course of those processes that may result in irreversible ischemic neuronal damage, as well as in the subsequent cell repair and plasticity underlying either permanent cerebral functional impairment or recovery as a result of intrinsic brain mechanisms or neuroprotective procedures. Thus, animal-related factors (species, strain, age, sex, co-morbidities), animalmodel-related factors (choice of ischemic model, anesthetic procedures, duration of ischemia, reperfusion, survival, possibility of monitoring of physiological parameters), selective vulnerability of specific neuron types in several brain structures, outcome assessment (histopathological, biochemical, functional, parameters of brain injury in specific cerebral structures), short- or long-term experimental design, pharmacological characteristics of the presumptive neuroprotective agent itself, timing and dose-response of neuroprotective drug administration with reference to starting and ending of the ischemic episode, may account for the relevance of results from these investigations.

Models of cerebral ischemia have been also developed in *in vitro* models, in particular brain tissue slices and neuronal cultures, allowing to study in detail the cellular phenomena leading either to neuronal damage or to neural recovery and plasticity after ischemia (Benítez-King, 2006; Goldberg & Choi, 1993; Kasai et al., 2003; Whittingham et al., 1984).

Several conditions have to be fulfilled by animal models of global cerebral ischemia in order to become appropriate counterparts of these pathophysiological conditions in human beings, as well as to yield reliable and valid results in supporting clinical therapeutic approaches. Thus**,** it could be expected that in animal models of global cerebral ischemia the ischemic episode can be induced in a constant and reproducible manner: low variation for the extent, temporal course, and magnitude of the resulting ischemic brain injury under specific experimental conditions, including duration of the ischemic episode; easy control of possible deviations of important physiological variables, feasible neurological, neuropathological, and functional evaluations; lack of influence of anesthetic drugs and surgical procedures on the mechanisms of brain injury, brain recovery and/or neuroprotection; short-, intermediate- and long-term follow up of the outcome; and economical, easily available experimental animals of those species better accepted by public animal welfare concerns to be used in experimental protocols of cerebral ischemia and neuroprotection.

### **2.1 Main animal models of global cerebral ischemia**

Models of global cerebral ischemia have been performed in both large (monkeys, sheep, dogs, pigs, cats, rabbits) and small animals (gerbils, rats, mice). Among these, both advantages and disadvantages can be recognized according to several practical aspects: main objectives of the model; monitoring procedures to be used; nature, number and timing of simultaneous parameters to be recorded in order to evaluate the ischemic brain injury and recovery; degree of similarity of structural and functional characteristics of brains of experimental animals to those of the human brain; and updated ethical outlines for the use of experimental animals in research protocols.

Since the whole brain is exposed to transient ischemia and reperfusion as a result of cardiac arrest and the subsequent cardiorespiratory resuscitation to allow survival in human beings, animal models of global cerebral ischemia have been designed attempting to totally or

Neuroprotection in Animal Models of Global Cerebral Ischemia 309

Gerbils usually lack of a common posterior communicating artery connecting the carotid and vertebro-basilar arterial system. Thus, the bilateral common carotid artery occlusion results in a reduction of global cerebral blood flow near to zero and injury of the most vulnerable brain structures (hippocampal CA1 pyramidal neurons after 5 min of ischemia) in most animals (Kirino, 1982). This model of forebrain global cerebral ischemia may fail in some animals in which a complete Willis circle persists, and the high susceptibility of gerbils

The four-vessel occlusion (4-VO) and the two-vessel occlusion with hypotension (2-VO) models in rats became, nowadays, the most widely used animal models that simulate the reduction of blood flow, as it would occur by effect of cardiac arrest, on the forebrain. The 4- VO model (Ginsberg & Busto, 1989; Pulsinelli & Brierley 1979; Pulsinelli & Buchan 1988; Pulsinelli & Duffy 1983; Pulsinelli et al., 1982) provides a method of reversible forebrain ischemia in awake, freely moving rats (but also in anesthetized rats). In a first step of the model procedures, vertebral arteries are permanently occluded and 24 or 48 hours later, the ischemia is produced through transient (10 – 20 min) occlusion of the common carotid arteries under light inhaled anesthesia so that the ischemic episode occurs while the animal is unanesthetized. Loss of the righting reflex, and unconsciousness persisting for at least 20 min after the onset of reperfusion have to occur for each animal to be included in the study. In this way, a reduction in cerebral blood flow to less than 5% of control values, which is followed by hyperemia during 5 to 15 min after reperfusion, and subsequent hypoperfusion lasting for 24 hr result in main ischemic neuronal damage in hippocampus, neocortex and striatum, along hours to days after ischemia, its magnitude relating to the duration of the ischemia. The effects of this insult are, however, quite variable between rat strains, as well as between those individuals surviving (survival rate, 50-75%) after having fulfilled the criteria required to be included in the experimental groups. Similar consequences in selectively vulnerable neurons in specific brain structures result from the 2-VO model of forebrain ischemia, in which bilateral common carotid artery occlusion and systemic hypotension (blood withdrawal and subsequent return with or without pharmacological procedures, leading to arterial blood pressure below 50 mm Hg) are combined to provoke reversible

forebrain ischemia (Eklof & Siesjo 1972a, 1972b; Smith et al., 1984a, 1984b).

carotid occlusion and controlled pulmonary ventilation (Traystman, 2003).

neuroprotection by itself.

Mouse models of global cerebral ischemia have been developed through bilateral common

It is known that animal models of global cerebral ischemia require adequate control of certain variables, such as careful control of animal's temperature and blood glucose concentration, in order to achieve consistent pathophysiological effects and brain injury (Colbourne & Corbett, 1994; Lipton, 1999; Siemkowicz, 1981; Siemkowicz.& Gjedde 1980). Hyperthermia and hyperglycemia increase brain injury**,** while hypothermia result**s** in

**3. Cellular mechanisms of neuronal injury, neuronal repair and plasticity**

Models of global cerebral ischemia in experimental animals, as well as *in vitro* models, in particular brain tissue slices and neuronal cultures, have allowed to study in detail the cellular phenomena leading either to neuronal damage, or to neural repair and plasticity after ischemia. From these studies it has been known that mechanisms of cellular damage, repair and plasticity may be the same, in general, both if reduction of blood flow to the brain tissue results from occlusion of one of the main cerebral arteries as would occur in focal

to seizures may influence the ischemic outcome.

partially mimic the consequences of this clinical condition on the brain (Ginsberg & Busto, 1989; Gupta & Briyal, 2004; Mc Bean & Kelly, 1998; Traystman, 2003), which are the main cause of neuronal injury to selective vulnerable brain regions, and neurological or cognitive impairment, in human beings.

Cardiac arrest (induced by injection of KCl, electric shock, thoracic compression, asphyxia, and mechanical obstruction of the ascending aorta) followed by cardiopulmonary resuscitation (by artificial ventilation, closed chest massage and electrical defibrillation), both in large experimental animals (formerly a common model, but nowadays rarely used) and also in rodents, has been a technique to produce global cerebral ischemia in an attempt to closely resemble the clinical situation of cardiac arrest, including complete ischemia and reperfusion in renal, splachnic and other peripheral organs. This technique seemed to be an excellent model of global cerebral ischemia, but it is expensive when large experimental animals are used, and intensive care (cardiopulmonary support under unconsciousness, control of blood pressure, pH, body fluids, and temperature) must be provided to the animals, especially during the first 24-48 h after the cardiac arrest. Complete acute global cerebral ischemia during cardiac arrest (8-20 min) and a variable period of incomplete cerebral ischemia during reperfusion, even after a successful cardiopulmonary resuscitation, as well as damage in those brain structures most vulnerable to ischemia, can be expected from this model (Berkowitz, et al., 1991; Bleyaert et al., 1978; Dave et al., 2004; Hossmann, 2008; Katz et al., 1995; Kofler et al., 2004; Radovsky et al., 1995; Safar et al., 1976; Todd et al., 1982). In particular, models of global cerebral ischemia in mice are currently of interest because of the availability of transgenic and knock-out strains for identification of cellular pathways of ischemic damage, and for neuroprotection studies.

Several other animal models of global cerebral ischemia have been designed in cats, monkeys, gerbils, mice, and rats, in order to circumscribe to the brain those harmful effects of the reduced blood flow that follows a cardiac arrest, avoiding affecting other vital organs in a whole body ischemia condition, as can be expected from animal models of cardiac arrest (Ginsberg & Busto, 1989).

Decapitation in small animals has been used as a model of global cerebral ischemia, only allowing the study of the immediate alterations of some biochemical and metabolic parameters elicited by ischemia in the brain contained into the head (Abe et al., 1983; Ikeda et al., 1986; Lowry et al., 1964; Yoshida et al., 1985).

A neck tourniquet or a neck cuff, whether they include or not arterial hypotension, have also been used to produce global cerebral ischemia in rats, cats, dogs, or monkeys. However, these techniques lead to variable ischemic outcomes since the produced ischemia may not be complete because of a remaining cerebral blood flow through the vertebral arteries, as well as complications due to vagal compression and venous congestion (Chopp et al., 1987, 1988; Grenell 1946; Nemoto et al., 1977; Sheller et al., 1992; Siemkowits & Gjedde, 1980; Siemkowitz & Hansen, 1978).

Reduction of cerebral blood flow near to zero has been accomplished in cats and monkeys, by occlusion of the innominate and left subclavian arteries near the aortic arch, and pharmacologically induced hypotension (below 80 mm Hg), without involvement of other organs in the ischemic phenomena**.** However, these experimental animals require intensive care procedures to their survival, and studies of long-term recovery are difficult to achieve (Bodsch et al., 1986; Clavier et al., 1994; Hossmann, 1971; Hossmann & Grose Ophoff, 1986; Zimmerman & Hossmann, 1975).

partially mimic the consequences of this clinical condition on the brain (Ginsberg & Busto, 1989; Gupta & Briyal, 2004; Mc Bean & Kelly, 1998; Traystman, 2003), which are the main cause of neuronal injury to selective vulnerable brain regions, and neurological or cognitive

Cardiac arrest (induced by injection of KCl, electric shock, thoracic compression, asphyxia, and mechanical obstruction of the ascending aorta) followed by cardiopulmonary resuscitation (by artificial ventilation, closed chest massage and electrical defibrillation), both in large experimental animals (formerly a common model, but nowadays rarely used) and also in rodents, has been a technique to produce global cerebral ischemia in an attempt to closely resemble the clinical situation of cardiac arrest, including complete ischemia and reperfusion in renal, splachnic and other peripheral organs. This technique seemed to be an excellent model of global cerebral ischemia, but it is expensive when large experimental animals are used, and intensive care (cardiopulmonary support under unconsciousness, control of blood pressure, pH, body fluids, and temperature) must be provided to the animals, especially during the first 24-48 h after the cardiac arrest. Complete acute global cerebral ischemia during cardiac arrest (8-20 min) and a variable period of incomplete cerebral ischemia during reperfusion, even after a successful cardiopulmonary resuscitation, as well as damage in those brain structures most vulnerable to ischemia, can be expected from this model (Berkowitz, et al., 1991; Bleyaert et al., 1978; Dave et al., 2004; Hossmann, 2008; Katz et al., 1995; Kofler et al., 2004; Radovsky et al., 1995; Safar et al., 1976; Todd et al., 1982). In particular, models of global cerebral ischemia in mice are currently of interest because of the availability of transgenic and knock-out strains for identification of cellular

Several other animal models of global cerebral ischemia have been designed in cats, monkeys, gerbils, mice, and rats, in order to circumscribe to the brain those harmful effects of the reduced blood flow that follows a cardiac arrest, avoiding affecting other vital organs in a whole body ischemia condition, as can be expected from animal models of cardiac arrest

Decapitation in small animals has been used as a model of global cerebral ischemia, only allowing the study of the immediate alterations of some biochemical and metabolic parameters elicited by ischemia in the brain contained into the head (Abe et al., 1983; Ikeda

A neck tourniquet or a neck cuff, whether they include or not arterial hypotension, have also been used to produce global cerebral ischemia in rats, cats, dogs, or monkeys. However, these techniques lead to variable ischemic outcomes since the produced ischemia may not be complete because of a remaining cerebral blood flow through the vertebral arteries, as well as complications due to vagal compression and venous congestion (Chopp et al., 1987, 1988; Grenell 1946; Nemoto et al., 1977; Sheller et al., 1992; Siemkowits & Gjedde, 1980;

Reduction of cerebral blood flow near to zero has been accomplished in cats and monkeys, by occlusion of the innominate and left subclavian arteries near the aortic arch, and pharmacologically induced hypotension (below 80 mm Hg), without involvement of other organs in the ischemic phenomena**.** However, these experimental animals require intensive care procedures to their survival, and studies of long-term recovery are difficult to achieve (Bodsch et al., 1986; Clavier et al., 1994; Hossmann, 1971; Hossmann & Grose Ophoff, 1986;

pathways of ischemic damage, and for neuroprotection studies.

et al., 1986; Lowry et al., 1964; Yoshida et al., 1985).

impairment, in human beings.

(Ginsberg & Busto, 1989).

Siemkowitz & Hansen, 1978).

Zimmerman & Hossmann, 1975).

Gerbils usually lack of a common posterior communicating artery connecting the carotid and vertebro-basilar arterial system. Thus, the bilateral common carotid artery occlusion results in a reduction of global cerebral blood flow near to zero and injury of the most vulnerable brain structures (hippocampal CA1 pyramidal neurons after 5 min of ischemia) in most animals (Kirino, 1982). This model of forebrain global cerebral ischemia may fail in some animals in which a complete Willis circle persists, and the high susceptibility of gerbils to seizures may influence the ischemic outcome.

The four-vessel occlusion (4-VO) and the two-vessel occlusion with hypotension (2-VO) models in rats became, nowadays, the most widely used animal models that simulate the reduction of blood flow, as it would occur by effect of cardiac arrest, on the forebrain. The 4- VO model (Ginsberg & Busto, 1989; Pulsinelli & Brierley 1979; Pulsinelli & Buchan 1988; Pulsinelli & Duffy 1983; Pulsinelli et al., 1982) provides a method of reversible forebrain ischemia in awake, freely moving rats (but also in anesthetized rats). In a first step of the model procedures, vertebral arteries are permanently occluded and 24 or 48 hours later, the ischemia is produced through transient (10 – 20 min) occlusion of the common carotid arteries under light inhaled anesthesia so that the ischemic episode occurs while the animal is unanesthetized. Loss of the righting reflex, and unconsciousness persisting for at least 20 min after the onset of reperfusion have to occur for each animal to be included in the study. In this way, a reduction in cerebral blood flow to less than 5% of control values, which is followed by hyperemia during 5 to 15 min after reperfusion, and subsequent hypoperfusion lasting for 24 hr result in main ischemic neuronal damage in hippocampus, neocortex and striatum, along hours to days after ischemia, its magnitude relating to the duration of the ischemia. The effects of this insult are, however, quite variable between rat strains, as well as between those individuals surviving (survival rate, 50-75%) after having fulfilled the criteria required to be included in the experimental groups. Similar consequences in selectively vulnerable neurons in specific brain structures result from the 2-VO model of forebrain ischemia, in which bilateral common carotid artery occlusion and systemic hypotension (blood withdrawal and subsequent return with or without pharmacological procedures, leading to arterial blood pressure below 50 mm Hg) are combined to provoke reversible forebrain ischemia (Eklof & Siesjo 1972a, 1972b; Smith et al., 1984a, 1984b).

Mouse models of global cerebral ischemia have been developed through bilateral common carotid occlusion and controlled pulmonary ventilation (Traystman, 2003).

It is known that animal models of global cerebral ischemia require adequate control of certain variables, such as careful control of animal's temperature and blood glucose concentration, in order to achieve consistent pathophysiological effects and brain injury (Colbourne & Corbett, 1994; Lipton, 1999; Siemkowicz, 1981; Siemkowicz.& Gjedde 1980). Hyperthermia and hyperglycemia increase brain injury**,** while hypothermia result**s** in neuroprotection by itself.

### **3. Cellular mechanisms of neuronal injury, neuronal repair and plasticity**

Models of global cerebral ischemia in experimental animals, as well as *in vitro* models, in particular brain tissue slices and neuronal cultures, have allowed to study in detail the cellular phenomena leading either to neuronal damage, or to neural repair and plasticity after ischemia. From these studies it has been known that mechanisms of cellular damage, repair and plasticity may be the same, in general, both if reduction of blood flow to the brain tissue results from occlusion of one of the main cerebral arteries as would occur in focal

Neuroprotection in Animal Models of Global Cerebral Ischemia 311

Several gene families such as immediate early genes, heat-shock proteins, and inflammation-and apoptosis-related genes, are known to be differentially expressed during cerebral ischemia, and some neuropathologic processes triggered by ischemia seem to be mediated in part by alterations of molecular transcriptional and translational activities

Activation of DNA fragmentation enzymes and energy-consuming DNA repair enzymes, finally lead to DNA breakdown, interruption of protein synthesis, and cell death (Iadecola &

In addition to the above mentioned cellular processes of ischemic damage, brain ischemia/reperfusion may also trigger cellular mechanisms for neuronal repair, and functional recovery through neuronal plasticity involving remaining neurons in vulnerable damaged or undamaged brain structures (Barone & Feuerstein, 1999; Bendel et al., 2005; Crepel et al., 2003; Hurtado et al., 2006; Jourdain et al., 2002; Ruan et al., 2006). The different ischemia/reperfusion induced cellular mechanisms leading either to brain injury and neuronal death, or to neuronal repair, as well as plasticity and brain functional recovery, may occur in a sequential or simultaneous manner. Their latencies and temporal course, from minutes to weeks, are important references in attempting to establish their differential relevance in those critical periods for neuronal damage and death, as well as the "window of opportunity" for specific neuroprotective procedures (Barone & Feuerstein, 1999; Lipton,

**3.2 Differential neuronal vulnerability in animal models of global cerebral ischemia**  Brain injury is expected to occur when cerebral blood flow is reduced to less than 10-20% of the normal value; the greater the reduction and/or longer lasting, the worst damage. Under these conditions, damage to specific brain structures due to immediate or delayed death of highly vulnerable neuronal groups, including the pyramidal neurons of the CA1 subfield of the hippocampus, and to a lesser degree those in layers 3 and 5 of the cerebral cortex, the Purkinje cells of the cerebellum, and spiny neurons in the striatum, take place after global cerebral ischemia (Ginsberg & Busto, 1989; Pulsinelli, 1985). Experimental models of global cerebral ischemia have allowed to know some neuronal characteristics that seem to account for selective vulnerability to ischemia, including a high density of excitatory glutamatergic synapses; low antioxidant enzyme reserves; high content of transition metals; increased expression of pro-apoptotic Bax protein; thus leading to differential susceptibility of some cell processes (Ca2+ homeostasis, oxidative-antioxidative balance, functional mitochondrial stability) to become out of physiological control under ischemia (Arai et al., 2001; Araki et al., 1989; Chen et al., 1996; Lipton 1999; Schmidt-Kastner et al., 2001; Sugawara et al., 1999). Brain injury after global ischemia/reperfusion is finally evidenced by neuronal death, affecting the neuronal population, circuit connectivity and functioning in specific brain structures involved in the neural integration of cognitive brain functions and behavior.

Cellular mechanisms of neuronal repair and plasticity have been observed to occur in vulnerable brain structures in which damage or death of neurons resulted from a sequence of pathophysiological phenomena triggered by global cerebral ischemia and the subsequent reperfusion. Thus, structural and functional characteristics of those neuronal components of circuits in the hippocampus and prefrontal cortex, which are identified, among others, as

(Mehta et al, 2007).

Alexander, 2001; Leker & Shohami, 2002).

1999; Leker & Shohami, 2002; Pulsinelli et al., 1997).

**3.3 Cellular mechanisms of neuronal plasticity and repair**

ischemia, and if it is the result of reduction of blood flow to the whole brain as it would occur after a cardiorrespiratory arrest.

### **3.1 Cellular mechanisms of neuronal injury**

Interruption of blood flow and hence, of glucose and oxygen supply to the brain, results in an immediate severe energy failure in terms of ATP depletion that leads to alterations of the cell membrane ionic gradients and a severe breakdown in cellular homeostasis. Several mechanisms of neuronal damage are triggered and evolve both in cascade and as parallel pathways (Gwag et al, 2002; Lakhan et al, 2009; Lipton, 1999; Mehta et al, 2007; Schneider et al, 2009; Sugawara et al, 2004; Warner et al., 2004). In particular, a massive accumulation of intracellular calcium and sodium occurs because of failure of their energy-dependent efflux processes, and anoxic depolarization. This further leads to accumulation of lactate and hydrogen ions, and as a consequence, to decreased pH.

As a result of anoxic depolarization, excitatory aminoacids such as glutamate and aspartate are released, activating ligand-gated calcium and sodium channels with a further influx of these ions into the cells. Calcium is also released from intracellular pools, and its excessive, unregulated intracellular overload causes direct Ca2+-dependent activation of lipases, proteases, and endonucleases leading to breakdown of structural and functional proteins, and damage to cytoskeleton and macromolecules including nucleic acids. A result of these phenomena is, among others, cell membrane lipoperoxidation.

Excessive intracellular calcium activate abnormal cell processes promoting functional derangements of mitochondria and an increased production of free radicals, exceeding the neuronal antioxidant reserves, and imposing risks to the structural and functional integrity of neuronal cells. The brain is highly susceptible to oxidative damage as a consequence of its high lipid and metal content, as well as other biochemical characteristics (Margaill et al., 2005; Reiter et al., 2005; Warner el al., 2004). Reperfusion and reoxygenation of the ischemic tissue, which must be reestablished within minutes in an effort to prevent severe neurological damage and favor survival of individuals, also may provide chemical substrates for further increasing cellular alterations, neuronal death and neurological deficits (Margaill et al., 2005).

Free radicals also contribute to the breakdown of the blood-brain barrier and brain edema. Reactive oxygen and nitrogen species including superoxide, hydroxyl free radical, and peroxylnitrite anion are also important mediators of inflammatory tissue damage, of activation and secretion of inflammatory cytokines such as tumor necrosis factor α, interleukin-1, and interleukin-6, and of expression of cyclo-oxigenase (COX)-2, and inducible nitric oxide synthase generating nitric oxide that also contributes to neuronal damage. These changes favor inflammatory reactions soon after cerebral ischemia/reperfusion (Barone & Feuerstein, 1999; Lakhan et al, 2009; Lipton, 1999; Mehta et al, 2007).

Calcium overload may additionally lead to mitochondrial damage and trigger an apoptotic cascade. The pro-apoptotic cascade involves nuclear factor κB- and p53-dependent pathways, changes in the Bcl-2 to Bax ratio, opening of the mitochondrial transition pore, release of cytochrome *c*, and activation of caspases (Chan, 2001; Chinopoulos & Adam-Vizi, 2006). In addition, caspase-independent pathways may also contribute to neuronal apoptosis.

ischemia, and if it is the result of reduction of blood flow to the whole brain as it would

Interruption of blood flow and hence, of glucose and oxygen supply to the brain, results in an immediate severe energy failure in terms of ATP depletion that leads to alterations of the cell membrane ionic gradients and a severe breakdown in cellular homeostasis. Several mechanisms of neuronal damage are triggered and evolve both in cascade and as parallel pathways (Gwag et al, 2002; Lakhan et al, 2009; Lipton, 1999; Mehta et al, 2007; Schneider et al, 2009; Sugawara et al, 2004; Warner et al., 2004). In particular, a massive accumulation of intracellular calcium and sodium occurs because of failure of their energy-dependent efflux processes, and anoxic depolarization. This further leads to accumulation of lactate and

As a result of anoxic depolarization, excitatory aminoacids such as glutamate and aspartate are released, activating ligand-gated calcium and sodium channels with a further influx of these ions into the cells. Calcium is also released from intracellular pools, and its excessive, unregulated intracellular overload causes direct Ca2+-dependent activation of lipases, proteases, and endonucleases leading to breakdown of structural and functional proteins, and damage to cytoskeleton and macromolecules including nucleic acids. A result of these

Excessive intracellular calcium activate abnormal cell processes promoting functional derangements of mitochondria and an increased production of free radicals, exceeding the neuronal antioxidant reserves, and imposing risks to the structural and functional integrity of neuronal cells. The brain is highly susceptible to oxidative damage as a consequence of its high lipid and metal content, as well as other biochemical characteristics (Margaill et al., 2005; Reiter et al., 2005; Warner el al., 2004). Reperfusion and reoxygenation of the ischemic tissue, which must be reestablished within minutes in an effort to prevent severe neurological damage and favor survival of individuals, also may provide chemical substrates for further increasing cellular alterations, neuronal death and neurological

Free radicals also contribute to the breakdown of the blood-brain barrier and brain edema. Reactive oxygen and nitrogen species including superoxide, hydroxyl free radical, and peroxylnitrite anion are also important mediators of inflammatory tissue damage, of activation and secretion of inflammatory cytokines such as tumor necrosis factor α, interleukin-1, and interleukin-6, and of expression of cyclo-oxigenase (COX)-2, and inducible nitric oxide synthase generating nitric oxide that also contributes to neuronal damage. These changes favor inflammatory reactions soon after cerebral ischemia/reperfusion (Barone & Feuerstein, 1999; Lakhan et al, 2009; Lipton, 1999; Mehta et

Calcium overload may additionally lead to mitochondrial damage and trigger an apoptotic cascade. The pro-apoptotic cascade involves nuclear factor κB- and p53-dependent pathways, changes in the Bcl-2 to Bax ratio, opening of the mitochondrial transition pore, release of cytochrome *c*, and activation of caspases (Chan, 2001; Chinopoulos & Adam-Vizi, 2006). In addition, caspase-independent pathways may also contribute to neuronal

occur after a cardiorrespiratory arrest.

deficits (Margaill et al., 2005).

al, 2007).

apoptosis.

**3.1 Cellular mechanisms of neuronal injury**

hydrogen ions, and as a consequence, to decreased pH.

phenomena is, among others, cell membrane lipoperoxidation.

Several gene families such as immediate early genes, heat-shock proteins, and inflammation-and apoptosis-related genes, are known to be differentially expressed during cerebral ischemia, and some neuropathologic processes triggered by ischemia seem to be mediated in part by alterations of molecular transcriptional and translational activities (Mehta et al, 2007).

Activation of DNA fragmentation enzymes and energy-consuming DNA repair enzymes, finally lead to DNA breakdown, interruption of protein synthesis, and cell death (Iadecola & Alexander, 2001; Leker & Shohami, 2002).

In addition to the above mentioned cellular processes of ischemic damage, brain ischemia/reperfusion may also trigger cellular mechanisms for neuronal repair, and functional recovery through neuronal plasticity involving remaining neurons in vulnerable damaged or undamaged brain structures (Barone & Feuerstein, 1999; Bendel et al., 2005; Crepel et al., 2003; Hurtado et al., 2006; Jourdain et al., 2002; Ruan et al., 2006). The different ischemia/reperfusion induced cellular mechanisms leading either to brain injury and neuronal death, or to neuronal repair, as well as plasticity and brain functional recovery, may occur in a sequential or simultaneous manner. Their latencies and temporal course, from minutes to weeks, are important references in attempting to establish their differential relevance in those critical periods for neuronal damage and death, as well as the "window of opportunity" for specific neuroprotective procedures (Barone & Feuerstein, 1999; Lipton, 1999; Leker & Shohami, 2002; Pulsinelli et al., 1997).

### **3.2 Differential neuronal vulnerability in animal models of global cerebral ischemia**

Brain injury is expected to occur when cerebral blood flow is reduced to less than 10-20% of the normal value; the greater the reduction and/or longer lasting, the worst damage. Under these conditions, damage to specific brain structures due to immediate or delayed death of highly vulnerable neuronal groups, including the pyramidal neurons of the CA1 subfield of the hippocampus, and to a lesser degree those in layers 3 and 5 of the cerebral cortex, the Purkinje cells of the cerebellum, and spiny neurons in the striatum, take place after global cerebral ischemia (Ginsberg & Busto, 1989; Pulsinelli, 1985). Experimental models of global cerebral ischemia have allowed to know some neuronal characteristics that seem to account for selective vulnerability to ischemia, including a high density of excitatory glutamatergic synapses; low antioxidant enzyme reserves; high content of transition metals; increased expression of pro-apoptotic Bax protein; thus leading to differential susceptibility of some cell processes (Ca2+ homeostasis, oxidative-antioxidative balance, functional mitochondrial stability) to become out of physiological control under ischemia (Arai et al., 2001; Araki et al., 1989; Chen et al., 1996; Lipton 1999; Schmidt-Kastner et al., 2001; Sugawara et al., 1999). Brain injury after global ischemia/reperfusion is finally evidenced by neuronal death, affecting the neuronal population, circuit connectivity and functioning in specific brain structures involved in the neural integration of cognitive brain functions and behavior.

### **3.3 Cellular mechanisms of neuronal plasticity and repair**

Cellular mechanisms of neuronal repair and plasticity have been observed to occur in vulnerable brain structures in which damage or death of neurons resulted from a sequence of pathophysiological phenomena triggered by global cerebral ischemia and the subsequent reperfusion. Thus, structural and functional characteristics of those neuronal components of circuits in the hippocampus and prefrontal cortex, which are identified, among others, as

Neuroprotection in Animal Models of Global Cerebral Ischemia 313

played by the CA1 region for the output of information flowing through the hippocampus, via the tri-synaptic circuit (Herreras et al., 1987). It is well known that the prefrontal cortex is directly involved in the organization of sequenced motor actions during working-memory performance (Fuster, 1999; I. Lee & Kesner, 2003), and that hippocampal projections supply of spatial information to the prefrontal cortex allowing suitability of motor responses in the spatial context (Jung et al., 1998). These phenomena may be altered not only by gross lesions of the prefrontal cortex, but fine alterations of its neuronal circuits may also result in impairment of the spatial working memory (Fritts et al., 1998; Lambe et al., 2000; I. Lee & Kesner, 2003; Olvera-Cortés et al., 2001; Taylor et al., 2003). Experimental data have shown that variations in cognitive behavioral performance are related to plastic changes in dendritic spines (Pérez-Vega et al., 2000). In addition, excitatory information flows mostly through dendritic spines-mediated synaptic contacts (Gray, 1959), which are highly sensitive to electrical stimulation and yet to mnemonic activity-related electrical phenomena

(Harris, 1999; Hartman et al., 2005; Onodera et al., 1990).

with neurons from intact, and ischemia melatonin treated rats.

(Modified from: García-Chávez et al., 2008).

Fig. 1. Photomicrographs of prefrontal third-layer pyramidal neurons of rats: intact (left), after global cerebral ischemia and neuroprotective melatonin (centre) or vehicle (right) treatment. Note the reduction in dendritic arborization protruding from the apical dendrite, and dendritic spine reduction (arrows) in the ischemic and non treated cell in comparison

highly vulnerable to ischemia, and their correlation with the integration of specific cerebral functions (mainly cognitive functions) after global cerebral ischemia, have been analyzed. In this sense, short- and long-term structural alterations have been shown to occur in the remaining pyramidal neurons of the hippocampus after ischemia; thus, axonal degeneration as well as reduction of dendritic length and arborizations, of number and shape of dendritic spines, and of number of synapses, are usually related to impairment of cognitive functions and recognized as degenerative changes. By contrast, cytoarchitectural adjustments such as axonal and dendritic sprouting, increase of number of dendritic spines and synapses, changes in the relative proportion of spine types, are interpreted as compensatory plastic responses of surviving neurons. They contribute to neuronal circuit remodeling and functional recovery, and have been correlated with preservation of cognitive functions after the ischemic insult, even in absence of neuroprotective procedures (Briones et al., 2006; Jourdain et al., 2002; Mudrick & Baimbridge, 1989; Neigh et al., 2004; Onodera et al., 1990; Ruan et al., 2006; Skibo & Nikonenko, 2010; Sorra & Harris, 2000). In addition, neurogenesis and integration of newly differentiated neurons into neuronal circuits in the Ammon's horn may contribute to recovery of hippocampal-dependent cognitive functions (Bendel et al., 2005; Bernabeu & Sharp, 2000).

Similarly, reductions of dendritic length, arborization, and dendritic spine density have also been described, among various cytoarchitectural adjustments, in sensorymotor cortex pyramidal neurons following global cerebral ischemia (Akulinin et al., 1997, 1998, 2004). These cytoarchitectural alterations could be influenced by the extent of neuronal remaining connections; thus, either reduction or increase of afferent connections may result in changes in dendritic arborizations and spine density (Fiala et al., 2002; Johansson & Belinchenko, 2002). It has been emphasized the functional relevance of neuronal connections from the hippocampus to the prefrontal cortex for synaptogenesis and neuronal plasticity accounting for learning and memory (González-Burgos, 2009; Laroche et al., 2000). Thus, a permanent deafferentation of pyramidal neurons at cortical layer V after the extensive reduction of pyramidal neuron population of the CA1 subfield of the Ammon's horn as expected to occur after global ischemia (Letechipía-Vallejo et al., 2007), may lead to changes in neuronal activity, which may in turn affect the cytoarchitectural characteristics of pyramidal prefrontal cortex neurons (García-Chávez et al., 2008; Wellman & Sengelaub, 1991).

These dendritic restructuring (Neigh el al., 2004; Ruan el al., 2006) and reactive synaptogenesis (Briones et al., 2005; Crepel et al., 2003; Jourdain et al., 2002, Kovalenko et al., 2006) among other phenomena including the activation of a variety of potential growth-promoting processes (Arvidsson et al., 2001; Gobbo & O´Mara, 2004; Schmidt-Kastner et al., 2001), that occur in neurons surviving to the ischemic insult in vulnerable brain structures, seem to be a part of mechanisms of adaptive changes, probably accounting for neuronal conditions favoring synaptic plasticity and functional recovery. In fact, a long-term progressive continuous plastic reorganization of the dendritic tree and dendritic spines, initially altered by acute global cerebral ischemia, has been shown to occur in pyramidal neurons at layers 3 and 5 of the sensorymotor cortex of the rat (Akulinin et al., 1997, 1998, 2004).

Thus, preservation or recovery of hippocampal- and pre-frontal cortex- dependent functions after global cerebral ischemia, may involve long-term cytoarchitectural modifications in those remaining hippocampal CA1 and prefronto-cortical (layers 3 and 5) pyramidal neurons, since their morpho-functional organization is critical for normal learning and memory performance (Block, 1999; McDonald & White, 1993; McNamara & Skelton, 1993; Olsen et al., 1994; Olvera-Cortés et al., 2002; Silva et al., 1998), on the basis of the major role

highly vulnerable to ischemia, and their correlation with the integration of specific cerebral functions (mainly cognitive functions) after global cerebral ischemia, have been analyzed. In this sense, short- and long-term structural alterations have been shown to occur in the remaining pyramidal neurons of the hippocampus after ischemia; thus, axonal degeneration as well as reduction of dendritic length and arborizations, of number and shape of dendritic spines, and of number of synapses, are usually related to impairment of cognitive functions and recognized as degenerative changes. By contrast, cytoarchitectural adjustments such as axonal and dendritic sprouting, increase of number of dendritic spines and synapses, changes in the relative proportion of spine types, are interpreted as compensatory plastic responses of surviving neurons. They contribute to neuronal circuit remodeling and functional recovery, and have been correlated with preservation of cognitive functions after the ischemic insult, even in absence of neuroprotective procedures (Briones et al., 2006; Jourdain et al., 2002; Mudrick & Baimbridge, 1989; Neigh et al., 2004; Onodera et al., 1990; Ruan et al., 2006; Skibo & Nikonenko, 2010; Sorra & Harris, 2000). In addition, neurogenesis and integration of newly differentiated neurons into neuronal circuits in the Ammon's horn may contribute to recovery of hippocampal-dependent cognitive functions (Bendel et al., 2005; Bernabeu & Sharp, 2000). Similarly, reductions of dendritic length, arborization, and dendritic spine density have also been described, among various cytoarchitectural adjustments, in sensorymotor cortex pyramidal neurons following global cerebral ischemia (Akulinin et al., 1997, 1998, 2004). These cytoarchitectural alterations could be influenced by the extent of neuronal remaining connections; thus, either reduction or increase of afferent connections may result in changes in dendritic arborizations and spine density (Fiala et al., 2002; Johansson & Belinchenko, 2002). It has been emphasized the functional relevance of neuronal connections from the hippocampus to the prefrontal cortex for synaptogenesis and neuronal plasticity accounting for learning and memory (González-Burgos, 2009; Laroche et al., 2000). Thus, a permanent deafferentation of pyramidal neurons at cortical layer V after the extensive reduction of pyramidal neuron population of the CA1 subfield of the Ammon's horn as expected to occur after global ischemia (Letechipía-Vallejo et al., 2007), may lead to changes in neuronal activity, which may in turn affect the cytoarchitectural characteristics of pyramidal

prefrontal cortex neurons (García-Chávez et al., 2008; Wellman & Sengelaub, 1991).

of the sensorymotor cortex of the rat (Akulinin et al., 1997, 1998, 2004).

These dendritic restructuring (Neigh el al., 2004; Ruan el al., 2006) and reactive synaptogenesis (Briones et al., 2005; Crepel et al., 2003; Jourdain et al., 2002, Kovalenko et al., 2006) among other phenomena including the activation of a variety of potential growth-promoting processes (Arvidsson et al., 2001; Gobbo & O´Mara, 2004; Schmidt-Kastner et al., 2001), that occur in neurons surviving to the ischemic insult in vulnerable brain structures, seem to be a part of mechanisms of adaptive changes, probably accounting for neuronal conditions favoring synaptic plasticity and functional recovery. In fact, a long-term progressive continuous plastic reorganization of the dendritic tree and dendritic spines, initially altered by acute global cerebral ischemia, has been shown to occur in pyramidal neurons at layers 3 and 5

Thus, preservation or recovery of hippocampal- and pre-frontal cortex- dependent functions after global cerebral ischemia, may involve long-term cytoarchitectural modifications in those remaining hippocampal CA1 and prefronto-cortical (layers 3 and 5) pyramidal neurons, since their morpho-functional organization is critical for normal learning and memory performance (Block, 1999; McDonald & White, 1993; McNamara & Skelton, 1993; Olsen et al., 1994; Olvera-Cortés et al., 2002; Silva et al., 1998), on the basis of the major role played by the CA1 region for the output of information flowing through the hippocampus, via the tri-synaptic circuit (Herreras et al., 1987). It is well known that the prefrontal cortex is directly involved in the organization of sequenced motor actions during working-memory performance (Fuster, 1999; I. Lee & Kesner, 2003), and that hippocampal projections supply of spatial information to the prefrontal cortex allowing suitability of motor responses in the spatial context (Jung et al., 1998). These phenomena may be altered not only by gross lesions of the prefrontal cortex, but fine alterations of its neuronal circuits may also result in impairment of the spatial working memory (Fritts et al., 1998; Lambe et al., 2000; I. Lee & Kesner, 2003; Olvera-Cortés et al., 2001; Taylor et al., 2003). Experimental data have shown that variations in cognitive behavioral performance are related to plastic changes in dendritic spines (Pérez-Vega et al., 2000). In addition, excitatory information flows mostly through dendritic spines-mediated synaptic contacts (Gray, 1959), which are highly sensitive to electrical stimulation and yet to mnemonic activity-related electrical phenomena (Harris, 1999; Hartman et al., 2005; Onodera et al., 1990).

Fig. 1. Photomicrographs of prefrontal third-layer pyramidal neurons of rats: intact (left), after global cerebral ischemia and neuroprotective melatonin (centre) or vehicle (right) treatment. Note the reduction in dendritic arborization protruding from the apical dendrite, and dendritic spine reduction (arrows) in the ischemic and non treated cell in comparison with neurons from intact, and ischemia melatonin treated rats. (Modified from: García-Chávez et al., 2008).

Neuroprotection in Animal Models of Global Cerebral Ischemia 315

should also be taken in account as to be compatible with their potential use in human

The time window of opportunity for the effective neuroprotective treatment is an important factor to be considered in preclinical models that may predict the timing of neuroprotective procedures in clinical situations with reference to the onset of global cerebral ischemia and subsequent reperfusion. The initial hypothesis that opportunity window for neuroprotective procedures would be limited to a short period after the ischemic episode has been changed in view of experimental evidence. Thus, different drugs or neuroprotective procedures having predominant mechanisms of action against specific cellular processes of ischemia damage occurring lately within the pathophysiological cascade, may allow to neuroprotection even when administered hours or days after ischemia. Besides, the opportunity time window may be further extended when it is expected that neuroprotective procedures act through promotion of cellular processes of neuronal repair and plasticity. In view of the multiple pathophysiological processes occurring both in sequence and simultaneously after ischemia and reperfusion, it is considered as an advantage for presumptive neuroprotective drugs to have multiple cellular or molecular mechanisms of action, as occurring with some originally endogenous compounds, namely melatonin, estradiol and progesterone (El-Abhar et al, 2002; Hurn et al, 1995; Jover-Mengual et al, 2010; Lebesgue et al, 2009; Reiter et al, 2005; Wang et al, 2008). By contrast, most synthetic drugs only have one mechanism of action accounting for neuroprotection. Attempting to counteract several mechanisms of ischemic brain injury would require the simultaneous administration of several drugs (Hicks et al, 1999; Matsumoto et al, 1993; Pazos et al, 1999; del Pilar Fernández et al, 1998; Sánchez-Casado et al, 2007; Zapater et al, 1997) (Table 1). Recommendations arisen from these consensuses of opinion have also highlighted the importance of long-term studies to identify whether functional preservation or recovery may be attributed to effects of the neuroprotective procedures, and/or to intrinsic mechanisms of plasticity and repair triggered by ischemia *per se*. Reliable parameters of long-term structural and functional outcome may allow to evaluate the final result of the neuroprotective procedures on cerebral structures vulnerable to ischemia. Thus, evaluation of neuronal population, cytoarchitectonic characteristics, and connectivity of the neural circuits in these vulnerable structures, as well as different aspects of cognitive functions depending on them should be included as a part of experimental designs of

It has been described that the neuronal population of remaining neurons in CA1 at survival times of 2-3 weeks may be less than that evaluated 3-4 months after the ischemic episode, suggesting that, without exogenous intervention, CA1 neurons may have been repopulate, became integrated to the hippocampal neuronal circuits, and contribute to functional recovery (Bendel, et al 2005; von Euler et al., 2006, Hartman et al, 2005, Nakatomi et al., 2002). Obviously, the potential repopulation complicates the interpretation of learning and memory studies after global cerebral ischemia, because short-term studies may not give an adequate end point of the cognitive alteration after global cerebral ischemia, which seems to

Experimental designs to evaluate the potential of neuroprotective drugs or hypothermia may have not met all requirements set by these consensuses in a single study, but integration of results of the many experimental studies may give enough information as to

beings.

neuroprotection.

require a long-term follow up.

support proposals for their clinical usefulness.

Since long-term preservation of the neuronal substrate in cerebral vulnerable structures underlying functional recovery after cerebral ischemia has been considered to be a major end point of neuroprotective strategies (STAIR, 1999) it can be expected that experimental designs for neuroprotection studies may lead to reliable interpretations of the efficiency of neuroprotective agents, in view of the proven capability of intrinsic cerebral mechanisms to promote , by themselves, neuronal repair and plasticity after ischemia.

Some neuronal proteins that are involved in structural and functional aspects of synaptic connectivity and neuronal circuits remodeling have been evaluated as parameters of ischemic damage and neuroprotection. In this sense, synaptophysin has been shown to be reduced in the frontal motor and temporal cortex of human beings that have been survived for 1 week to 1 year after a cardiac arrest (Akulinin et al., 1998). Besides, a reduction of synaptophysin 2, Munc-18-interacting proteins, 1-3 days after global cerebral ischemia in mice has been related to delayed neuronal death (Nishimura et al., 2000). On the other hand it has been proposed that progesterone-induced increase (3-35 days after ischemia) in the expression of synaptophysin and growth-associated protein 43, and the effects of venlafaxine preventing the decrease of synaptophysin, in the rat hippocampus are evidences of the neuroprotective effects of these drugs (Fang et al., 2010; Zhao et al., 2011).

### **4. Approaches to neuroprotection in animal models of global cerebral ischemia**

The experimental approach to neuroprotection aimed to influence, through pharmacological and non pharmacological procedures, those early and late neural phenomena accounting either for brain damage or for neuronal repair, plasticity and functional recovery after global cerebral ischemia and reperfusion, has resulted in a considerable amount of reliable information along the last 40 years.

Different strategies of neuroprotection attempting to prevent, reduce, or stop the progress of the ischemic brain damage have been assayed in animal models of global cerebral ischemia, under the premise of an opposition relationship between the mechanism(s) of action of the presumptive neuroprotective drugs or non pharmacological procedures, and the pathophysiological mechanisms of brain damage, which has been maintained as targets of neuroprotective strategies.

Neuroprotection studies in animal models of global cerebral ischemia have maintained the main objective of support proposals of pharmacological and non-pharmacological neuroprotective procedures to be incorporated as a matter for clinical trials aimed to a better management of human beings exposed to global cerebral ischemia, frequently as a consequence of a cardiorespiratory arrest. Translation of knowledge about neuroprotection obtained from models in experimental animals, to clinical practice has not been successful. This situation has been also observed in the case of focal cerebral ischemia, leading to consensus meetings (Fisher et al., 2009; STAIR, 1999) attempting to establish the better conditions for preclinical studies of neuroprotection as to give reliable results to be applied in clinical conditions. If opinion of these consensuses may be recognized as applicable to preclinical studies of global cerebral ischemia, it is apparent that some factors must be taken in account for designing and carrying of the respective experimental protocols. Thus, studies in animal models of global cerebral ischemia should give information on effective neuroprotective doses in the case of drugs being tested; hence, dose-response relationships should be investigated. Routes of drug administration and pharmacokinetic characteristics

Since long-term preservation of the neuronal substrate in cerebral vulnerable structures underlying functional recovery after cerebral ischemia has been considered to be a major end point of neuroprotective strategies (STAIR, 1999) it can be expected that experimental designs for neuroprotection studies may lead to reliable interpretations of the efficiency of neuroprotective agents, in view of the proven capability of intrinsic cerebral mechanisms to

Some neuronal proteins that are involved in structural and functional aspects of synaptic connectivity and neuronal circuits remodeling have been evaluated as parameters of ischemic damage and neuroprotection. In this sense, synaptophysin has been shown to be reduced in the frontal motor and temporal cortex of human beings that have been survived for 1 week to 1 year after a cardiac arrest (Akulinin et al., 1998). Besides, a reduction of synaptophysin 2, Munc-18-interacting proteins, 1-3 days after global cerebral ischemia in mice has been related to delayed neuronal death (Nishimura et al., 2000). On the other hand it has been proposed that progesterone-induced increase (3-35 days after ischemia) in the expression of synaptophysin and growth-associated protein 43, and the effects of venlafaxine preventing the decrease of synaptophysin, in the rat hippocampus are evidences

promote , by themselves, neuronal repair and plasticity after ischemia.

of the neuroprotective effects of these drugs (Fang et al., 2010; Zhao et al., 2011).

**ischemia** 

information along the last 40 years.

neuroprotective strategies.

**4. Approaches to neuroprotection in animal models of global cerebral** 

The experimental approach to neuroprotection aimed to influence, through pharmacological and non pharmacological procedures, those early and late neural phenomena accounting either for brain damage or for neuronal repair, plasticity and functional recovery after global cerebral ischemia and reperfusion, has resulted in a considerable amount of reliable

Different strategies of neuroprotection attempting to prevent, reduce, or stop the progress of the ischemic brain damage have been assayed in animal models of global cerebral ischemia, under the premise of an opposition relationship between the mechanism(s) of action of the presumptive neuroprotective drugs or non pharmacological procedures, and the pathophysiological mechanisms of brain damage, which has been maintained as targets of

Neuroprotection studies in animal models of global cerebral ischemia have maintained the main objective of support proposals of pharmacological and non-pharmacological neuroprotective procedures to be incorporated as a matter for clinical trials aimed to a better management of human beings exposed to global cerebral ischemia, frequently as a consequence of a cardiorespiratory arrest. Translation of knowledge about neuroprotection obtained from models in experimental animals, to clinical practice has not been successful. This situation has been also observed in the case of focal cerebral ischemia, leading to consensus meetings (Fisher et al., 2009; STAIR, 1999) attempting to establish the better conditions for preclinical studies of neuroprotection as to give reliable results to be applied in clinical conditions. If opinion of these consensuses may be recognized as applicable to preclinical studies of global cerebral ischemia, it is apparent that some factors must be taken in account for designing and carrying of the respective experimental protocols. Thus, studies in animal models of global cerebral ischemia should give information on effective neuroprotective doses in the case of drugs being tested; hence, dose-response relationships should be investigated. Routes of drug administration and pharmacokinetic characteristics should also be taken in account as to be compatible with their potential use in human beings.

The time window of opportunity for the effective neuroprotective treatment is an important factor to be considered in preclinical models that may predict the timing of neuroprotective procedures in clinical situations with reference to the onset of global cerebral ischemia and subsequent reperfusion. The initial hypothesis that opportunity window for neuroprotective procedures would be limited to a short period after the ischemic episode has been changed in view of experimental evidence. Thus, different drugs or neuroprotective procedures having predominant mechanisms of action against specific cellular processes of ischemia damage occurring lately within the pathophysiological cascade, may allow to neuroprotection even when administered hours or days after ischemia. Besides, the opportunity time window may be further extended when it is expected that neuroprotective procedures act through promotion of cellular processes of neuronal repair and plasticity.

In view of the multiple pathophysiological processes occurring both in sequence and simultaneously after ischemia and reperfusion, it is considered as an advantage for presumptive neuroprotective drugs to have multiple cellular or molecular mechanisms of action, as occurring with some originally endogenous compounds, namely melatonin, estradiol and progesterone (El-Abhar et al, 2002; Hurn et al, 1995; Jover-Mengual et al, 2010; Lebesgue et al, 2009; Reiter et al, 2005; Wang et al, 2008). By contrast, most synthetic drugs only have one mechanism of action accounting for neuroprotection. Attempting to counteract several mechanisms of ischemic brain injury would require the simultaneous administration of several drugs (Hicks et al, 1999; Matsumoto et al, 1993; Pazos et al, 1999; del Pilar Fernández et al, 1998; Sánchez-Casado et al, 2007; Zapater et al, 1997) (Table 1).

Recommendations arisen from these consensuses of opinion have also highlighted the importance of long-term studies to identify whether functional preservation or recovery may be attributed to effects of the neuroprotective procedures, and/or to intrinsic mechanisms of plasticity and repair triggered by ischemia *per se*. Reliable parameters of long-term structural and functional outcome may allow to evaluate the final result of the neuroprotective procedures on cerebral structures vulnerable to ischemia. Thus, evaluation of neuronal population, cytoarchitectonic characteristics, and connectivity of the neural circuits in these vulnerable structures, as well as different aspects of cognitive functions depending on them should be included as a part of experimental designs of neuroprotection.

It has been described that the neuronal population of remaining neurons in CA1 at survival times of 2-3 weeks may be less than that evaluated 3-4 months after the ischemic episode, suggesting that, without exogenous intervention, CA1 neurons may have been repopulate, became integrated to the hippocampal neuronal circuits, and contribute to functional recovery (Bendel, et al 2005; von Euler et al., 2006, Hartman et al, 2005, Nakatomi et al., 2002). Obviously, the potential repopulation complicates the interpretation of learning and memory studies after global cerebral ischemia, because short-term studies may not give an adequate end point of the cognitive alteration after global cerebral ischemia, which seems to require a long-term follow up.

Experimental designs to evaluate the potential of neuroprotective drugs or hypothermia may have not met all requirements set by these consensuses in a single study, but integration of results of the many experimental studies may give enough information as to support proposals for their clinical usefulness.

Neuroprotection in Animal Models of Global Cerebral Ischemia 317

References

Pentoxifylline Sirin et al., 1998; Tuong et al., 1994. Edaravone Kubo et al., 2009 ; Otani et al., 2005.

Melatonin Cervantes et al., 2008; Cho et al, 1997;

Other Bashkatova et al, 2001; Fang et al, 2010;

Warner et al, 2004.

Wappler et al., 2010.

Zani et al., 2007.

Growth Factors BDNF D'Cruz et al., 2002; Kiprianova et al., 1999a, 1999b;

Hypothermia Asai et al., 2000; Baumann et al, 2009;

González-Burgos et al., 2007; Letechipía-Vallejo et al., 2001; Letechipía-Vallejo et al., 2007; Rennie et al., 2008; Weil et al., 2009.

Estradiol Dai et al., 2007 ; He et al., 2002 ; Hurn et al., 1995 ;

Plamondon & Roberge, 2008.

Erythropoietin Cotena et al, 2008 ; Givehchian et al., 2010;

Lu et al., 2002 ; Wang et al., 2006 ;

Gaur & Kumar, 2010; Nanri et al, 1998; Pazos et al., 1999; Sinha et al., 2001;

Jover-Mengual et al, 2010; Koh et al., 2006 ;

Blondeau et al., 2002; Fernandes et al., 2008; Lauritzen et al., 2000; Ma et al., 2008;

Incagnioli et al., 2009; Zhang et al, 2007.

Chopp et al, 1988; Colbourne & Corbett, 1994;

Silasi & Colbourne, 2011; Webster et al., 2009; Zhang H. et al., 2010; Zhang Z, et al., 2001.

Larsson et al., 1999; Popp et al., 2004.

Dong et al, 2001; Noguchi et al., 2011;

Lebesgue et al., 2009 ; Littleton-Kearney et al, 2005 ;

Selakovic et al., 2010; Stevens & Yaksh, 1990.

El-Abhar et al., 2002; García-Chávez et al., 2008;

Antioxidants Tirilazad Li et al., 2010; del Pilar Fernández et al., 2008;

Methylene blue Wiklund et al., 2007.

Human albumin Belayev et al., 1999.

Neuroprotective

Agent

Main Mechanism

Antiapoptotic agents

Cell proliferation stimulants

Reduction of: cerebral

excitatory aminoacids, apoptosis, inflammatory reactions. Enhancement of

BDNF

metabolism and oxygen demands, reactive oxygen species, release of

Other mechanisms Delta 9-

NON-PHARMACOLOGICAL AGENTS

tetrahydrocannabinol

Linoleic acid and other PUFA's

of Action


References

Levemopamil Block & Schwarz 1998. Dantrolene Nakayama et al, 2002 Flunarizine Lee Y.S. et al., 1999.

Linoleic acid Blondeau et al., 2002.

Lamotrigine Conroy et al., 1999 ; Crumrine et al., 1997;

Lubeluzole Koinig et al., 2001; Mueller et al., 2003;

MgSO4 Meloni et al., 2009; Miles et al., 2001;

Zinc Matsushita et al., 1996.

GABAergic agents Clomethiazole Clarkson et al., 2005; Chaulk et al, 2003; Cross

Dextromethorphan Block & Schwarz, 1998.

Dizocilpine Bernabeu, R., & Sharp, 2000;

Kwon et al., 2000; Montero et al., 2007; Stevens & Yaksh, 1990;

Haseldonckx et al., 1997

Sirin et al., 1998.

Stepień et al., 2005.

Sydserff et al., 2000. Diacepam Corbett et al, 2008; Dowden et al, 1999;

Schwartz et al, 1995. Thiopental Kofke et al., 1979; Pappas & Mironovich, 1981;

> Moralí et al., 2011a, 2011b; Ozacmak & Sayan, 2009 ;

Todd et al, 1982 . Propofol Cai et al., 2011 ; Cervantes et al., 1995 ; Ergün et al, 2002.

Creatine Lensman et al., 2006; Otellin et al., 2003.

Nimodipine Cervantes et al., 1992; Choi SK et al., 2011;

Lazarewicz et al., 1993;

del Pilar Fernández et al., 1998;

Hicks et al., 1999; Janac et al., 2008;

Selakovic et al., 2010; Zhang et al., 2009.

Lee Y.S. et al., 1999 ; Morimoto et al., 2002 ; Shuaib et al., 1995b ; Wiard et al., 1995.

et al, 1995; Liang et al, 1997; Shuaib et al., 1995a;

Hall et al, 1998; Johansen FF, Diemer, 1991;

Aggarwal et al., 2008 ; Cervantes et al., 2002 ; González-Vidal et al., 1998 ; Moralí et al., 2005 ;

J.M. Wang et al, 2008 ; Zhao et al, 2011.

Haddon et al., 1988; Lazarewicz et al., 1990;

Rami & Krieglstein, 1994; Zornow et al, 1996.

Neuroprotective

Agent

PHARMACOLOGICAL AGENTS

Antiepileptic

Progesterone,

agents

allopregnanolone

Main Mechanism

Increase of energy

Calcium channel

of Action

reserve

blockers

K+ channel activators

Glutamate antagonists


Neuroprotection in Animal Models of Global Cerebral Ischemia 319

anti-inflammatory processes, among others (Lakhan et al, 2009; Lipton, 1999; Mehta et al,

Neurological, behavioral, electrophysiological and histopathological correlates of the outcome after global cerebral ischemia being end points of cellular processes triggered by

Global cerebral ischemia usually does not result in long lasting focal neurological deficits in rats. Thus neurological deficit scores resulting from sensorimotor tests assessing motorsensory functions in rats, including placement reactions, righting and flexion reflexes, equilibrium, spontaneous motility, among others may be altered shortly after (24 h) global cerebral ischemia, but they appear recovered 7 days after ischemia. These transient neurological deficits have been interpreted as functional alteration of hippocampus and striatum; though correlation between neurological deficit scores and ischemic neuronal damage in these structures, not always were found (Block, 1999; Hartman et al., 2005; Kofler

Elevated, four (two open and two closed) arms plus maze, and open field tests have been used, among other to evaluate anxiety after global cerebral ischemia especially in rodents. Thus scores of latency to enter to open arms, the number of open and closed arms entries and rears are taken as parameters of anxiety in the elevated plus maze, while in the open field (circular arena 80 cm in diameter, three concentric rings and lines radiating from the center) tests, the number of segments entered with all the four paws, the number of rears,

Since the clinical consequences of cardiac arrest, as the main cause of global cerebral ischemia, have been consistently described as long-term alterations of cognitive functions, it can be expected that similar cognitive deficits may be elicited by global cerebral ischemia in experimental animals. In fact, the most vulnerable neurons to ischemia are located in brain structures involved in cognitive processes (Ginsberg & Busto, 1989; Gionet et al., 1991; Pulsinelli, 1985); thus, evaluation of cognitive functions mainly dependent on hippocampus, striatum and prefrontal cortex, and its electrophysiological and morphological correlates may be reliable parameters of brain injury and neuroprotection after global cerebral ischemia. The magnitude and type of cognitive deficits in experimental animals submitted to global cerebral ischemia may vary considerably depending on the animal model, the survival times of testing, and the specific behavioral tests that could have been used. Among these procedures to evaluate cognitive functions, the Morris water maze, the eight-arms radial Olton maze, and the T maze, have been widely used in assessing learning and memory in both 2VO and 4VO models in rats, and its correlation with neuronal loss (Block, 1999; Hartmann et al., 2005; Olsen et al., 1994; Volpe et al., 1984), and functional and morphological characteristics of the neural substrate underlying cognitive functions in brain structures vulnerable to ischemia. Novel object recognition tests have been shown to be a reliable index of cognitive functions since rats or mice normally spend more time exploring novel objects, whereas animals with recognition memory deficits will explore novel and

and the number of *faecal boli* are indexes of anxiety (Nelson et al., 1997).

ischemia, give information about ischemic brain injury and neuroprotection.

2007; Schneider et al, 2009).

**4.1.1 Neurological assessment** 

**4.1.2 Mood and behavioral assessment** 

**4.1.3 Cognitive functions assessment** 

et al, 2004).


Table 1. Main pharmacological and non-pharmacological agents showing neuroprotective effects through molecular, biochemical , histopathological, behavioral, neurologic, and cognitive parameters.

These strategies have allowed identifying the neuroprotective characteristics of many agents, including non-pharmacological procedures like hypothermia, that have been tested in animal models of global cerebral ischemia from the knowledge of an opposition relationship between their mechanism(s) of action, and the nature of the pathophysiological phenomena of ischemic damage. They may be grouped in relation to their main predominant mechanism of action against ischemic damage: calcium channel blockers, glutamate antagonists, GABAergic drugs, antioxidant agents, anti-inflammatory compounds, etc. Many of these compounds are products of chemical synthesis; but endogenous compounds (melatonin, estradiol, progesterone, allopregnanolone, etc.) playing important physiological roles in mammals, have also been shown to exert potent neuroprotective effects. Table 1 presents some examples of the various groups of neuroprotective agents.

### **4.1 Outcome assessment of brain injury and neuroprotection in animal models of global cerebral ischemia**

Assessment of brain injury and neuroprotection in animal models of global cerebral ischemia can be effected at different levels of biological organization of the central nervous system, from molecular and cellular phenomena to brain functions requiring highly integrated, behavioral expressions. In general, parameters of cellular and molecular processes leading to ischemic brain damage or neuroprotection require obtaining brain tissue samples at a selected time point after ischemia for these phenomena to be evaluated. On the other hand, a follow-up of damage and/or recovery through repeated bioelectrical, behavioral, and cognitive measurements is possible to be done in the same animal along extended periods. Parameters that allow evaluating the presence and magnitude of ischemic brain injury at the different levels of biological organization are also reliable indexes of neuroprotective actions, as they are induced by ischemia and may be counteracted by neuroprotective procedures. A similar consideration can be done regarding cell repair and plasticity mechanisms triggered by the ischemic insult, which are expected to be favored by neuroprotective agents.

Measurements have been done of parameters of each of the various phenomena affected by ischemia which constitute the starting point of ischemic brain injury. These include timely and topographically appropriate evaluation of ionic changes, release of neurotransmitters, modification of receptor molecular structure, excitotoxicy, morphological and functional mitochondrial alterations, reactive oxygen and nitrogen species, antioxidant enzymes and lipoperoxidation, activation of pro- and antiapoptotic cascades, DNA breakdown, pro- and anti-inflammatory processes, among others (Lakhan et al, 2009; Lipton, 1999; Mehta et al, 2007; Schneider et al, 2009).

Neurological, behavioral, electrophysiological and histopathological correlates of the outcome after global cerebral ischemia being end points of cellular processes triggered by ischemia, give information about ischemic brain injury and neuroprotection.

### **4.1.1 Neurological assessment**

318 Advances in the Preclinical Study of Ischemic Stroke

References

ASSOCIATION OF PHARMACOLOGICAL AND NON-PHARMACOLOGICAL AGENTS

Table 1. Main pharmacological and non-pharmacological agents showing neuroprotective effects through molecular, biochemical , histopathological, behavioral, neurologic, and

These strategies have allowed identifying the neuroprotective characteristics of many agents, including non-pharmacological procedures like hypothermia, that have been tested in animal models of global cerebral ischemia from the knowledge of an opposition relationship between their mechanism(s) of action, and the nature of the pathophysiological phenomena of ischemic damage. They may be grouped in relation to their main predominant mechanism of action against ischemic damage: calcium channel blockers, glutamate antagonists, GABAergic drugs, antioxidant agents, anti-inflammatory compounds, etc. Many of these compounds are products of chemical synthesis; but endogenous compounds (melatonin, estradiol, progesterone, allopregnanolone, etc.) playing important physiological roles in mammals, have also been shown to exert potent neuroprotective effects. Table 1 presents some examples of the various groups of

**4.1 Outcome assessment of brain injury and neuroprotection in animal models of** 

Assessment of brain injury and neuroprotection in animal models of global cerebral ischemia can be effected at different levels of biological organization of the central nervous system, from molecular and cellular phenomena to brain functions requiring highly integrated, behavioral expressions. In general, parameters of cellular and molecular processes leading to ischemic brain damage or neuroprotection require obtaining brain tissue samples at a selected time point after ischemia for these phenomena to be evaluated. On the other hand, a follow-up of damage and/or recovery through repeated bioelectrical, behavioral, and cognitive measurements is possible to be done in the same animal along extended periods. Parameters that allow evaluating the presence and magnitude of ischemic brain injury at the different levels of biological organization are also reliable indexes of neuroprotective actions, as they are induced by ischemia and may be counteracted by neuroprotective procedures. A similar consideration can be done regarding cell repair and plasticity mechanisms triggered by the ischemic insult, which are expected to be favored by

Measurements have been done of parameters of each of the various phenomena affected by ischemia which constitute the starting point of ischemic brain injury. These include timely and topographically appropriate evaluation of ionic changes, release of neurotransmitters, modification of receptor molecular structure, excitotoxicy, morphological and functional mitochondrial alterations, reactive oxygen and nitrogen species, antioxidant enzymes and lipoperoxidation, activation of pro- and antiapoptotic cascades, DNA breakdown, pro- and

Meloni et al., 2009.

Sánchez Casado et al., 2007

Neuroprotective

Hypothermia +

Hypothermia + MgSO4 + tirilazad

Agent

MgSO4

Main Mechanism

cognitive parameters.

neuroprotective agents.

**global cerebral ischemia** 

neuroprotective agents.

of Action

Global cerebral ischemia usually does not result in long lasting focal neurological deficits in rats. Thus neurological deficit scores resulting from sensorimotor tests assessing motorsensory functions in rats, including placement reactions, righting and flexion reflexes, equilibrium, spontaneous motility, among others may be altered shortly after (24 h) global cerebral ischemia, but they appear recovered 7 days after ischemia. These transient neurological deficits have been interpreted as functional alteration of hippocampus and striatum; though correlation between neurological deficit scores and ischemic neuronal damage in these structures, not always were found (Block, 1999; Hartman et al., 2005; Kofler et al, 2004).

### **4.1.2 Mood and behavioral assessment**

Elevated, four (two open and two closed) arms plus maze, and open field tests have been used, among other to evaluate anxiety after global cerebral ischemia especially in rodents. Thus scores of latency to enter to open arms, the number of open and closed arms entries and rears are taken as parameters of anxiety in the elevated plus maze, while in the open field (circular arena 80 cm in diameter, three concentric rings and lines radiating from the center) tests, the number of segments entered with all the four paws, the number of rears, and the number of *faecal boli* are indexes of anxiety (Nelson et al., 1997).

### **4.1.3 Cognitive functions assessment**

Since the clinical consequences of cardiac arrest, as the main cause of global cerebral ischemia, have been consistently described as long-term alterations of cognitive functions, it can be expected that similar cognitive deficits may be elicited by global cerebral ischemia in experimental animals. In fact, the most vulnerable neurons to ischemia are located in brain structures involved in cognitive processes (Ginsberg & Busto, 1989; Gionet et al., 1991; Pulsinelli, 1985); thus, evaluation of cognitive functions mainly dependent on hippocampus, striatum and prefrontal cortex, and its electrophysiological and morphological correlates may be reliable parameters of brain injury and neuroprotection after global cerebral ischemia.

The magnitude and type of cognitive deficits in experimental animals submitted to global cerebral ischemia may vary considerably depending on the animal model, the survival times of testing, and the specific behavioral tests that could have been used. Among these procedures to evaluate cognitive functions, the Morris water maze, the eight-arms radial Olton maze, and the T maze, have been widely used in assessing learning and memory in both 2VO and 4VO models in rats, and its correlation with neuronal loss (Block, 1999; Hartmann et al., 2005; Olsen et al., 1994; Volpe et al., 1984), and functional and morphological characteristics of the neural substrate underlying cognitive functions in brain structures vulnerable to ischemia. Novel object recognition tests have been shown to be a reliable index of cognitive functions since rats or mice normally spend more time exploring novel objects, whereas animals with recognition memory deficits will explore novel and

Neuroprotection in Animal Models of Global Cerebral Ischemia 321

Spatial working memory can be evaluated by using the 8-arms Olton radial maze (Myhrer, 2003; Olton**,** 1983, 1987; Olton et al., 1982; Shibata et al., 2007). For a daily standard evaluation all eight arms are baited and the rat is allowed to collect food from each arm; the number of errors, defined as a re-entry into an arm that had already been visited, is recorded in order to evaluate withholding and updating of information about each arm visited and rewarding obtained. An alternative maze configuration in which only some of the eight arms are baited allows to evaluate reference memory besides working memory through recording of the number of reference memory errors (number of entries into unbaited arms) and working memory errors (re-entry into an already visited arm). Performance in the Olton maze requires an adequate functioning of hippocampalprefrontocortical neuronal circuits, and is a reliable parameter of morpho-functional integrity of these brain structures after ischemia and neuroprotection (Cassel et al., 1998; Fritts et al., 1998; Izaki et al., 2008; Kolb, 1990, Kolb et al 1982; Laroche et al., 2000; Olton et al., 1982; Seamans et al., 1995; Winocur, 1982). An aquatic version of the 8-arm radial maze has also been described (Kolb et al, 1982), and used to correlate hippocampal pyramidal

Neuronal population of different neuron types in brain vulnerable structures has been considered as a reliable parameter of ischemia brain damage and neuroprotection. Thus, pyramidal neuron population in the Ammon´s horn of the hippocampus and in the neocortex (Bleayert et al, 1978; Colbourne & Corbett, 1994; García-Chávez et al., 2008; Hartman et al, 2005; Johansen & Diemer, 1991; Kirino, 1982; Letechipía-Vallejo et al., 2007; Moralí et al., 2011b; Pulsinelli, 1985; Schmidt-Kastner & Freund, 1991; Shuaib et al, 1995), or different neuron types in other brain vulnerable structures (Block & Schwartz, 1998; Cervantes et al., 2002), have been evaluated through the number and proportion of surviving neurons. However, most of these studies deal with histopathological assessment of the hippocampus, the highest vulnerable brain region to global cerebral ischemia. Usually four separate counts of surviving neurons in selected areas of the Ammon´s horn are obtained from each of five coronal sections of the hippocampus per rat, stained with cresyl violet for a total of 20 counts per animal, under the different experimental conditions (Hartman et al., 2005). Similar procedures are followed for neuronal counting in other brain

Immunohistochemical staining techniques have been also used in animal models of global cerebral ischemia and neuroprotection in order to identify specific proteins or fluorescent DNA labels that may selectively mark cells undergoing an acute necrotic or apoptotic process, as well as the activation of specific cellular processes involved in neuronal damage or repair and survival. Immunohistochemical marks (c-fos/c-jun, heat shock proteins, Bcl-2/Bax immunoreactivity, among others) allow to identify neuron types and neuroanatomical regions where ischemia-induced phenomena take place. Besides, immunohistochemical markers of glial fibrillary acidic protein (GFAP) as well as microglia cell surface components lead to identification of reactive gliosis in the hippocampus, as a consequence of global cerebral ischemia and ischemic neuronal death, which elicited activation of microglial cells and interleukine 1 release that may trigger an astrocyte reaction mainly located in the *stratum lacunosum-moleculare, stratum moleculare*, and *hilus*, and

neurons damage and working memory performance (Nelson et al. 1997).

**4.1.4 Histopathological assessment** 

structures vulnerable to ischemia.

familiar objects equally (Hartman et al., 2005). Cognitive functions have also been assessed in rodents through conditioned avoidance tasks (Block, 1999; Kofler et al., 2004; Langdon et al., 2008).

Several paradigms in the Morris water maze and in the eight-arms radial Olton maze, that have been used in most of neuroprotection studies in which cognitive functions are assessed, have proven to be useful for testing hippocampal, striatum and prefrontal cortex functioning as end points of brain damage or neuroprotection after global cerebral ischemia (Morris, 1984; Olton et al, 1982).

Hippocampal functioning has been evaluated in rats and mice through some behavioral paradigms that require the integrity of this brain structure and related structures in the temporal lobe (Barnes, 1979; Morris et al., 1982, 1990), in order to configure cognitive spatial representations, i.e., a cognitive spatial map (Cassels, 1998; Jarrad, 1993; McDonald and White, 1994; 1995; Moser et al, 1993). Thus parameters of spatial learning training to locate a hidden platform, (escape latency: time spent by the animal to reach the platform; swimming path length: distance swam until reaching the platform; searching strategy: pattern of the swimming path towards the platform) and probe trial to evaluate retention of spatial learning (time spent, or the distance traveled by the animal in each of the four quadrants of the maze; number of crossings over the former platform location) in the Morris water maze including extra maze spatial clues, have been used in testing the morpho-functional state of the hippocampus (Dalm et al 2000; D'Hooge & De Deyn, 2001; Eichenbaum et al, 1990; Morris, 1984; Myhrer, 2003)..

Under these training conditions and since there are no intra maze clues to guide the animal's behavior, it is assumed that, to achieve the goal, the animal has to build the cognitive map and thus, a hippocampal processing of information occurs (Gallagher and Pelleymounter, 1988, O'Keffe & Nadel 1978). For this reason, studies of neuroprotection use the spatial learning in the Morris water maze paradigm, as a reliable index of the hippocampal functioning.

However, in addition to place learning, spatial navigation in the water maze may occur through at least, two additional strategies not depending on the hippocampus but on the striatum: signal learning and egocentric learning (Brandeis et al 1989; Gallagher & Pelleymounter, 1988; O'Keefe & Nadel 1978). Signal learning is displayed when the animal reaches a visible platform, or a visible stimulus indicating (signaling) the location of the platform within the maze. Learning of the association between the stimulus and the response is established and depends on the functioning of the striatum (McDonald & White, 1994). The egocentric learning occurs when the animal develops stereotyped motor patterns to locate the invisible platform on the basis of the proprioceptive information provided by its own movement. It is also an ability that depends on the memory system to which the striatum belongs (McDonald & White, 1994; McDonald & White 1995; Oliveira et al., 1997). Results obtained when evaluating both adult and aged male rats, show that some adult rats may use either place, hippocampal dependent allocentric, or striatum-dependent, egocentric strategies; on the other hand, aged rats use egocentric, as their main swimming strategy to solve the task (Dalm et al., 2000; Olvera-Cortés et al, 2011). Thus, deficits in the performance of this task may indicate an alteration of any of these two abilities, place and egocentric learning, so that different parameters should be evaluated to assess the mechanism underlying the observed deficit (D'Hooge & De Deyn, 2001). A qualitative analysis of the swimming paths both during the training period and the probe trial may allow a better determining of the strategy used by the rat in solving the task in the water maze.

familiar objects equally (Hartman et al., 2005). Cognitive functions have also been assessed in rodents through conditioned avoidance tasks (Block, 1999; Kofler et al., 2004; Langdon et

Several paradigms in the Morris water maze and in the eight-arms radial Olton maze, that have been used in most of neuroprotection studies in which cognitive functions are assessed, have proven to be useful for testing hippocampal, striatum and prefrontal cortex functioning as end points of brain damage or neuroprotection after global cerebral ischemia

Hippocampal functioning has been evaluated in rats and mice through some behavioral paradigms that require the integrity of this brain structure and related structures in the temporal lobe (Barnes, 1979; Morris et al., 1982, 1990), in order to configure cognitive spatial representations, i.e., a cognitive spatial map (Cassels, 1998; Jarrad, 1993; McDonald and White, 1994; 1995; Moser et al, 1993). Thus parameters of spatial learning training to locate a hidden platform, (escape latency: time spent by the animal to reach the platform; swimming path length: distance swam until reaching the platform; searching strategy: pattern of the swimming path towards the platform) and probe trial to evaluate retention of spatial learning (time spent, or the distance traveled by the animal in each of the four quadrants of the maze; number of crossings over the former platform location) in the Morris water maze including extra maze spatial clues, have been used in testing the morpho-functional state of the hippocampus (Dalm et al 2000; D'Hooge & De Deyn, 2001; Eichenbaum et al, 1990;

Under these training conditions and since there are no intra maze clues to guide the animal's behavior, it is assumed that, to achieve the goal, the animal has to build the cognitive map and thus, a hippocampal processing of information occurs (Gallagher and Pelleymounter, 1988, O'Keffe & Nadel 1978). For this reason, studies of neuroprotection use the spatial learning in the Morris water maze paradigm, as a reliable index of the

However, in addition to place learning, spatial navigation in the water maze may occur through at least, two additional strategies not depending on the hippocampus but on the striatum: signal learning and egocentric learning (Brandeis et al 1989; Gallagher & Pelleymounter, 1988; O'Keefe & Nadel 1978). Signal learning is displayed when the animal reaches a visible platform, or a visible stimulus indicating (signaling) the location of the platform within the maze. Learning of the association between the stimulus and the response is established and depends on the functioning of the striatum (McDonald & White, 1994). The egocentric learning occurs when the animal develops stereotyped motor patterns to locate the invisible platform on the basis of the proprioceptive information provided by its own movement. It is also an ability that depends on the memory system to which the striatum belongs (McDonald & White, 1994; McDonald & White 1995; Oliveira et al., 1997). Results obtained when evaluating both adult and aged male rats, show that some adult rats may use either place, hippocampal dependent allocentric, or striatum-dependent, egocentric strategies; on the other hand, aged rats use egocentric, as their main swimming strategy to solve the task (Dalm et al., 2000; Olvera-Cortés et al, 2011). Thus, deficits in the performance of this task may indicate an alteration of any of these two abilities, place and egocentric learning, so that different parameters should be evaluated to assess the mechanism underlying the observed deficit (D'Hooge & De Deyn, 2001). A qualitative analysis of the swimming paths both during the training period and the probe trial may allow a better

determining of the strategy used by the rat in solving the task in the water maze.

al., 2008).

(Morris, 1984; Olton et al, 1982).

Morris, 1984; Myhrer, 2003)..

hippocampal functioning.

Spatial working memory can be evaluated by using the 8-arms Olton radial maze (Myhrer, 2003; Olton**,** 1983, 1987; Olton et al., 1982; Shibata et al., 2007). For a daily standard evaluation all eight arms are baited and the rat is allowed to collect food from each arm; the number of errors, defined as a re-entry into an arm that had already been visited, is recorded in order to evaluate withholding and updating of information about each arm visited and rewarding obtained. An alternative maze configuration in which only some of the eight arms are baited allows to evaluate reference memory besides working memory through recording of the number of reference memory errors (number of entries into unbaited arms) and working memory errors (re-entry into an already visited arm). Performance in the Olton maze requires an adequate functioning of hippocampalprefrontocortical neuronal circuits, and is a reliable parameter of morpho-functional integrity of these brain structures after ischemia and neuroprotection (Cassel et al., 1998; Fritts et al., 1998; Izaki et al., 2008; Kolb, 1990, Kolb et al 1982; Laroche et al., 2000; Olton et al., 1982; Seamans et al., 1995; Winocur, 1982). An aquatic version of the 8-arm radial maze has also been described (Kolb et al, 1982), and used to correlate hippocampal pyramidal neurons damage and working memory performance (Nelson et al. 1997).

### **4.1.4 Histopathological assessment**

Neuronal population of different neuron types in brain vulnerable structures has been considered as a reliable parameter of ischemia brain damage and neuroprotection. Thus, pyramidal neuron population in the Ammon´s horn of the hippocampus and in the neocortex (Bleayert et al, 1978; Colbourne & Corbett, 1994; García-Chávez et al., 2008; Hartman et al, 2005; Johansen & Diemer, 1991; Kirino, 1982; Letechipía-Vallejo et al., 2007; Moralí et al., 2011b; Pulsinelli, 1985; Schmidt-Kastner & Freund, 1991; Shuaib et al, 1995), or different neuron types in other brain vulnerable structures (Block & Schwartz, 1998; Cervantes et al., 2002), have been evaluated through the number and proportion of surviving neurons. However, most of these studies deal with histopathological assessment of the hippocampus, the highest vulnerable brain region to global cerebral ischemia. Usually four separate counts of surviving neurons in selected areas of the Ammon´s horn are obtained from each of five coronal sections of the hippocampus per rat, stained with cresyl violet for a total of 20 counts per animal, under the different experimental conditions (Hartman et al., 2005). Similar procedures are followed for neuronal counting in other brain structures vulnerable to ischemia.

Immunohistochemical staining techniques have been also used in animal models of global cerebral ischemia and neuroprotection in order to identify specific proteins or fluorescent DNA labels that may selectively mark cells undergoing an acute necrotic or apoptotic process, as well as the activation of specific cellular processes involved in neuronal damage or repair and survival. Immunohistochemical marks (c-fos/c-jun, heat shock proteins, Bcl-2/Bax immunoreactivity, among others) allow to identify neuron types and neuroanatomical regions where ischemia-induced phenomena take place. Besides, immunohistochemical markers of glial fibrillary acidic protein (GFAP) as well as microglia cell surface components lead to identification of reactive gliosis in the hippocampus, as a consequence of global cerebral ischemia and ischemic neuronal death, which elicited activation of microglial cells and interleukine 1 release that may trigger an astrocyte reaction mainly located in the *stratum lacunosum-moleculare, stratum moleculare*, and *hilus*, and

Neuroprotection in Animal Models of Global Cerebral Ischemia 323

neuroprotection are possible alternatives to prevent or reduce the risk of ischemic neuronal damage (Savitz & Fisher, 2007; Weigl et al, 2005). This has stimulated designing of experimental studies on prophylactic neuroprotection to assess the effectiveness of several agents and their clinical potential. Some neuroprotective agents have proven to be more effective when applied before the ischemic insult than when given later in time, in particular those agents affecting the early cellular phenomena induced by ischemia, such as calcium channel blockers, GABAergic and anti-excitotoxic agents, as well as antioxidant drugs (Weigl et al, 2005). Pharmacological treatments (antihypertensive, antidiabetic, antithrombotic, antiatherogenic drugs) effective in modifying in the long term the risk for cardiac arrest or cardiac infarct which may result in global cerebral ischemia or in severe hypoperfusion have also been proposed as prophylactic neuroprotection procedures (Savitz

Though an increasing number of drugs have proven to be effective neuroprotective agents in experimental models of global cerebral ischemia, data supporting proposals for their clinical use have not been enough to influence clinical management and outcome of patients exposed to global cerebral ischemia in clinical trials. However, after its evaluation in animal models of global cerebral ischemia, special interest has been paid to carry out clinical trials with a non-pharmacological procedure, hypothermia, as a part of the intensive care of patients after a cardiorespiratory arrest. Nevertheless, the wide perspectives to gain information on neuroprotection through experimental designs including animal models of global cerebral ischemia are maintained to date, despite the tendency to preferentially conduct studies on rodents; in particular if differences between experimental animals and human beings are taken into account, and attention is paid to reproduce those components

Partially supported by Instituto Mexicano del Seguro Social, MEXICO (2006/1A/I/029;

Abe, K.; Yoshida, S.; Watson, B.D.; Busto, R.; Kogure, K. & Ginsberg, M.D. (1983). Alpha-

Aggarwal, R.; Medhi, B.; Pathak, A.; Dhawan, V. & Chakrabarti, A. (2008). Neuroprotective

Akulinin, V.A.; Belichenko, P.V. & Dahlstrom, A. (1998). Quantitative Analysis of

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Tocopherol and Ubiquinones in Rat Brain Subjected to Decapitation Ischemia. *Brain* 

Effect of Progesterone on Acute Phase Changes Induced by Partial Global Cerebral Ischaemia in Mice. *Journal of Pharmacy and Pharmacology,*Vol.60, No.6, (May 2008),

Synaptophysin Immunoreactivity in Human Neocortex after Cardiac Arrest: Confocal Laser Scanning Microscopy Study. *Resuscitation,*Vol.39, No.3, (March

mainly accounting for brain damage after global cerebral ischemia.

& Fisher, 2007).

**5. Conclusion** 

**6. Acknowledgement** 

FIS/IMSS/PROT/196).

pp. 731-737, ISSN 0022-3573

1999), pp. 207-213, ISSN 0300-9572

**7. References** 

persisting for weeks after ischemia (Buffo et al., 2010; Choi JS et al, 2008; Mori et al, 2008; Morioka et al., 1991, 1992; Nikonenko et al., 2009; Petito & Halaby, 1993). The efficacy of neuroprotective agents can also be determined on the basis of the success in preventing the occurrence of necrosis, apoptosis, heat shock expression, gliosis, etc., as indicated by the immunohistochemical biomarkers (Scallet, 1995). Different parameters of the glial reaction elicited by global cerebral ischemia have been used as indexes of brain damage or neuroprotection (Cervantes et al., 2002; de Yebra et al., 2006; Duan et al., 2011; Korzhevskii et al., 2005; Piao et al., 2002; Soltys et al., 2003).

Neuronal cytoarchitecture and fine structure parameters of synaptic connectivity have also been used for histopathological assessment after brain damage and neuroprotection (Briones et al., 2006; García-Chávez et al., 2008; González-Burgos et al., 2007; Johansson & Belichenko, 2002; Kovalenko et al., 2006, Moralí et al., 2011a; Nikonenko et al., 2009; Ruan et al., 2006).

### **4.2 Therapeutic opportunity window in animal models of global cerebral ischemia**

In any case, recognition of a "therapeutic opportunity window" or "therapeutic time window" in relation to the timing of the ischemic episode, the temporal course of the mechanisms of brain damage and/or repair, and the exerting of actions of presumptive pharmacological or non pharmacological neuroprotective agents, has been a relevant aspect in the approach to neuroprotection in experimental models of global cerebral ischemia (Pulsinelli et al., 1997; Barone & Feuerstein, 1999). In these, the beginning and the extent of this therapeutic window can be expected to be different according to the actions of neuroprotective procedures against immediate or late cellular mechanisms of brain damage, or in favor of later long-lasting cerebral processes of repair and plasticity.

Thus optimal neuroprotective effectiveness may require a schedule of drug administration in which drug actions are coincident with the therapeutic opportunity window, that have to be established for different drugs according to their specific mechanisms of action and pharmacokinetic characteristics. In this sense, counteracting of immediate cell mechanisms of neuronal damage may require the administration of neuroprotective drugs before the ischemic episode, though its administration has to be continued afterwards for variable periods. By contrast, drug-promoting repair or plasticity processes admit the starting of neuroprotective treatment hours or days after ischemia.

Accordingly, designs of neuroprotective studies in experimental animals in supporting proposals of neuroprotection for patients exposed to global cerebral ischemia due to cardiorespiratory arrest, should take in account that this clinical condition usually occurs unexpectedly, and requires cardiorespiratory resuscitation maneuvers; thus neuroprotection procedures have to be installed soon, but after the ischemic episode. Experimental designs of neuroprotection studies assessing neuroprotective procedures against late neuronal damage processes or promoting neuronal repair and plasticity, favoring functional preservation and recovery, may lead supporting to a wideness of the therapeutic opportunity window, for neuroprotection in human beings.

### **4.2.1 Prophylactic neuroprotection**

Transient global cerebral ischemia can occur during certain clinical situations which can either be anticipated, occur during intraoperative emergencies, or even induced, like extracorporeal circulation for cardiac surgery. Under these conditions, prophylactic neuroprotection as that provided by intraoperative hypothermia and pharmacological neuroprotection are possible alternatives to prevent or reduce the risk of ischemic neuronal damage (Savitz & Fisher, 2007; Weigl et al, 2005). This has stimulated designing of experimental studies on prophylactic neuroprotection to assess the effectiveness of several agents and their clinical potential. Some neuroprotective agents have proven to be more effective when applied before the ischemic insult than when given later in time, in particular those agents affecting the early cellular phenomena induced by ischemia, such as calcium channel blockers, GABAergic and anti-excitotoxic agents, as well as antioxidant drugs (Weigl et al, 2005). Pharmacological treatments (antihypertensive, antidiabetic, antithrombotic, antiatherogenic drugs) effective in modifying in the long term the risk for cardiac arrest or cardiac infarct which may result in global cerebral ischemia or in severe hypoperfusion have also been proposed as prophylactic neuroprotection procedures (Savitz & Fisher, 2007).

### **5. Conclusion**

322 Advances in the Preclinical Study of Ischemic Stroke

persisting for weeks after ischemia (Buffo et al., 2010; Choi JS et al, 2008; Mori et al, 2008; Morioka et al., 1991, 1992; Nikonenko et al., 2009; Petito & Halaby, 1993). The efficacy of neuroprotective agents can also be determined on the basis of the success in preventing the occurrence of necrosis, apoptosis, heat shock expression, gliosis, etc., as indicated by the immunohistochemical biomarkers (Scallet, 1995). Different parameters of the glial reaction elicited by global cerebral ischemia have been used as indexes of brain damage or neuroprotection (Cervantes et al., 2002; de Yebra et al., 2006; Duan et al., 2011; Korzhevskii

Neuronal cytoarchitecture and fine structure parameters of synaptic connectivity have also been used for histopathological assessment after brain damage and neuroprotection (Briones et al., 2006; García-Chávez et al., 2008; González-Burgos et al., 2007; Johansson & Belichenko, 2002; Kovalenko et al., 2006, Moralí et al., 2011a; Nikonenko et al., 2009; Ruan et al., 2006).

**4.2 Therapeutic opportunity window in animal models of global cerebral ischemia** In any case, recognition of a "therapeutic opportunity window" or "therapeutic time window" in relation to the timing of the ischemic episode, the temporal course of the mechanisms of brain damage and/or repair, and the exerting of actions of presumptive pharmacological or non pharmacological neuroprotective agents, has been a relevant aspect in the approach to neuroprotection in experimental models of global cerebral ischemia (Pulsinelli et al., 1997; Barone & Feuerstein, 1999). In these, the beginning and the extent of this therapeutic window can be expected to be different according to the actions of neuroprotective procedures against immediate or late cellular mechanisms of brain damage,

Thus optimal neuroprotective effectiveness may require a schedule of drug administration in which drug actions are coincident with the therapeutic opportunity window, that have to be established for different drugs according to their specific mechanisms of action and pharmacokinetic characteristics. In this sense, counteracting of immediate cell mechanisms of neuronal damage may require the administration of neuroprotective drugs before the ischemic episode, though its administration has to be continued afterwards for variable periods. By contrast, drug-promoting repair or plasticity processes admit the starting of

Accordingly, designs of neuroprotective studies in experimental animals in supporting proposals of neuroprotection for patients exposed to global cerebral ischemia due to cardiorespiratory arrest, should take in account that this clinical condition usually occurs unexpectedly, and requires cardiorespiratory resuscitation maneuvers; thus neuroprotection procedures have to be installed soon, but after the ischemic episode. Experimental designs of neuroprotection studies assessing neuroprotective procedures against late neuronal damage processes or promoting neuronal repair and plasticity, favoring functional preservation and recovery, may lead supporting to a wideness of the therapeutic

Transient global cerebral ischemia can occur during certain clinical situations which can either be anticipated, occur during intraoperative emergencies, or even induced, like extracorporeal circulation for cardiac surgery. Under these conditions, prophylactic neuroprotection as that provided by intraoperative hypothermia and pharmacological

or in favor of later long-lasting cerebral processes of repair and plasticity.

neuroprotective treatment hours or days after ischemia.

opportunity window, for neuroprotection in human beings.

**4.2.1 Prophylactic neuroprotection** 

et al., 2005; Piao et al., 2002; Soltys et al., 2003).

Though an increasing number of drugs have proven to be effective neuroprotective agents in experimental models of global cerebral ischemia, data supporting proposals for their clinical use have not been enough to influence clinical management and outcome of patients exposed to global cerebral ischemia in clinical trials. However, after its evaluation in animal models of global cerebral ischemia, special interest has been paid to carry out clinical trials with a non-pharmacological procedure, hypothermia, as a part of the intensive care of patients after a cardiorespiratory arrest. Nevertheless, the wide perspectives to gain information on neuroprotection through experimental designs including animal models of global cerebral ischemia are maintained to date, despite the tendency to preferentially conduct studies on rodents; in particular if differences between experimental animals and human beings are taken into account, and attention is paid to reproduce those components mainly accounting for brain damage after global cerebral ischemia.

### **6. Acknowledgement**

Partially supported by Instituto Mexicano del Seguro Social, MEXICO (2006/1A/I/029; FIS/IMSS/PROT/196).

### **7. References**


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**15** 

*Velasco Suárez* 

*México* 

**Nrf2 Activation, an Innovative Therapeutic** 

*Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía Manuel* 

Cerebrovascular disease is the second cause of death and the most frequent cause of nontraumatic disability in adults worldwide, according to the World Health Organization (WHO, 2005). Noteworthy, acute ischemic stroke accounts for about 85% of all cases (Diez-Tejedor et al., 2001). The most common cause of stroke is a sudden occlusion of a blood vessel, resulting in activation of a series of biochemical events eventually leading to neuronal death (Dirgnal et al., 1999). Although return of blood flow (reperfusion) in ischemic brain tissue is essential for restoring normal function, paradoxically it can result in a secondary damage, where oxidative stress mediators play a critical role (Wong & Crack,

Antioxidant therapies have been used to determine whether oxidative stress may constitute a valuable therapeutic target in cerebral ischemia. Indeed, free radical scavengers (direct antioxidants) and agents that decrease free radicals production reduce damage in experimental models of cerebral ischemia. Despite experimental evidence supports the concept that free radicals production represents a valuable therapeutic target in stroke, negative results have been obtained in a number of clinical trials when some direct antioxidant agents have been evaluated (Aguilera et al., 2007). At present, this discrepancy is unclear; however, administration of treatment outside the temporal window of efficacy and difficulties in the establishment of the onset of ischemia and reperfusion in humans (Hsu et al., 2000) are factors that likely contributing to these differences. Clearly, development of preclinical testing must consider these factors in order to improve

NF-E2-Related Factor-2 (Nrf2) is a transcription factor that play a crucial role in the cellular protection against oxidative stress. Nrf2 is referred to as the "master regulator" of the antioxidant response due to the fact that it modulates the expression of several genes including phase 2 and antioxidant enzymes playing an important role in detoxification of reactive oxygen species (ROS) and electrophilic species, including heme oxygenase-1, NAD(P)H:quinone oxidoreductase, glutathione-S-transferase, gamma-glutamyl cysteine ligase, glutathione reductase, etc. Recent studies demonstrate that dysfunction of Nrf2-

driven pathways impairs cellular redox state thus oxidative stress.

**1. Introduction** 

successful transition to clinical studies.

2008).

**Alternative in Cerebral Ischemia** 

Ana L. Colín-González and Perla D. Maldonado

Carlos Silva-Islas, Ricardo A. Santana,

Hippocampal Neuronal Death after Transient Global Cerebral Ischemia. *Neurobiology of Disease,* Vol.25, No.1, (September 2006), pp. 45-53, ISSN 0969-9961


### **Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia**

 Carlos Silva-Islas, Ricardo A. Santana, Ana L. Colín-González and Perla D. Maldonado *Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez México* 

### **1. Introduction**

346 Advances in the Preclinical Study of Ischemic Stroke

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Cerebrovascular disease is the second cause of death and the most frequent cause of nontraumatic disability in adults worldwide, according to the World Health Organization (WHO, 2005). Noteworthy, acute ischemic stroke accounts for about 85% of all cases (Diez-Tejedor et al., 2001). The most common cause of stroke is a sudden occlusion of a blood vessel, resulting in activation of a series of biochemical events eventually leading to neuronal death (Dirgnal et al., 1999). Although return of blood flow (reperfusion) in ischemic brain tissue is essential for restoring normal function, paradoxically it can result in a secondary damage, where oxidative stress mediators play a critical role (Wong & Crack, 2008).

Antioxidant therapies have been used to determine whether oxidative stress may constitute a valuable therapeutic target in cerebral ischemia. Indeed, free radical scavengers (direct antioxidants) and agents that decrease free radicals production reduce damage in experimental models of cerebral ischemia. Despite experimental evidence supports the concept that free radicals production represents a valuable therapeutic target in stroke, negative results have been obtained in a number of clinical trials when some direct antioxidant agents have been evaluated (Aguilera et al., 2007). At present, this discrepancy is unclear; however, administration of treatment outside the temporal window of efficacy and difficulties in the establishment of the onset of ischemia and reperfusion in humans (Hsu et al., 2000) are factors that likely contributing to these differences. Clearly, development of preclinical testing must consider these factors in order to improve successful transition to clinical studies.

NF-E2-Related Factor-2 (Nrf2) is a transcription factor that play a crucial role in the cellular protection against oxidative stress. Nrf2 is referred to as the "master regulator" of the antioxidant response due to the fact that it modulates the expression of several genes including phase 2 and antioxidant enzymes playing an important role in detoxification of reactive oxygen species (ROS) and electrophilic species, including heme oxygenase-1, NAD(P)H:quinone oxidoreductase, glutathione-S-transferase, gamma-glutamyl cysteine ligase, glutathione reductase, etc. Recent studies demonstrate that dysfunction of Nrf2 driven pathways impairs cellular redox state thus oxidative stress.

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 349

phosphorylation, proteolysis, and mitochondrial damage.

Fig. 1. Vascular obstruction of cerebral blood flow (ischemia) is accompanied by an

the infiltration of neutrophils and other cells (see below) (Chan, 2001).

viable for a variable period of time.

immediate drop in neurological activity ultimately leading to cell death (*infarct zone*). Infarct core is surrounded by an area supplied with oxygen and glucose by collateral blood vessels (*penumbra zone*). Cells from the penumbra area are not functional; however, they remain

4. *Generation of free radicals and oxidative stress.* Reactive oxygen (ROS) and nitrogen (RNS) species generation is increased during ischemia, but particularly during reperfusion, and they eventually lead to oxidative stress. ROS and RNS cause lipid peroxidation, membrane injury, disruption of cellular processes, and DNA damage. Moreover, oxidative stress contributes to the disruption of the blood-brain barrier, hence allowing

5. *Inflammation and apoptosis*. Cerebral injury is a potent triggering of inflammatory cytokines and proteases secretion by microglia, leukocytes and resident cells of the neurovascular unit. Once the neurovascular barriers are breached, multiple neuroinflammatory cascades are activated, further leading to secondary brain injury

Ca2+ levels. In turn, voltage gated Ca2+ channels together with reverse operation of the Na+/Ca2+ exchanger also increase intracellular Ca2+ levels (Figure 3). Once in the cytoplasmic domain, Ca2+ activates a variety of Ca2+ dependent enzymes, including protein kinase C, phospholipase A2, phospholipase C, cyclooxygenase-2, Ca2+ dependent nitric oxide synthase, proteases and endonucleases, hence triggering protein

Since ischemia and reperfusion insults generate an oxidative stress state, and considering that up to date there is no effective treatment to reverse morphological and behavioral alterations induced by stroke, it is conceivable that administration of antioxidants may limit oxidative damage and ameliorate progression of the disease. In this context, Nrf2 inducers are promising indirect antioxidant agents that are effective to attenuate oxidative stress and tissue/cell damage in different *in vivo* and *in vitro* experimental paradigms; therefore, here we review some compounds capable of inducing cellular antioxidant responses in order to understand their usefulness in prevention and treatment of cerebral ischemia-induced damage through activation of the Nrf2/ARE pathway.

### **2. Mechanism related to cerebral ischemic damage**

Brain tissue requires high and constant supply of oxygen and glucose provided for the vascular system to maintain its viability and normal functions. Vascular obstruction – either transitory or permanent - of cerebral blood flow (ischemia) is accompanied by an immediate drop in neurological activity ultimately leading to cell death. The brain is not affected homogeneously and so, cerebral ischemia generates differentially damaged areas. Complete loss of blood flow produces an *infarct zone* where necrotic cell death is observed. The infarct area is surrounded by a *penumbra zone*, which is located between the *infarct zone* and the non-damaged area, or normally irrigated tissue. Cells belonging to the *penumbra zone* are still irrigated by collateral arteries, which maintain them viable for a variable period of time, although not functional (Figure 1). This is the area that shall be rescued, and the potential target for intervention with neuroprotective treatments (Dirgnal et al., 1999).

The return of blood flow (reperfusion) is associated with a decrease in the infarct size and clinical outcome. Although reperfusion is determinant for cell function recovery, after prolonged periods of ischemia, it also exerts negative side-effects. If blood flow is not restored within hours, the penumbra region will become part of the infarct zone. In some patients, reperfusion may exacerbate brain injury (*e.g.,* some patients show edema or intracranial hemorrhage) (Kuroda & Siesjo, 1997). In animal models, reperfusion can induce larger infarct areas that can be associated with permanent vessel occlusion (Aronowski et al., 1997).

The reduction and return of blood flow triggers a cascade of events further leading to neuronal death (Dirgnal et al., 1999; Durukan & Tatlisumak, 2007). Such sequence includes:


Since ischemia and reperfusion insults generate an oxidative stress state, and considering that up to date there is no effective treatment to reverse morphological and behavioral alterations induced by stroke, it is conceivable that administration of antioxidants may limit oxidative damage and ameliorate progression of the disease. In this context, Nrf2 inducers are promising indirect antioxidant agents that are effective to attenuate oxidative stress and tissue/cell damage in different *in vivo* and *in vitro* experimental paradigms; therefore, here we review some compounds capable of inducing cellular antioxidant responses in order to understand their usefulness in prevention and treatment of cerebral ischemia-induced

Brain tissue requires high and constant supply of oxygen and glucose provided for the vascular system to maintain its viability and normal functions. Vascular obstruction – either transitory or permanent - of cerebral blood flow (ischemia) is accompanied by an immediate drop in neurological activity ultimately leading to cell death. The brain is not affected homogeneously and so, cerebral ischemia generates differentially damaged areas. Complete loss of blood flow produces an *infarct zone* where necrotic cell death is observed. The infarct area is surrounded by a *penumbra zone*, which is located between the *infarct zone* and the non-damaged area, or normally irrigated tissue. Cells belonging to the *penumbra zone* are still irrigated by collateral arteries, which maintain them viable for a variable period of time, although not functional (Figure 1). This is the area that shall be rescued, and the potential target for intervention with neuroprotective treatments

The return of blood flow (reperfusion) is associated with a decrease in the infarct size and clinical outcome. Although reperfusion is determinant for cell function recovery, after prolonged periods of ischemia, it also exerts negative side-effects. If blood flow is not restored within hours, the penumbra region will become part of the infarct zone. In some patients, reperfusion may exacerbate brain injury (*e.g.,* some patients show edema or intracranial hemorrhage) (Kuroda & Siesjo, 1997). In animal models, reperfusion can induce larger infarct areas that can be associated with permanent vessel occlusion (Aronowski et al.,

The reduction and return of blood flow triggers a cascade of events further leading to neuronal death (Dirgnal et al., 1999; Durukan & Tatlisumak, 2007). Such sequence

1. *Energy failure.* This is the first event of the ischemic cascade. Cells need oxygen and glucose to undergo oxidative phosphorylation for energy production, consequently

2. *Depolarization of membrane*. The impairment of ATP production disrupts Na+/K+- ATPase and Ca2+/H+-ATPase pumps and reverses the Na+/Ca2+-transporter. Upon these conditions, cells are unable to maintain membrane potential and Ca2+ voltagedependent channels are activated, leading to depolarization of cellular membrane

3. *Excitotoxicity and increase in intracellular Ca2+ levels*. After depolarization, excitotoxic amino acids - mostly glutamate - are released to the synaptic cleft. Glutamate activates N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and metabotropic glutamate receptors, thereby increasing intracellular

during ischemia ATP production is decreased (Figure 2).

damage through activation of the Nrf2/ARE pathway.

(Dirgnal et al., 1999).

1997).

includes:

(Figure 3).

**2. Mechanism related to cerebral ischemic damage** 

Ca2+ levels. In turn, voltage gated Ca2+ channels together with reverse operation of the Na+/Ca2+ exchanger also increase intracellular Ca2+ levels (Figure 3). Once in the cytoplasmic domain, Ca2+ activates a variety of Ca2+ dependent enzymes, including protein kinase C, phospholipase A2, phospholipase C, cyclooxygenase-2, Ca2+ dependent nitric oxide synthase, proteases and endonucleases, hence triggering protein phosphorylation, proteolysis, and mitochondrial damage.

Fig. 1. Vascular obstruction of cerebral blood flow (ischemia) is accompanied by an immediate drop in neurological activity ultimately leading to cell death (*infarct zone*). Infarct core is surrounded by an area supplied with oxygen and glucose by collateral blood vessels (*penumbra zone*). Cells from the penumbra area are not functional; however, they remain viable for a variable period of time.


Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 351

**ISCHEMIA** 

Fig. 3. Reduction of blood flow decreases ATP production, disrupts ATP-dependent pumps () and reverses the Na+/Ca2+ transporter (). Upon these conditions, cells are unable to maintain membrane potential *(Depolarization of membrane)*. After depolarization, glutamate (GLUT) is released and activates N-methyl-D-aspartic acid (NMDAr) and α-amino-3 hydroxy-5-methylisoxazole-4-propionic acid (AMPAr) receptors*(*, *Excitotoxicity)*, hence directly increasing intracellular Ca2+ levels (). On one hand, GLUT activates metabotropic

ROS and RNS produce cellular damage through lipid peroxidation, nucleic acid alteration and inactivation of enzymes (Figure 4); they also modify cellular signaling and gene regulation, contributing to breakdown of the blood-brain barrier and edema generation (Moro et al., 2005). Oxidative stress can ultimately induce neuronal damage, leading to

The brain is particularly sensitive to oxidative stress since 20% of the total oxygen consumed by the body is used by this organ, which constitutes only 2% of the total body weight. This

glutamate receptors (mGLUr) (), which releases inositol 1,4,5-triphosphate (IP3), a molecule that binds to its receptor at the endoplasmatic reticulum to release more Ca2+ (, *Increase of intracellular Ca2+ level*). On the other hand, voltage gated Ca2+ channels (VDCC) and the reverse operation of the Na+/Ca2+-exchanger increase intracellular Ca2+ levels. Energy disruption also affects astrocytes, causing a deficient activity of glutamate

transporters (EAAT1 and EAAT2) ().

neuronal death by apoptosis or necrosis (Loh et al., 2006).

(Danton & Dietrich, 2003). Post-ischemic inflammation contributes to brain injury and has been linked to apoptosis. Cell death in cerebral ischemia is mainly dependent of the localization of the cells. For instance, in the core region, cell death is caused mainly by necrosis, while apoptosis predominates in the penumbra area.

Fig. 2. The reduction of blood flow decreases oxygen and glucose levels; consequently, ATP production *(Energy failure)* (), glycolysis() and ATP-dependent processes are blocked. Upon these conditions, oxidative damage is generated by residual oxygen in mitochondria. Pathways that are inhibited during ischemia are crossed out in the image. TCA cycle, tricarboxylic acid cycle; nNOS, neuronal nitric oxide synthase.

### **3. Oxidative stress is one of the most important events in ischemia/reperfusion-induced cerebral damage**

In cells, the predominant ROS and RNS produced are superoxide anion (O2– ), hydrogen peroxide (H2O2), hydroxyl radical ( OH), nitric oxide ( NO), peroxynitrite anion (ONOO–), and nitrogen dioxide ( NO2). In normal conditions, natural defense against ROS and RNS is provided by antioxidant molecules such as glutathione (GSH), ascorbic acid, α-tocopherol, and a number of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). SOD converts O2 – to H2O2, whereas GPx and CAT convert H2O2 to H2O. However, an imbalance in the formation and clearance of ROS and RNS can lead to oxidative stress and subsequent changes affecting the cell dynamics (Aguilera et al., 2007; Margaill et al., 2005).

**ISCHEMIA** 

Fig. 2. The reduction of blood flow decreases oxygen and glucose levels; consequently, ATP production *(Energy failure)* (), glycolysis() and ATP-dependent processes are blocked. Upon these conditions, oxidative damage is generated by residual oxygen in mitochondria. Pathways that are inhibited during ischemia are crossed out in the image. TCA cycle,

OH), nitric oxide (

provided by antioxidant molecules such as glutathione (GSH), ascorbic acid, α-tocopherol, and a number of antioxidant enzymes, including superoxide dismutase (SOD), glutathione

convert H2O2 to H2O. However, an imbalance in the formation and clearance of ROS and RNS can lead to oxidative stress and subsequent changes affecting the cell dynamics

NO2). In normal conditions, natural defense against ROS and RNS is

), hydrogen

NO), peroxynitrite anion (ONOO–),

to H2O2, whereas GPx and CAT

tricarboxylic acid cycle; nNOS, neuronal nitric oxide synthase.

**ischemia/reperfusion-induced cerebral damage** 

peroxidase (GPx), and catalase (CAT). SOD converts O2–

peroxide (H2O2), hydroxyl radical (

(Aguilera et al., 2007; Margaill et al., 2005).

and nitrogen dioxide (

**3. Oxidative stress is one of the most important events in** 

In cells, the predominant ROS and RNS produced are superoxide anion (O2–

necrosis, while apoptosis predominates in the penumbra area.

(Danton & Dietrich, 2003). Post-ischemic inflammation contributes to brain injury and has been linked to apoptosis. Cell death in cerebral ischemia is mainly dependent of the localization of the cells. For instance, in the core region, cell death is caused mainly by

Fig. 3. Reduction of blood flow decreases ATP production, disrupts ATP-dependent pumps () and reverses the Na+/Ca2+ transporter (). Upon these conditions, cells are unable to maintain membrane potential *(Depolarization of membrane)*. After depolarization, glutamate (GLUT) is released and activates N-methyl-D-aspartic acid (NMDAr) and α-amino-3 hydroxy-5-methylisoxazole-4-propionic acid (AMPAr) receptors*(*, *Excitotoxicity)*, hence directly increasing intracellular Ca2+ levels (). On one hand, GLUT activates metabotropic glutamate receptors (mGLUr) (), which releases inositol 1,4,5-triphosphate (IP3), a molecule that binds to its receptor at the endoplasmatic reticulum to release more Ca2+ (, *Increase of intracellular Ca2+ level*). On the other hand, voltage gated Ca2+ channels (VDCC) and the reverse operation of the Na+/Ca2+-exchanger increase intracellular Ca2+ levels. Energy disruption also affects astrocytes, causing a deficient activity of glutamate transporters (EAAT1 and EAAT2) ().

ROS and RNS produce cellular damage through lipid peroxidation, nucleic acid alteration and inactivation of enzymes (Figure 4); they also modify cellular signaling and gene regulation, contributing to breakdown of the blood-brain barrier and edema generation (Moro et al., 2005). Oxidative stress can ultimately induce neuronal damage, leading to neuronal death by apoptosis or necrosis (Loh et al., 2006).

The brain is particularly sensitive to oxidative stress since 20% of the total oxygen consumed by the body is used by this organ, which constitutes only 2% of the total body weight. This

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 353

**REPERFUSION** 

–

follows: mitochondrial respiratory chain ; cyclooxygenase-2 (COX-2) ; NADPH oxidase (NOX) ; xanthine oxidase (XO) ; and nitric oxide synthase (NOS), responsible for nitric

NO to generate peroxynytrite anion (ONOO–) , or be degraded by superoxide

However, the distinction between direct and indirect antioxidants is complicated by a close reciprocal relation between these two types of agents, as is showed in the following

1. Whilst glutathione is the main protective direct antioxidant present in high concentrations (mM) in tissues, its rate of synthesis is controlled by -glutamate cysteine ligase (GCL), a typical phase 2 enzyme that is upregulated by phase 2 inducers which are, by definition, indirect antioxidants. The complexity of this reciprocal relation is further enhanced by the mandatory participation of glutathione in activities of several antioxidant enzymes (glutathione peroxidase, glutathione-S-transferases, glutathione

2. At least one phase 2 enzyme, heme oxygenase-1 (HO-1) generates carbon monoxide and

3. Some direct antioxidants are inducers of the phase 2 response; e.g., the vicinal dithiol

lipoic acid and reduced Michale reaction acceptors such as hydroquinones.

biliverdin/biliruvin, which are small direct antioxidant molecules.

dismutase (SOD) to hydrogen peroxide (H2O2) . Then, H2O2 can be catabolized by glutathione peroxidase (GPx) or catalase (CAT) to H2O , or react with Fe2+ to form

) during reperfusion are summarized as

can react

if tetrahydrobiopterin (BH4) is deficient . O2–

OH (**11**), responsible for damaging lipids, proteins and DNA.

OH) via the Fenton reaction . ONOO– can be degraded to nitrogen

Fig. 4. Main sources of superoxide anion (O2

NO) formation , or O2–

 ) and

examples (Dinkova-Kostova et al., 2007):

oxide (

hydroxyl radicals (

dioxide radical (NO2

reductase).

with

feature makes the brain the major generator of ROS and RNS when compared with other organs (Dringen, 2000). Moreover, in brain there are numerous conditions favoring ROS and RNS production, including: 1) a high unsaturated lipid content, 2) chemical reactions involving dopamine oxidation (Heiss, 2002; Hou & MacManus, 2002), 3) high concentrations of iron in various regions, and 4) lower antioxidant systems than other organs such as kidney or liver (Dringen, 2000).

As previously described, physiopathological mechanisms leading to neuronal injury in cerebral stroke are complex and multifactorial. However, several studies suggest that oxidative stress, secondary to ROS and RNS production, actively participates during postischemic brain damage (Peters et al., 1998; Rodrigo et al., 2005). During ischemia, free radical production in the infarct zone decreases or remains without change, while it increases during reperfusion. However, free radical production in the penumbral zone increases during both events (Liu et al., 2003). Despite the low oxygen tension produced during ischemia, exist an increase in ROS formation after 1.6 h of ischemia, the highest ROS production (489 ± 330% of control) occurs after 20 min of reperfusion, and remains increased at least for 3 h (Peters et al., 1998). Christensen et al. (1994) reported that ROS production is maximal during the first hour of reperfusion.

Main sources of ROS, RNS, and free radicals during reperfusion are summarized as follows (Aguilera et al., 2007; Margaill et al., 2005):


### **4. Direct and indirect antioxidants**

Living systems have developed multiple lines of defense against oxidative stress. Cellular protection against oxidative stress is a process more complex than cellular protection against electrophiles. In this process two types of molecules participate (Dinkova-Kostova et al., 2007):


feature makes the brain the major generator of ROS and RNS when compared with other organs (Dringen, 2000). Moreover, in brain there are numerous conditions favoring ROS and RNS production, including: 1) a high unsaturated lipid content, 2) chemical reactions involving dopamine oxidation (Heiss, 2002; Hou & MacManus, 2002), 3) high concentrations of iron in various regions, and 4) lower antioxidant systems than other organs such as

As previously described, physiopathological mechanisms leading to neuronal injury in cerebral stroke are complex and multifactorial. However, several studies suggest that oxidative stress, secondary to ROS and RNS production, actively participates during postischemic brain damage (Peters et al., 1998; Rodrigo et al., 2005). During ischemia, free radical production in the infarct zone decreases or remains without change, while it increases during reperfusion. However, free radical production in the penumbral zone increases during both events (Liu et al., 2003). Despite the low oxygen tension produced during ischemia, exist an increase in ROS formation after 1.6 h of ischemia, the highest ROS production (489 ± 330% of control) occurs after 20 min of reperfusion, and remains increased at least for 3 h (Peters et al., 1998). Christensen et al. (1994) reported that ROS production is

Main sources of ROS, RNS, and free radicals during reperfusion are summarized as follows

– .

an important regulator of NOS function because it is required to maintain enzymatic coupling. Loss or oxidation of BH4 to 7,8-dihydrobiopterin (BH2) is associated with

Living systems have developed multiple lines of defense against oxidative stress. Cellular protection against oxidative stress is a process more complex than cellular protection against electrophiles. In this process two types of molecules participate (Dinkova-Kostova et al., 2007): 1. *Direct antioxidants*. Compounds of low molecular weight (ascorbate, glutathione, tocopherols, lipoid acid, ubiquinones, carotenes) that can undergo redox reactions and

). Direct antioxidants are consumed or modified in the process of their antioxidant action

2. *Indirect antioxidants*. These agents may or may not have redox activity, and exert many of their effects through upregulation of phase 2 and antioxidant enzymes. In turn, theses enzymes act catalytically, exhibit long half-lives, and display a wide variety of

scavenge reactive oxidation products (peroxides), as well as ROS and RNS (

antioxidant activities, in addition to their capacities to detoxify electrophiles.

(ROS scavenger). Thus, it is necessary to replenish or regenerate them.

during NADPH oxidation.

and generate the strong oxidant ONOO–. Tetrahydrobiopterin (BH4) is

NO in normal conditions.

when it catalyzes oxidation of hypoxhantine to uric

during oxidative metabolism of arachidonic

rather than

NO produced can

NO (Crabtree &

OH, ONOO–

kidney or liver (Dringen, 2000).

maximal during the first hour of reperfusion.

3. Cyclooxygenase 2 (COX-2) produces O2–

4. NADPH oxidase (NOX) produces O2–

5. Nitric oxide synthases (NOS) produce

acid, a delayed process in ischemia reperfusion.

NOS uncoupling, resulting in the production of O2–

(Aguilera et al., 2007; Margaill et al., 2005): 1. Mitochondrial respiratory chain generates O2

2. Xanthine oxidase produces O2–

Channon, 2011) (Figure 4).

**4. Direct and indirect antioxidants** 

acid.

react with O2–

Fig. 4. Main sources of superoxide anion (O2– ) during reperfusion are summarized as follows: mitochondrial respiratory chain ; cyclooxygenase-2 (COX-2) ; NADPH oxidase (NOX) ; xanthine oxidase (XO) ; and nitric oxide synthase (NOS), responsible for nitric oxide ( NO) formation , or O2– if tetrahydrobiopterin (BH4) is deficient . O2– can react with NO to generate peroxynytrite anion (ONOO–) , or be degraded by superoxide dismutase (SOD) to hydrogen peroxide (H2O2) . Then, H2O2 can be catabolized by glutathione peroxidase (GPx) or catalase (CAT) to H2O , or react with Fe2+ to form hydroxyl radicals ( OH) via the Fenton reaction . ONOO– can be degraded to nitrogen dioxide radical (NO2 ) and OH (**11**), responsible for damaging lipids, proteins and DNA.

However, the distinction between direct and indirect antioxidants is complicated by a close reciprocal relation between these two types of agents, as is showed in the following examples (Dinkova-Kostova et al., 2007):


Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 355

at low basal levels, but can be markedly elevated by various small molecules (indirect

Using an oligonucleotide microarray analysis, Lee et al. (2003a) reported that *tert*butylhydroquinone (t-BHQ), a well know Nrf2 inducer, stimulated a group of genes responsible for conferring protection against oxidative stress or inflammation in primary cortical astrocytes. The major functional categories are detoxification enzymes, antioxidant proteins, NADPH-producing proteins, growth factors, defense/immune/inflammationrelated proteins, and signaling proteins (Table 1). It has been proposed that proteins within these functional categories are vital to cell's defense system, suggesting that an orchestrated change in the modulation of Nrf2/ARE pathway would stimulate a synergistic protective

Proteins and enzymes directly related with an antioxidant protective effect can be divided

*Group 1.* Genes involved in glutathione (GSH) homeostasis. GSTs catalyze the nucleophilic addition of GSH to an electrophilic group of a broad spectrum of xenobiotic compounds. GPx and PRx metabolize H2O2 to H2O and oxidized GSH (GSSG), and GR regenerates GSH. Ideally, in association with an increased utilization of GSH, there would also be an increased production of GSH. The rate-limiting step in the GSH biosynthesis is mediated by GCLM/GCLC. The coordinate regulation of these genes can evoke a synergistic effect in the maintenance of GSH levels, as well as in detoxification of reactive intermediates

*Group 2.* Genes involved in H2O2 detoxification and iron homeostasis. SOD and HO-1 are

H2O2, and HO-1 generates a potent radical scavenger, bilirubin. However, SOD and HO-1 can induce more oxidative stress because they increase the cellular concentrations of H2O2 and free iron, respectively; which together can generate OH through the Fenton reaction. For complete detoxification of superoxide, H2O2 should be further metabolized to H2O by GPx, CAT, or PRx. CAT directly detoxifies H2O2, whereas PRx uses GSH (Figure 6) and/or thioredoxin (Trx) as an electron donor for peroxidation of H2O2, resulting in generation of GSSG or oxidized thioredoxin, respectively (Figure 6). GSSG and oxidized thioredoxin are converted to their reduced forms by GR and TXNRD1, respectively. In addition, proper management of free iron is also important for minimizing oxidative stress, and this can be best achieved by ferritin. Ferritin converts Fe2+ to Fe3+ (ferroxidase activity) and sequesters it, thereby avoiding the participation of Fe2+ in the Fenton reaction (Orino et al., 2001). Thus, up-regulation of HO-1 together with ferritin constitutes a physiological strategy to increase

OH formation is minimized. *Group 3.* Genes involved in NADPH homeostasis. NQO1, GR, and TXNRD1 are important in detoxifying quinones and maintaining the cellular redox balance. One common feature of these proteins is the fact that they use NADPH as an electron donor. So, for efficient detoxification and maintenance of cellular redox status, it would be beneficial to up-regulate these proteins together with the appropriate reducing potential (NADPH) to support enzymatic reactions. G6PD/malic enzyme can directly generate NADPH, and transketolase/transaldolase can increase NADPH production by regenerating substrates for G6PD (Figure 7). These Nrf2-dependent genes would also contribute to cell's detoxification

resulting

very important for cellular defense against oxidative stress. SOD detoxifies O2–

antioxidants).

effect.

(Figure 5).

into 3 major groups (Lee et al., 2003a):

the antioxidant potential while

potential and cellular redox balance.

4. Phase 2 enzymes NADPH:quinone oxidoreductase-1 (NQO1) and glutathione reductase are responsible for regeneration of reduced and active forms of oxidized tocopherols, and ubiquinone and glutathione, respectively.

### **5. Indirect antioxidants induce a cytoprotective phase 2 response**

Aerobic cells have developed an elaborated mechanism for their protection against oxidative stress, known as "phase 2 response" (Dinkova-Kostova & Talaly, 2008; Kensler et al., 2007; Kobayashi & Yamamoto, 2006; Motohashi & Yamamoto, 2004). Phase 2 response involves a group of genes that are regulated by a common molecular signaling pathway depending of the transcription factor Nrf2, and can be coordinately induced by a variety of synthetic and natural agents (Dinkova-Kostova et al., 2005a; Talalay, 2000). Extensive studies on chemistry of inducers have disclosed that all are chemically reactive without having common structural features (Dinkova-Kostova et al., 2004), and all react with sulfhydryl groups (Dinkova-Kostova et al., 2001) of highly reactive cysteine residues of Keap1, the cellular sensor that is integrally involved in the mechanism of induction (Itoh et al., 2003; Wakabayashi et al., 2004). The known inducers belong to at least nine chemical classes (Dinkova-Kostova et al., 2004): (i) diphenols, phenylenediamines and quinones; (ii) Michael reaction acceptors; (iii) isothiocyanates/dithiocarbamates; (iv) 1,2-dithiole-3 thiones/oxathiolene oxides; (v) hydroperoxides; (vi) trivalent arsenicals; (vii) heavy metals; (viii) vicinal dimercaptans; and (ix) carotenoids.

It is now widely recognized that the up-regulation of the phase 2 response is a powerful, highly efficient and promising strategy for protection against several diseases including ischemic stroke (Alfieri et al., 2011; Talalay, 2000). Experimental evidence shows the powerful protective effects of phase 2 response: (*i*) its up-regulation protects cells, animals, and humans against a wide variety of damaging agents including ROS, RNS, carcinogens, electrophiles, and radiation (Kensler et al., 2007; Kobayashi & Yamamoto, 2006; Motohashi & Yamamoto, 2004; Talalay et al., 2007); (*ii*) when the phase 2 response is disrupted, cells are much more susceptible to oxidative damage; and (*iii*) numerous anticarcinogens have been identified and isolated from natural sources by bioassays that monitor induction of Nrf2 dependent enzymes such as NAD(P)H:quinone oxidoreductase (NQO1) (Kang & Pezzuto, 2004; Zhang et al., 1992).

### **5.1 Phase 2 proteins and enzymes**

In the past, enzymatic protection against oxidants focused largely on classical enzymes such as SOD, CAT, and various types of peroxidases (Halliwell & Gutteridge, 1999), now this is changing. Phase 2 proteins were originally perceived as only promoters of xenobiotic conjugation with endogenous ligands (e.g., glutathione, glucuronic acid) to generate more water-soluble and easily excretable products. This restricted view of the nature and functions of phase 2 proteins and enzymes has gradually been expanded. Nowadays, several genes are considered part of the phase 2 response. Enzymes encoded by these genes have chemically versatile antioxidant properties, share common regulatory mechanisms, and are highly inducible by a variety of agents including dietary components (Ramos-Gomez et al., 2001; Talalay, 2000).

Phase 2 proteins catalyze diverse reactions that collectively result in broad protection against the continuous damaging effects of ROS, RNS and electrophiles. They are expressed

4. Phase 2 enzymes NADPH:quinone oxidoreductase-1 (NQO1) and glutathione reductase are responsible for regeneration of reduced and active forms of oxidized tocopherols,

Aerobic cells have developed an elaborated mechanism for their protection against oxidative stress, known as "phase 2 response" (Dinkova-Kostova & Talaly, 2008; Kensler et al., 2007; Kobayashi & Yamamoto, 2006; Motohashi & Yamamoto, 2004). Phase 2 response involves a group of genes that are regulated by a common molecular signaling pathway depending of the transcription factor Nrf2, and can be coordinately induced by a variety of synthetic and natural agents (Dinkova-Kostova et al., 2005a; Talalay, 2000). Extensive studies on chemistry of inducers have disclosed that all are chemically reactive without having common structural features (Dinkova-Kostova et al., 2004), and all react with sulfhydryl groups (Dinkova-Kostova et al., 2001) of highly reactive cysteine residues of Keap1, the cellular sensor that is integrally involved in the mechanism of induction (Itoh et al., 2003; Wakabayashi et al., 2004). The known inducers belong to at least nine chemical classes (Dinkova-Kostova et al., 2004): (i) diphenols, phenylenediamines and quinones; (ii) Michael reaction acceptors; (iii) isothiocyanates/dithiocarbamates; (iv) 1,2-dithiole-3 thiones/oxathiolene oxides; (v) hydroperoxides; (vi) trivalent arsenicals; (vii) heavy metals;

It is now widely recognized that the up-regulation of the phase 2 response is a powerful, highly efficient and promising strategy for protection against several diseases including ischemic stroke (Alfieri et al., 2011; Talalay, 2000). Experimental evidence shows the powerful protective effects of phase 2 response: (*i*) its up-regulation protects cells, animals, and humans against a wide variety of damaging agents including ROS, RNS, carcinogens, electrophiles, and radiation (Kensler et al., 2007; Kobayashi & Yamamoto, 2006; Motohashi & Yamamoto, 2004; Talalay et al., 2007); (*ii*) when the phase 2 response is disrupted, cells are much more susceptible to oxidative damage; and (*iii*) numerous anticarcinogens have been identified and isolated from natural sources by bioassays that monitor induction of Nrf2 dependent enzymes such as NAD(P)H:quinone oxidoreductase (NQO1) (Kang & Pezzuto,

In the past, enzymatic protection against oxidants focused largely on classical enzymes such as SOD, CAT, and various types of peroxidases (Halliwell & Gutteridge, 1999), now this is changing. Phase 2 proteins were originally perceived as only promoters of xenobiotic conjugation with endogenous ligands (e.g., glutathione, glucuronic acid) to generate more water-soluble and easily excretable products. This restricted view of the nature and functions of phase 2 proteins and enzymes has gradually been expanded. Nowadays, several genes are considered part of the phase 2 response. Enzymes encoded by these genes have chemically versatile antioxidant properties, share common regulatory mechanisms, and are highly inducible by a variety of agents including dietary components (Ramos-

Phase 2 proteins catalyze diverse reactions that collectively result in broad protection against the continuous damaging effects of ROS, RNS and electrophiles. They are expressed

**5. Indirect antioxidants induce a cytoprotective phase 2 response** 

and ubiquinone and glutathione, respectively.

(viii) vicinal dimercaptans; and (ix) carotenoids.

2004; Zhang et al., 1992).

**5.1 Phase 2 proteins and enzymes** 

Gomez et al., 2001; Talalay, 2000).

at low basal levels, but can be markedly elevated by various small molecules (indirect antioxidants).

Using an oligonucleotide microarray analysis, Lee et al. (2003a) reported that *tert*butylhydroquinone (t-BHQ), a well know Nrf2 inducer, stimulated a group of genes responsible for conferring protection against oxidative stress or inflammation in primary cortical astrocytes. The major functional categories are detoxification enzymes, antioxidant proteins, NADPH-producing proteins, growth factors, defense/immune/inflammationrelated proteins, and signaling proteins (Table 1). It has been proposed that proteins within these functional categories are vital to cell's defense system, suggesting that an orchestrated change in the modulation of Nrf2/ARE pathway would stimulate a synergistic protective effect.

Proteins and enzymes directly related with an antioxidant protective effect can be divided into 3 major groups (Lee et al., 2003a):

*Group 1.* Genes involved in glutathione (GSH) homeostasis. GSTs catalyze the nucleophilic addition of GSH to an electrophilic group of a broad spectrum of xenobiotic compounds. GPx and PRx metabolize H2O2 to H2O and oxidized GSH (GSSG), and GR regenerates GSH. Ideally, in association with an increased utilization of GSH, there would also be an increased production of GSH. The rate-limiting step in the GSH biosynthesis is mediated by GCLM/GCLC. The coordinate regulation of these genes can evoke a synergistic effect in the maintenance of GSH levels, as well as in detoxification of reactive intermediates (Figure 5).

*Group 2.* Genes involved in H2O2 detoxification and iron homeostasis. SOD and HO-1 are very important for cellular defense against oxidative stress. SOD detoxifies O2– resulting H2O2, and HO-1 generates a potent radical scavenger, bilirubin. However, SOD and HO-1 can induce more oxidative stress because they increase the cellular concentrations of H2O2 and free iron, respectively; which together can generate OH through the Fenton reaction. For complete detoxification of superoxide, H2O2 should be further metabolized to H2O by GPx, CAT, or PRx. CAT directly detoxifies H2O2, whereas PRx uses GSH (Figure 6) and/or thioredoxin (Trx) as an electron donor for peroxidation of H2O2, resulting in generation of GSSG or oxidized thioredoxin, respectively (Figure 6). GSSG and oxidized thioredoxin are converted to their reduced forms by GR and TXNRD1, respectively. In addition, proper management of free iron is also important for minimizing oxidative stress, and this can be best achieved by ferritin. Ferritin converts Fe2+ to Fe3+ (ferroxidase activity) and sequesters it, thereby avoiding the participation of Fe2+ in the Fenton reaction (Orino et al., 2001). Thus, up-regulation of HO-1 together with ferritin constitutes a physiological strategy to increase the antioxidant potential while OH formation is minimized.

*Group 3.* Genes involved in NADPH homeostasis. NQO1, GR, and TXNRD1 are important in detoxifying quinones and maintaining the cellular redox balance. One common feature of these proteins is the fact that they use NADPH as an electron donor. So, for efficient detoxification and maintenance of cellular redox status, it would be beneficial to up-regulate these proteins together with the appropriate reducing potential (NADPH) to support enzymatic reactions. G6PD/malic enzyme can directly generate NADPH, and transketolase/transaldolase can increase NADPH production by regenerating substrates for G6PD (Figure 7). These Nrf2-dependent genes would also contribute to cell's detoxification potential and cellular redox balance.

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 357

Fig. 5. Genes involved in glutathione (GSH) homeostasis are indicated in black boxes. GST, glutathione-S-transferase; GCLM, -glutamate cysteine ligase modifier subunit; GCLC, -glutamate cysteine ligase catalytic subunit; GPx, glutathione peroxidase; PRx,

Fig. 6. Genes involved in H2O2 detoxification and iron homeostasis are indicated in black boxes. SOD, superoxide dismutase; CAT, catalase; PRx, peroxiredoxin; Trx, thioredoxin;

HO-1, hemo oxygenase-1; TXNRD1, thioredoxin reductase-1.

peroxiredoxin; GR, glutathione reductase.


aKnown to contain or to potentially have an ARE sequence. Modified of Lee et al., 2003a.

Table 1. Nrf2-dependent genes induced by *tert*-butylhydroquinone in primary cortical astrocytes

 -glutamate cysteine ligase modifier subunit (GCLM)a -glutamate cysteine ligase catalytic subunit (GCLC)a Hemo oxygenase-1 (HO-1) (decycling)a

*Transcription*

4

*Defense/immune/ inflammation* 

> lectin EST, similar to dithiolethioneinducible-1 PAF acetylhydrolase

 P lysozyme structural Lysozyme M Prostaglandinendoperoxide synthase-2

Matrix

12

metalloproteinase-

 Proliferin Proliferin-2 Nerve growth factor- β Platelet-derived growth factor-α

*Growth* 

 CCAAT/enhancerbinding protein-β Zinc finger protein of cerebellum-2 TG-interacting factor MafG Activating

transcription factor-

Macrophage C-type

*Antioxidant/reducing*

 Thioredoxin reductase-1 (TXNRD-1) Thioredoxin (Trx)a Ferritin light chain-

Ferritin H subunita

peroxiredoxin (PRx) 1-Cys PRx protein-2 Transferrin receptor Cu, Zn superoxide dismutase (CuZnSOD)a Catalase-1 (CAT) Glutathione

peroxidase-4 (GPx)

reductase-1 (GR)

dehydrogenase (G-6PD), X-linked

Glutathione

 Glucose-6 phosphate

 G-6PDH-2 Transaldolase-1 Transketolase Solute carrier family-1/4 Glycine transporter- Malic enzyme, supernatanta

Table 1. Nrf2-dependent genes induced by *tert*-butylhydroquinone in primary cortical

1a

Type I

**GENE GENE GENE** 

*potential* 

*Detoxification* 

 NAD(P)H:quinone oxidoreductase-1

transferase (GST) A4a

glycosyltransferase

Epoxide hydrolase-1a

dehydrogenase-2

dehydrogenase-9 Aldehyde oxidase-1 Cytochrome P450 1B1

cAMP-dependent regulatory, type Iβ AW125016 4 1.9 0.07

 Mitogen-activated protein kinase-10

aKnown to contain or to potentially have an ARE sequence.

(NQO1)a Glutathione-S-

 GST Pi2a GST Mu1a GST Mu3a GST Omega1a GST microsomal-1a

UDP

1A6a

Aldehyde

Aldehyde

Protein kinase,

NR

Modified of Lee et al., 2003a.

astrocytes

*Signaling* 

Fig. 5. Genes involved in glutathione (GSH) homeostasis are indicated in black boxes. GST, glutathione-S-transferase; GCLM, -glutamate cysteine ligase modifier subunit; GCLC, -glutamate cysteine ligase catalytic subunit; GPx, glutathione peroxidase; PRx, peroxiredoxin; GR, glutathione reductase.

Fig. 6. Genes involved in H2O2 detoxification and iron homeostasis are indicated in black boxes. SOD, superoxide dismutase; CAT, catalase; PRx, peroxiredoxin; Trx, thioredoxin; HO-1, hemo oxygenase-1; TXNRD1, thioredoxin reductase-1.

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 359

degradation (McMahon et al., 2004). Neh3 domain is required for transcriptional activation of the protein (Nioi et al., 2005). Neh4 and Neh5 domains are required for its binding to ARE

Fig. 8. Nrf2 and Keap1 domains. *Upper panel*: in Nrf2, Neh1 is the basic DNA binding

(kelch-like ECH-associated protein 1) binding domain. Neh3 is required for transcriptional activation of the protein. Neh4 and Neh5 domains are required for the binding to ARE. Neh6 is essential for both Nrf2 turnover in stressed cells and for its degradation. *Lower panel*: in Keap1, BTB domain functions as a substrate adaptor protein for a

Cul3-dependent ubiquitin ligase complex. IVR domain is a domain of intervention which is distinguished for its high number of cysteine residues. DGR domain is associated with actin

Under oxidant conditions, Nrf2 binds with high affinity to the *cis*-acting enhancer sequence called Antioxidant Response Element (ARE, 5´-GTGACnnnGC-3´), located in the 5´-flanking regions of a broad range of antioxidant and cytoprotective genes that act against oxidative/electrophilic damage (Nguyen et al., 2004; Rushmore et al., 1991). The binding of Nrf2 to ARE requires its heterodimerization with small Maf proteins (Katsuoka et al., 2005), which stimulates transcription of downstream genes, with participation of transcriptional co-activators - mainly CREB-binding protein (CBP) -, through the Neh4 and Neh5 domains (Figure 8, *upper panel*) in the transcription factor. These co-activators act synergistically to

Nrf2 activity is primarily regulated by suppressor protein Keap1 (Figure 8, *lower panel*), a member of the BTB (Broad complex/Tramtrack/Bric-a-brac)-Kelch protein family (Cullinan et al., 2004), that under normal conditions (unstressed) forms a complex with Nrf2 within the cytosol. This complex is associated with actin filaments through its double glycine repeat

domain and the leucine zipper for dimerization. Neh2 is the Keap1

filaments, giving stability to Keap1.

attain maximum its activity (Katoh et al., 2001).

**7. Regulation of Nrf2: Keap1 (ARE elements)** 

(Figure 8, *upper panel*).

Together, these coordinately regulated gene clusters presented in Figures 5, 6 and 7 strongly support the hypothesis that Nrf2-dependent gene expression is crucial for an efficient detoxification of reactive metabolites and ROS, as well as for the cellular capacity to counteract stressing events such as inflammation.

Fig. 7. Genes involved in NADPH homeostasis are indicated in black boxes. P450, cytochrome P450; GST, glutathione-S-transferase; TXNRD1, thioredoxin reductase-1; NQO1, NAD(P)H:quinone oxidoreductase-1; GR, glutathione reductase; G6PD, glucose-6-phosphate dehydrogenase.

### **6. Nrf2 characteristics**

The transcription factor Nrf2 (Nuclear factor-E2-related factor 2) is the guardian of redox homeostasis because it regulates basal and inducible expression of array ride of antioxidant and cytoprotective genes, providing a level of protection required for normal cellular activities and against various oxidative stress-related pathologies, including ischemic stroke (Cho & Kleeberger, 2009; Nguyen et al., 2004; Van Muiswinkel & Kuiperij, 2005). Nrf2 is highly expressed in detoxification organs - such as liver and kidney - and organs exposed to the external environment - such as skin, lung and digestive tract - (Motohashi et al., 2002), whereas in the brain its levels are low (Moi et al., 1994).

Nrf2 is a member of the cap 'n' collar (CNC) family basic region-leucine zipper transcription factor (Katsuoka et al., 2005; Sykiotis & Bohmann, 2010). Nrf2 protein has six highly conserved regions, called Nrf2-ECH homology (Neh) domains. Neh1 is located in the half Cterminal of the molecule and constitutes the basic DNA binding domain and the leucine zipper for dimerization. Neh2 domain is located in the proximal N-terminus of Nrf2 and represents the region through which Nrf2 associates with the cytoplasmic protein Keap1 (kelch-like ECH-associated protein 1) (Itoh et al., 1999). Neh6 is a redox-insensitive degron, which is essential for maximal turnover of Nrf2 in stressed cells, as well as for its

Together, these coordinately regulated gene clusters presented in Figures 5, 6 and 7 strongly support the hypothesis that Nrf2-dependent gene expression is crucial for an efficient detoxification of reactive metabolites and ROS, as well as for the cellular capacity to

Fig. 7. Genes involved in NADPH homeostasis are indicated in black boxes. P450, cytochrome P450; GST, glutathione-S-transferase; TXNRD1, thioredoxin reductase-1; NQO1, NAD(P)H:quinone oxidoreductase-1; GR, glutathione reductase; G6PD,

The transcription factor Nrf2 (Nuclear factor-E2-related factor 2) is the guardian of redox homeostasis because it regulates basal and inducible expression of array ride of antioxidant and cytoprotective genes, providing a level of protection required for normal cellular activities and against various oxidative stress-related pathologies, including ischemic stroke (Cho & Kleeberger, 2009; Nguyen et al., 2004; Van Muiswinkel & Kuiperij, 2005). Nrf2 is highly expressed in detoxification organs - such as liver and kidney - and organs exposed to the external environment - such as skin, lung and digestive tract - (Motohashi et al., 2002),

Nrf2 is a member of the cap 'n' collar (CNC) family basic region-leucine zipper transcription factor (Katsuoka et al., 2005; Sykiotis & Bohmann, 2010). Nrf2 protein has six highly conserved regions, called Nrf2-ECH homology (Neh) domains. Neh1 is located in the half Cterminal of the molecule and constitutes the basic DNA binding domain and the leucine zipper for dimerization. Neh2 domain is located in the proximal N-terminus of Nrf2 and represents the region through which Nrf2 associates with the cytoplasmic protein Keap1 (kelch-like ECH-associated protein 1) (Itoh et al., 1999). Neh6 is a redox-insensitive degron, which is essential for maximal turnover of Nrf2 in stressed cells, as well as for its

counteract stressing events such as inflammation.

glucose-6-phosphate dehydrogenase.

whereas in the brain its levels are low (Moi et al., 1994).

**6. Nrf2 characteristics** 

degradation (McMahon et al., 2004). Neh3 domain is required for transcriptional activation of the protein (Nioi et al., 2005). Neh4 and Neh5 domains are required for its binding to ARE (Figure 8, *upper panel*).

Fig. 8. Nrf2 and Keap1 domains. *Upper panel*: in Nrf2, Neh1 is the basic DNA binding domain and the leucine zipper for dimerization. Neh2 is the Keap1 (kelch-like ECH-associated protein 1) binding domain. Neh3 is required for transcriptional activation of the protein. Neh4 and Neh5 domains are required for the binding to ARE. Neh6 is essential for both Nrf2 turnover in stressed cells and for its degradation. *Lower panel*: in Keap1, BTB domain functions as a substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. IVR domain is a domain of intervention which is distinguished for its high number of cysteine residues. DGR domain is associated with actin filaments, giving stability to Keap1.

Under oxidant conditions, Nrf2 binds with high affinity to the *cis*-acting enhancer sequence called Antioxidant Response Element (ARE, 5´-GTGACnnnGC-3´), located in the 5´-flanking regions of a broad range of antioxidant and cytoprotective genes that act against oxidative/electrophilic damage (Nguyen et al., 2004; Rushmore et al., 1991). The binding of Nrf2 to ARE requires its heterodimerization with small Maf proteins (Katsuoka et al., 2005), which stimulates transcription of downstream genes, with participation of transcriptional co-activators - mainly CREB-binding protein (CBP) -, through the Neh4 and Neh5 domains (Figure 8, *upper panel*) in the transcription factor. These co-activators act synergistically to attain maximum its activity (Katoh et al., 2001).

### **7. Regulation of Nrf2: Keap1 (ARE elements)**

Nrf2 activity is primarily regulated by suppressor protein Keap1 (Figure 8, *lower panel*), a member of the BTB (Broad complex/Tramtrack/Bric-a-brac)-Kelch protein family (Cullinan et al., 2004), that under normal conditions (unstressed) forms a complex with Nrf2 within the cytosol. This complex is associated with actin filaments through its double glycine repeat

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 361

exhibit different affinity for Keap1; the affinity of ETGE is greater than DLG (Tong et al., 2006b). The term "hinge" indicates that the interaction of high affinity is not affected by inducers; in contrast, inducers abolish the low-affinity interaction mediated by the "latch", thereby disrupting the presentation of Nrf2 to the ubiquitination machinery of Keap1 (Li & Kong, 2009) (Figure 10, *right panel*). Other models that describe the interaction between Nrf2 and Keap1 have provided conflicting information when contrasted with the "hinge and

Fig. 10. Effect of sulforaphane on Nrf2/Keap1 complex. *Left panel*: Upon unstressed conditions, this complex is dissociated and Nrf2 can either suffer proteosomal degradation or respond to stimuli typical of basal cell metabolism. In the later, Nrf2 is phosphorylated and translocated to the nucleus forming heterodimers with Maf and acting on ARE. *Right panel*: Under stress oxidative conditions, or in the presence of inducers, several cysteine residues suffer changes inducing its Nrf2 dissociation and further translocation of

Sulforaphane induces a phase 2 response as a result of gene expression modulation through Nrf2/ARE pathway. ARE-driven targets include NAD(P)H:quinone oxidereductase (NQO1), heme oxygenase-1 (HO-1) and -glutamylcysteine ligase (GCL). The induction of these enzymes has been observed both in *in vivo* and *in vitro* experiments after sulforaphane

this factor to nucleus, where it will induce phase 2 genes transcription.

treatment.

latch" model (Lo & Hannink, 2006; 2008).

(DGR) domain (Figure 10, *left panel*), which plays an important role in retention of Nrf2 (Kang et al., 2004).

BTB domain of Keap1 functions as an adaptor for Cul3-dependent E3 ubiquitin ligase complex that interacts with the seven lysine residues located in the Neh2 domain of Nrf2, promoting its ubiquitination (Kobayashi et al., 2004; Zhang et al., 2004) and its continuous degradation by 26S proteasome (Nguyen et al., 2003). This is supported by the relatively short half-life of Nrf2 (10-30 min) in absence of cellular stress (McMahon et al., 2003). Upon oxidative stress conditions, the interaction between Nrf2 and Keap1 is disrupted through changes in certain domains of Keap1, hence promoting the release of Nrf2 (Eggler et al., 2005).

The human Keap1 protein contains 27 cysteine residues, some of which are highly reactive to a wide variety of chemical stimuli. Furthermore, a large amount of evidence has emerged suggesting that certain cysteines of Keap1 may be targets of Nrf2 inducers such as sulforaphane, which reacts with thiol groups of Keap1 to form resistant thionoacyl adducts by hydrolysis and transacylation reactions (Hong et al., 2005) (Figure 9).

Fig. 9. Formation of adducts between sulforaphane and Keap1.

It has been reported that Cys151 in BTB domain of Keap1 is required for inhibition of Keap1-dependent Nrf2 degradation stimulated by sulforaphane and oxidative stress (Zhang & Hannink, 2003). Cys273 and Cys288, located in the IVR domain of Keap1, are essential for its repressive activity under basal conditions. It has been suggested that this effect also responds to sulforaphane (Kobayashi et al., 2006). On the other hand, it has been reported that Cys489, Cys583, and Cys624 were most reactive toward sulforaphane (Hong et al., 2005). Therefore, the responsiveness of Nrf2 to inducers, such as sulforaphane, involves redox-dependent alterations of thiol groups in several domains of Keap1, which acts like a sensor responding to oxidative and environment stress through dynamic changes in cystein reducing status (Jung & Kwak, 2010). In turn, Keap1 is considered as a zinc metalloprotein because the chemical modification of critical cysteine residues is modulated by thiol-bound zinc (approximately 1 mol per subunit), which is displaced by the reaction with inducers or other classical sulfhydryl reagents, such as sulforaphane (Dinkova-Kostova et al., 2005b).

Another important event in the activation of Nrf2 may be its phosphorylation. The protein kinase-dependent signal transduction pathways have been implicated in the release of Nrf2 from Keap1-mediated repression, mainly by protein kinase C, whose target is a single serine residue, Ser40 (Bloom & Jaiswal, 2003; Huang et al., 2002). To explain how Keap1/Nrf2 complex respond to basal or inducible stimuli, it has been proposed the "hinge and latch" model (Tong et al., 2006a), which suggests that a single Nrf2 molecule makes contacts with two domains of Keap1 homodimer (McMahon et al., 2006; Tong et al., 2006a). Neh2 domain of Nrf2 contains two sites for Keap1 binding, termed motifs DLG and ETGE. These motifs

(DGR) domain (Figure 10, *left panel*), which plays an important role in retention of Nrf2

BTB domain of Keap1 functions as an adaptor for Cul3-dependent E3 ubiquitin ligase complex that interacts with the seven lysine residues located in the Neh2 domain of Nrf2, promoting its ubiquitination (Kobayashi et al., 2004; Zhang et al., 2004) and its continuous degradation by 26S proteasome (Nguyen et al., 2003). This is supported by the relatively short half-life of Nrf2 (10-30 min) in absence of cellular stress (McMahon et al., 2003). Upon oxidative stress conditions, the interaction between Nrf2 and Keap1 is disrupted through changes in certain domains of Keap1, hence promoting the release of Nrf2 (Eggler et al.,

The human Keap1 protein contains 27 cysteine residues, some of which are highly reactive to a wide variety of chemical stimuli. Furthermore, a large amount of evidence has emerged suggesting that certain cysteines of Keap1 may be targets of Nrf2 inducers such as sulforaphane, which reacts with thiol groups of Keap1 to form resistant thionoacyl adducts

It has been reported that Cys151 in BTB domain of Keap1 is required for inhibition of Keap1-dependent Nrf2 degradation stimulated by sulforaphane and oxidative stress (Zhang & Hannink, 2003). Cys273 and Cys288, located in the IVR domain of Keap1, are essential for its repressive activity under basal conditions. It has been suggested that this effect also responds to sulforaphane (Kobayashi et al., 2006). On the other hand, it has been reported that Cys489, Cys583, and Cys624 were most reactive toward sulforaphane (Hong et al., 2005). Therefore, the responsiveness of Nrf2 to inducers, such as sulforaphane, involves redox-dependent alterations of thiol groups in several domains of Keap1, which acts like a sensor responding to oxidative and environment stress through dynamic changes in cystein reducing status (Jung & Kwak, 2010). In turn, Keap1 is considered as a zinc metalloprotein because the chemical modification of critical cysteine residues is modulated by thiol-bound zinc (approximately 1 mol per subunit), which is displaced by the reaction with inducers or other classical sulfhydryl reagents, such as sulforaphane (Dinkova-Kostova et al., 2005b). Another important event in the activation of Nrf2 may be its phosphorylation. The protein kinase-dependent signal transduction pathways have been implicated in the release of Nrf2 from Keap1-mediated repression, mainly by protein kinase C, whose target is a single serine residue, Ser40 (Bloom & Jaiswal, 2003; Huang et al., 2002). To explain how Keap1/Nrf2 complex respond to basal or inducible stimuli, it has been proposed the "hinge and latch" model (Tong et al., 2006a), which suggests that a single Nrf2 molecule makes contacts with two domains of Keap1 homodimer (McMahon et al., 2006; Tong et al., 2006a). Neh2 domain of Nrf2 contains two sites for Keap1 binding, termed motifs DLG and ETGE. These motifs

by hydrolysis and transacylation reactions (Hong et al., 2005) (Figure 9).

Fig. 9. Formation of adducts between sulforaphane and Keap1.

(Kang et al., 2004).

2005).

exhibit different affinity for Keap1; the affinity of ETGE is greater than DLG (Tong et al., 2006b). The term "hinge" indicates that the interaction of high affinity is not affected by inducers; in contrast, inducers abolish the low-affinity interaction mediated by the "latch", thereby disrupting the presentation of Nrf2 to the ubiquitination machinery of Keap1 (Li & Kong, 2009) (Figure 10, *right panel*). Other models that describe the interaction between Nrf2 and Keap1 have provided conflicting information when contrasted with the "hinge and latch" model (Lo & Hannink, 2006; 2008).

Fig. 10. Effect of sulforaphane on Nrf2/Keap1 complex. *Left panel*: Upon unstressed conditions, this complex is dissociated and Nrf2 can either suffer proteosomal degradation or respond to stimuli typical of basal cell metabolism. In the later, Nrf2 is phosphorylated and translocated to the nucleus forming heterodimers with Maf and acting on ARE. *Right panel*: Under stress oxidative conditions, or in the presence of inducers, several cysteine residues suffer changes inducing its Nrf2 dissociation and further translocation of this factor to nucleus, where it will induce phase 2 genes transcription.

Sulforaphane induces a phase 2 response as a result of gene expression modulation through Nrf2/ARE pathway. ARE-driven targets include NAD(P)H:quinone oxidereductase (NQO1), heme oxygenase-1 (HO-1) and -glutamylcysteine ligase (GCL). The induction of these enzymes has been observed both in *in vivo* and *in vitro* experiments after sulforaphane treatment.

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 363

an infarct volume and neurological deficit significantly larger than in wild type mice (Shah

Taking together, these data suggest that Nrf2 is upregulated in permanent ischemia and ischemic/reperfusion, an augment that is related with a decreased expression of Keap1 and an altered expression of antioxidant proteins. Thus, this upregulation may be due to an alteration in the redox state, a mechanism through which cells active an antioxidant response to protect themselves from future oxidant damage. Moreover, it has been demonstrated that Nrf2 activation induces the expression of the Nrf2 gene itself (Lee et al., 2005), indicating that the administration of Nrf2 inducers may be an important

A wide range of dietary phytochemicals or supplements with medicinal properties have been reported to activate adaptive stress responses related with the induction of cytoprotective genes through Nrf2 induction (Surh et al., 2008). The mechanism of action of such phytochemicals can therefore be considered as a form of hormesis where a stressor triggers an adaptive response which increases resistance to more severe stress and disease (Calabrese et al., 2007). Unfortunately, few of these compounds have been tested in brain ischemic models; some of them are sulforaphane, curcumin and ter-butilhydroquinone,

Sulforaphane is a natural dietary isothiocyanate present in cruciferous vegetables of the genus *Brassica* such as broccoli, brussel sprouts, cauliflower, cabbage, etc. Several studies have shown the neuroprotective properties of sulforaphane against ischemia/reperfusion damage. It has been found that sulforaphane (5 mg/kg) reduced the cerebral infarct volume in a carotid/middle cerebral artery occlusion common model in rodents when it was administered 15 min after injury (Zhao et al., 2006). Other groups reported that an injection of sulforaphane (5 mg/kg) 30 min before the onset of ischemia reduced the infarct size in a neonatal hypoxia-ischemia model (Ping et al., 2010). In both studies, the protective effects of sulforaphane were associated with its well-known capacity to induce the expression of

Other *in vivo* studies support the ability of sulforaphane as inducer of phase II enzymes in brain increasing HO-1, NQO1 and GST mRNA levels (Chen et al., 2011). It has also shown in *in vitro* studies that pretreatment and post-treatment with sulforaphane reduced hippocampal death of astrocytes and neurons induced by transient exposure to O2 and glucose deprivation. This protective effect was associated with nuclear accumulation of Nrf2 accompanied by an increase in NQO1, HO-1 and GCL mRNA levels, and a decrease in DNA oxidation (Danilov et al., 2009; Soane et al., 2010). Altogether, these studies indicate that sulforaphane could be considered as a useful tool for pre- and post-treatment of brain injury

Curcumin is a diferuloylmethane derived from the rhizomes of turmeric (*Curcuma longa* Linn, Zingiberaceae) widely used in Indian curry with a favorable safe profile. Its chemopreventive effects have been related with its antioxidant and anti-inflammatory

neuroprotective antioxidant mechanism that can limit stroke damage.

**9. Effect of Nrf2 inducers in cerebral ischemia** 

HO-1 mRNA and protein through Nrf2/ARE pathway.

due its well-know capacity as inducer of Nrf2.

et al., 2007).

among others. *Sulforaphane* 

*Curcumin* 

### **8. Nrf2 in cerebral ischemia**

Nrf2 has been detected in neuronal and glial cells (Chen et al., 2011; Li et al., 2011; Shah et al., 2010; Yang et al., 2009). Previous studies using gel-shift assay found that ischemic brains selectively upregulates ARE-mediated gene expression, whereas binding activities of other stress response elements were unchanged, including metal response element, interleukin-6, and STAT (signal transducer and activator of transcription) response elements (Campage et al., 2000).

Middle cerebral artery occlusion (permanent or transient) is a classical and wellcharacterized model inducing cerebral ischemia in rats that involves a cytotoxic response occurring within few minutes from the onset of cerebral ischemia, and encompasses oxidative stress, pro-inflammatory responses and cell death (Ikeda et al., 2003; Longa et al., 1989; Simonyi et al., 2005). Yang et al. (2009) used permanent focal ischemia to detect the expression of Nrf2. They found that Nrf2 protein and mRNA were upregulated when is compared with normal control, showing a peak at 24 h and localizing with nuclei and cytoplasm of neurons and astrocytes. Alternatively, Nrf2 was presented in the injured regions of cortices with cerebral ischemic/reperfusion, and markedly increased in both cytoplasm and nuclei (Li et al., 2011). Meanwhile, Keap1 immunoreactivity was significantly reduced. Besides, an altered expression of thioredoxin, glutathione, and heme oxigenase was detected (Tanaka et al., 2011).

Oligemia is another model that was used to determine Nrf2 localization. It consists in a reduction in the mean arterial pressure to 30-40 mm Hg, resulting in a 50% reduction in cerebral blood flow after reperfusion. This blood flow reduction presents an increase in oxidative stress through lipid peroxidation (Heim et al., 1995; Läer et al., 1993) and an augmented OH production during the reperfusion phase (Heim et al., 2000). In this model, Nrf2 was specifically upregulated 1 h after the surgery. Nrf2-positive neurons were found in the Purkinje cells of the cerebellar cortex and in the pyramidal neurons of the cingulate cortex (Liverman et al., 2004).

Additionally, Nrf2 knockout (Nrf2-/-) mice have been used to understand the role of Nrf2 during ischemia-mediated oxidative brain insult.

*In vitro* studies showed that neurons and astrocytes from Nrf2 knockout (Nrf2-/-) mice were more sensitive to oxidative stress, Ca2+ influx and mitochondrial toxicity than neurons and astrocytes from wild type animals; however, when the cells were transfected with a functional Nrf2 construct, they became less prone to oxidative stress (Kraft et al., 2004; Lee et al., 2003a; Lee and Johnson, 2004). Consistent with these results, dominant negative-Nrf2 stable cells and Nrf2-sensitized neuroblastoma cells silenced with siRNA were more amenable to apoptosis induced by nitric oxide (Dhakshinamoorthy & Porter, 2004). Also, increasing Nrf2 activity in mixed neuronal/glial cultures was highly neuroprotective in *in vitro* models that simulated components of stroke damage, such as oxidative glutamate toxicity, H2O2 exposure, metabolic inhibition by rotenone, and Ca2+ overload (Duffy et al., 1998; Kraft et al., 2004; Lee et al., 2003b; Murphy et al., 1991; Shih et al., 2003).

*In vivo,* using permanent middle cerebral artery occlussion by cauterization, Shih et al. (2005) did not observe significant difference in infarct size between Nrf2-/- and Nrf2+/+ mice 24 h after stroke. However, 7 days after permanent focal ischemia, they observed a two-fold increase in infarct volume with Nrf2-/- mice, while the infarct size of Nrf2 +/+ mice did not increase in size between 24 h and 7 days. On the other hand, Nrf2 knockout (Nrf2-/-) mice subjected to 90 min middle cerebral artery occlusion followed by 24 h reperfusion, showed an infarct volume and neurological deficit significantly larger than in wild type mice (Shah et al., 2007).

Taking together, these data suggest that Nrf2 is upregulated in permanent ischemia and ischemic/reperfusion, an augment that is related with a decreased expression of Keap1 and an altered expression of antioxidant proteins. Thus, this upregulation may be due to an alteration in the redox state, a mechanism through which cells active an antioxidant response to protect themselves from future oxidant damage. Moreover, it has been demonstrated that Nrf2 activation induces the expression of the Nrf2 gene itself (Lee et al., 2005), indicating that the administration of Nrf2 inducers may be an important neuroprotective antioxidant mechanism that can limit stroke damage.

### **9. Effect of Nrf2 inducers in cerebral ischemia**

A wide range of dietary phytochemicals or supplements with medicinal properties have been reported to activate adaptive stress responses related with the induction of cytoprotective genes through Nrf2 induction (Surh et al., 2008). The mechanism of action of such phytochemicals can therefore be considered as a form of hormesis where a stressor triggers an adaptive response which increases resistance to more severe stress and disease (Calabrese et al., 2007). Unfortunately, few of these compounds have been tested in brain ischemic models; some of them are sulforaphane, curcumin and ter-butilhydroquinone, among others.

### *Sulforaphane*

362 Advances in the Preclinical Study of Ischemic Stroke

Nrf2 has been detected in neuronal and glial cells (Chen et al., 2011; Li et al., 2011; Shah et al., 2010; Yang et al., 2009). Previous studies using gel-shift assay found that ischemic brains selectively upregulates ARE-mediated gene expression, whereas binding activities of other stress response elements were unchanged, including metal response element, interleukin-6, and STAT (signal transducer and activator of transcription) response elements (Campage et

Middle cerebral artery occlusion (permanent or transient) is a classical and wellcharacterized model inducing cerebral ischemia in rats that involves a cytotoxic response occurring within few minutes from the onset of cerebral ischemia, and encompasses oxidative stress, pro-inflammatory responses and cell death (Ikeda et al., 2003; Longa et al., 1989; Simonyi et al., 2005). Yang et al. (2009) used permanent focal ischemia to detect the expression of Nrf2. They found that Nrf2 protein and mRNA were upregulated when is compared with normal control, showing a peak at 24 h and localizing with nuclei and cytoplasm of neurons and astrocytes. Alternatively, Nrf2 was presented in the injured regions of cortices with cerebral ischemic/reperfusion, and markedly increased in both cytoplasm and nuclei (Li et al., 2011). Meanwhile, Keap1 immunoreactivity was significantly reduced. Besides, an altered expression of thioredoxin, glutathione, and heme oxigenase

Oligemia is another model that was used to determine Nrf2 localization. It consists in a reduction in the mean arterial pressure to 30-40 mm Hg, resulting in a 50% reduction in cerebral blood flow after reperfusion. This blood flow reduction presents an increase in oxidative stress through lipid peroxidation (Heim et al., 1995; Läer et al., 1993) and an

Nrf2 was specifically upregulated 1 h after the surgery. Nrf2-positive neurons were found in the Purkinje cells of the cerebellar cortex and in the pyramidal neurons of the cingulate

Additionally, Nrf2 knockout (Nrf2-/-) mice have been used to understand the role of Nrf2

*In vitro* studies showed that neurons and astrocytes from Nrf2 knockout (Nrf2-/-) mice were more sensitive to oxidative stress, Ca2+ influx and mitochondrial toxicity than neurons and astrocytes from wild type animals; however, when the cells were transfected with a functional Nrf2 construct, they became less prone to oxidative stress (Kraft et al., 2004; Lee et al., 2003a; Lee and Johnson, 2004). Consistent with these results, dominant negative-Nrf2 stable cells and Nrf2-sensitized neuroblastoma cells silenced with siRNA were more amenable to apoptosis induced by nitric oxide (Dhakshinamoorthy & Porter, 2004). Also, increasing Nrf2 activity in mixed neuronal/glial cultures was highly neuroprotective in *in vitro* models that simulated components of stroke damage, such as oxidative glutamate toxicity, H2O2 exposure, metabolic inhibition by rotenone, and Ca2+ overload (Duffy et al.,

*In vivo,* using permanent middle cerebral artery occlussion by cauterization, Shih et al. (2005) did not observe significant difference in infarct size between Nrf2-/- and Nrf2+/+ mice 24 h after stroke. However, 7 days after permanent focal ischemia, they observed a two-fold increase in infarct volume with Nrf2-/- mice, while the infarct size of Nrf2 +/+ mice did not increase in size between 24 h and 7 days. On the other hand, Nrf2 knockout (Nrf2-/-) mice subjected to 90 min middle cerebral artery occlusion followed by 24 h reperfusion, showed

1998; Kraft et al., 2004; Lee et al., 2003b; Murphy et al., 1991; Shih et al., 2003).

OH production during the reperfusion phase (Heim et al., 2000). In this model,

**8. Nrf2 in cerebral ischemia** 

was detected (Tanaka et al., 2011).

cortex (Liverman et al., 2004).

during ischemia-mediated oxidative brain insult.

al., 2000).

augmented

Sulforaphane is a natural dietary isothiocyanate present in cruciferous vegetables of the genus *Brassica* such as broccoli, brussel sprouts, cauliflower, cabbage, etc. Several studies have shown the neuroprotective properties of sulforaphane against ischemia/reperfusion damage. It has been found that sulforaphane (5 mg/kg) reduced the cerebral infarct volume in a carotid/middle cerebral artery occlusion common model in rodents when it was administered 15 min after injury (Zhao et al., 2006). Other groups reported that an injection of sulforaphane (5 mg/kg) 30 min before the onset of ischemia reduced the infarct size in a neonatal hypoxia-ischemia model (Ping et al., 2010). In both studies, the protective effects of sulforaphane were associated with its well-known capacity to induce the expression of HO-1 mRNA and protein through Nrf2/ARE pathway.

Other *in vivo* studies support the ability of sulforaphane as inducer of phase II enzymes in brain increasing HO-1, NQO1 and GST mRNA levels (Chen et al., 2011). It has also shown in *in vitro* studies that pretreatment and post-treatment with sulforaphane reduced hippocampal death of astrocytes and neurons induced by transient exposure to O2 and glucose deprivation. This protective effect was associated with nuclear accumulation of Nrf2 accompanied by an increase in NQO1, HO-1 and GCL mRNA levels, and a decrease in DNA oxidation (Danilov et al., 2009; Soane et al., 2010). Altogether, these studies indicate that sulforaphane could be considered as a useful tool for pre- and post-treatment of brain injury due its well-know capacity as inducer of Nrf2.

### *Curcumin*

Curcumin is a diferuloylmethane derived from the rhizomes of turmeric (*Curcuma longa* Linn, Zingiberaceae) widely used in Indian curry with a favorable safe profile. Its chemopreventive effects have been related with its antioxidant and anti-inflammatory

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 365

Gupta et al. (2003) found that garlic oil administration 90 min before the ischemia/reperfusion diminished the infarct area and associated this effect to its antioxidant properties. Saleem et al. (2006) showed that aqueous garlic extract treatment increased neurobehavioral score, decreased malondialdehyde levels, increased GSH content, and prevented the depletion in GPx, GR, GST and Na+/K+-ATPasa activities. Moreover, CAT and SOD activities were increased by aqueous garlic extract. Aguilera et al. (2010) reported that the major protective effect exerted by aged garlic extract was observed when it was administered at the onset of reperfusion. In this work, aged garlic extract prevented the ischemia/reperfusion-induced increase in nitrotyrosine levels and the decrease in GPx, SOD

Numagami et al. (1996) demonstrated that aged garlic extract compounds that present a thioallyl group (particularly S-allylcysteine) exhibited a strong antioxidant capacity in a model of cerebral ischemia in rats. Indeed, S-allylcysteine reduced the infarct volume and brain edema, while prevented ONOO– formation and lipid peroxidation (Numagami & Ohnishi, 2001). More recently, S-allylcysteine (300 mg/kg, i.p.) produced a protective effect on cerebral ischemic injury in rats due to the inhibition of extracellular signal-regulated kinase activity (Kim et al., 2006a). The fact that S-allylcysteine can cross the blood-brain barrier turned it soon of potential interest to be tested in neurotoxic models. In fact, the prophylactic impact and rescue properties of S-allylcysteine in ischemia/reperfusion injury are being recently discussed and reinforced (Sener et al., 2007). In addition, S-allylcysteine is a stable compound (Lawson, 1998) and is easily absorbed by gastrointestinal tract after oral administration (Kodera et al., 2002). One of its advantages in regard to other garlic compounds, such as allicin and dialyl sulfide, is its limited toxicity established by its higher lethal oral dose (Amagase et al., 2001). Pharmacokinetic studies demonstrate fast absorption and distribution phases followed by a slow elimination phase for oral administration, as well as fast distribution and slow elimination phases for i.v. administration (Nagae et al., 1994; Yan & Zeng, 2005). Pharmacokinetics of S-allylcysteine in humans by oral garlic administration revealed a half-life of 10 h and clearance time of 30 h (Kodera et al., 2002), suggesting a high bioavailability. After its oral administration, S-allylcysteine is absorbed by gastrointestinal tract, and its higher concentrations are detected in plasma and kidney up to

On the other hand, garlic oil-derived organosulfur compounds such as diallyl trisulfide, dialyl disulfide, and dialyl sulfide provide significant protection against carcinogenesis, and this protection is likely related with their antioxidant properties (Maldonado et al., 2009). Moreover, the lipophilic characteristics of these compounds allow crossing the blood-brain barrier as follows: dialyl sulfide crosses the blood-brain barrier easier than dialyl disulfide >

**10. Presumable protective effect of garlic compounds in cerebral ischemia**  Numerous studies have shown that garlic and its compounds exhibit a diverse biological activity, including anti-tumorigenic, anti-atherosclerosis, detoxification, anti-inflammatory, and antioxidant (Aguilera et al., 2010; Ali et al., 2000; Fisher et al., 2007; Fukushima et al., 1997; Mathew & Biju, 2008). The effect of different garlic preparations (aged garlic extract, aqueous garlic extract, garlic oil) and isolated compounds (S-allylcysteine) in cerebral ischemia, has been associated to its ability to scavenge ROS, acting as direct antioxidants

(Kim et al., 2006a).

and CAT activities both in cortex and striatum.

8 h post-intake (Nagae et al., 1994; Yan & Zeng, 2005).

diallyl trisulfide > S-allylcysteine (Kim et al., 2006b).

properties (Surh & Chun, 2007; Thangapazham et al., 2006). However, its mechanism of action is still poorly understood.

Curcumin has a protective effect against neurodegeneration in cerebral ischemia through the preservation of the blood-brain barrier integrity, and a decrease of the ischemiainduced lipid peroxidation, mitochondrial dysfunction and anti-apoptotic effects (Sun et al., 2008).

Yang et al., (2009) observed that the systematic administration of curcumin (100 mg/kg) 15 min after middle cerebral artery permanent occlusion increased Nrf2 nuclear translocation and Nrf2 and HO-1 gene and protein levels at 24 h onset of reperfusion. Curcumin reduced neurologic deficit, brain edema and infarct volume at 24 h after stroke. These results show that curcumin maybe an effective therapeutic drug for the treatment of brain injury toward a potential mechanism of upregulation Nrf2/ARE pathway at gene and protein levels.

However, the bioavailability of curcumin is very limited due to poor absorption, rapid metabolism and quick systemic elimination. Moreover, it has a poor blood-brain barrier penetration following acute administration. To improve its bioavailability, pharmacokinetics and interaction with multiple viable targets, new curcumin derivatives are being synthesized (Lapchak, 2001).

### *tert-Butylhydroquinone (t-BHQ)*

*tert*-butylhydroquinone (t-BHQ), a metabolite of the widely used food antioxidant butylated hydroxyanisole, has already been approved for human use (Food and Agriculture Organization of the United Nations/World Health Organization, 1999; National Toxicology Program, 1997). t-BHQ possesses an oxidizable 1,4 diphenolic structure that confers its potent ability to dissociate Keap1/Nrf2 complex (Van Ommen et al., 1992). T-BHQ can protect neuronal cells against the oxidative insult initiated by dopamine, H2O2, *tert*-butyl hydroperoxide, NMDA and glutamate (Duffy et al., 1998; Kraft et al., 2004; Li et al., 2002; Murphy et al., 1991; Shah et al., 2007).

Shih et al., (2005) determined the neuroprotective effect of tBHQ in ischemic injury in two different ischemia/reperfusion models - middle cerebral artery occlusion and endothelin-1 vasoconstriction - in rats and mice, using different routes of administration: intacerebroventricular, intraperitoneal, and dietary. Intracerebroventricular administration of t-BHQ (1 µL/h) during 3 days before rats were subjected to 1.5 h of ischemia and 24 h reperfusion showed a significant reduction of infarction in the cortex and a significant reduction in the neuronal scores. Intraperitoneal administration of t-BHQ (16.7 mg/Kg; 3 times/8h) 24 h before middle cerebral artery occlusion improved functional recovery up to 1 month after MCAO, showing a long-term benefit in ischemic damage and sensimotor deficit. Nrf2+/+ and Nrf2+/- mice fed with 1% t-BHQ during one week before permanent focal ischemia did not show changes in infarct area after 7 days, while Nrf2-/- mice were less tolerant to the diet, losing 20% body weight and showing a continuous growth of infarct area, thus suggesting that loss of Nrf2 function promotes peri-infact zone. Finally, Nrf2+/+ and Nrf2-/- mice were fed with t-BHQ after endothelin-1 administration into cortical parenchyma. Nrf2+/+ mice showed a decrease in endothelin-1-induced infarction while Nrf2-/- mice showed an exacerbated injury (Shih et al., 2003; 2005).

Collectively, these data suggest that t-BHQ may have a therapeutic potential for ischemic injury by increasing brain antioxidant capacity though the up-regulation of Nrf2 expression.

properties (Surh & Chun, 2007; Thangapazham et al., 2006). However, its mechanism of

Curcumin has a protective effect against neurodegeneration in cerebral ischemia through the preservation of the blood-brain barrier integrity, and a decrease of the ischemiainduced lipid peroxidation, mitochondrial dysfunction and anti-apoptotic effects (Sun et

Yang et al., (2009) observed that the systematic administration of curcumin (100 mg/kg) 15 min after middle cerebral artery permanent occlusion increased Nrf2 nuclear translocation and Nrf2 and HO-1 gene and protein levels at 24 h onset of reperfusion. Curcumin reduced neurologic deficit, brain edema and infarct volume at 24 h after stroke. These results show that curcumin maybe an effective therapeutic drug for the treatment of brain injury toward a potential mechanism of upregulation Nrf2/ARE pathway at gene

However, the bioavailability of curcumin is very limited due to poor absorption, rapid metabolism and quick systemic elimination. Moreover, it has a poor blood-brain barrier penetration following acute administration. To improve its bioavailability, pharmacokinetics and interaction with multiple viable targets, new curcumin derivatives are being

*tert*-butylhydroquinone (t-BHQ), a metabolite of the widely used food antioxidant butylated hydroxyanisole, has already been approved for human use (Food and Agriculture Organization of the United Nations/World Health Organization, 1999; National Toxicology Program, 1997). t-BHQ possesses an oxidizable 1,4 diphenolic structure that confers its potent ability to dissociate Keap1/Nrf2 complex (Van Ommen et al., 1992). T-BHQ can protect neuronal cells against the oxidative insult initiated by dopamine, H2O2, *tert*-butyl hydroperoxide, NMDA and glutamate (Duffy et al., 1998; Kraft et al., 2004; Li et al., 2002;

Shih et al., (2005) determined the neuroprotective effect of tBHQ in ischemic injury in two different ischemia/reperfusion models - middle cerebral artery occlusion and endothelin-1 vasoconstriction - in rats and mice, using different routes of administration: intacerebroventricular, intraperitoneal, and dietary. Intracerebroventricular administration of t-BHQ (1 µL/h) during 3 days before rats were subjected to 1.5 h of ischemia and 24 h reperfusion showed a significant reduction of infarction in the cortex and a significant reduction in the neuronal scores. Intraperitoneal administration of t-BHQ (16.7 mg/Kg; 3 times/8h) 24 h before middle cerebral artery occlusion improved functional recovery up to 1 month after MCAO, showing a long-term benefit in ischemic damage and sensimotor deficit. Nrf2+/+ and Nrf2+/- mice fed with 1% t-BHQ during one week before permanent focal ischemia did not show changes in infarct area after 7 days, while Nrf2-/- mice were less tolerant to the diet, losing 20% body weight and showing a continuous growth of infarct area, thus suggesting that loss of Nrf2 function promotes peri-infact zone. Finally, Nrf2+/+ and Nrf2-/- mice were fed with t-BHQ after endothelin-1 administration into cortical parenchyma. Nrf2+/+ mice showed a decrease in endothelin-1-induced infarction while

Collectively, these data suggest that t-BHQ may have a therapeutic potential for ischemic injury by increasing brain antioxidant capacity though the up-regulation of Nrf2 expression.

Nrf2-/- mice showed an exacerbated injury (Shih et al., 2003; 2005).

action is still poorly understood.

al., 2008).

and protein levels.

synthesized (Lapchak, 2001). *tert-Butylhydroquinone (t-BHQ)* 

Murphy et al., 1991; Shah et al., 2007).

### **10. Presumable protective effect of garlic compounds in cerebral ischemia**

Numerous studies have shown that garlic and its compounds exhibit a diverse biological activity, including anti-tumorigenic, anti-atherosclerosis, detoxification, anti-inflammatory, and antioxidant (Aguilera et al., 2010; Ali et al., 2000; Fisher et al., 2007; Fukushima et al., 1997; Mathew & Biju, 2008). The effect of different garlic preparations (aged garlic extract, aqueous garlic extract, garlic oil) and isolated compounds (S-allylcysteine) in cerebral ischemia, has been associated to its ability to scavenge ROS, acting as direct antioxidants (Kim et al., 2006a).

Gupta et al. (2003) found that garlic oil administration 90 min before the ischemia/reperfusion diminished the infarct area and associated this effect to its antioxidant properties. Saleem et al. (2006) showed that aqueous garlic extract treatment increased neurobehavioral score, decreased malondialdehyde levels, increased GSH content, and prevented the depletion in GPx, GR, GST and Na+/K+-ATPasa activities. Moreover, CAT and SOD activities were increased by aqueous garlic extract. Aguilera et al. (2010) reported that the major protective effect exerted by aged garlic extract was observed when it was administered at the onset of reperfusion. In this work, aged garlic extract prevented the ischemia/reperfusion-induced increase in nitrotyrosine levels and the decrease in GPx, SOD and CAT activities both in cortex and striatum.

Numagami et al. (1996) demonstrated that aged garlic extract compounds that present a thioallyl group (particularly S-allylcysteine) exhibited a strong antioxidant capacity in a model of cerebral ischemia in rats. Indeed, S-allylcysteine reduced the infarct volume and brain edema, while prevented ONOO– formation and lipid peroxidation (Numagami & Ohnishi, 2001). More recently, S-allylcysteine (300 mg/kg, i.p.) produced a protective effect on cerebral ischemic injury in rats due to the inhibition of extracellular signal-regulated kinase activity (Kim et al., 2006a). The fact that S-allylcysteine can cross the blood-brain barrier turned it soon of potential interest to be tested in neurotoxic models. In fact, the prophylactic impact and rescue properties of S-allylcysteine in ischemia/reperfusion injury are being recently discussed and reinforced (Sener et al., 2007). In addition, S-allylcysteine is a stable compound (Lawson, 1998) and is easily absorbed by gastrointestinal tract after oral administration (Kodera et al., 2002). One of its advantages in regard to other garlic compounds, such as allicin and dialyl sulfide, is its limited toxicity established by its higher lethal oral dose (Amagase et al., 2001). Pharmacokinetic studies demonstrate fast absorption and distribution phases followed by a slow elimination phase for oral administration, as well as fast distribution and slow elimination phases for i.v. administration (Nagae et al., 1994; Yan & Zeng, 2005). Pharmacokinetics of S-allylcysteine in humans by oral garlic administration revealed a half-life of 10 h and clearance time of 30 h (Kodera et al., 2002), suggesting a high bioavailability. After its oral administration, S-allylcysteine is absorbed by gastrointestinal tract, and its higher concentrations are detected in plasma and kidney up to 8 h post-intake (Nagae et al., 1994; Yan & Zeng, 2005).

On the other hand, garlic oil-derived organosulfur compounds such as diallyl trisulfide, dialyl disulfide, and dialyl sulfide provide significant protection against carcinogenesis, and this protection is likely related with their antioxidant properties (Maldonado et al., 2009). Moreover, the lipophilic characteristics of these compounds allow crossing the blood-brain barrier as follows: dialyl sulfide crosses the blood-brain barrier easier than dialyl disulfide > diallyl trisulfide > S-allylcysteine (Kim et al., 2006b).

Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 367

Aguilera, P.; Chánez-Cárdenas, M.E. & Maldonado, P.D. (2007). Recent Advances in the Use

Aguilera, P.; Chánez-Cárdenas, M.E.; Ortiz-Plata, A.; León-Aparicio, D.; Barrera, D.;

*Phytomedicine*, Vol.17, No.4-3, (March), pp. 241-247, ISSN 0944-7113 (Print) Alfieri, A.; Srivastava, S.; Siow, R.C.; Modo, M.; Fraser, P.A. & Mann, G.E. (2011). Targeting

Ali, M.; Al-Qattan, K.K.; Al-Enezi, F.; Khanafer, R.M. & Mustafa, T. (2000). Effect of Allicin

Amagase, H.; Petesch, B.L.; Matsuura, H.; Kasuga, S. & Itakura, Y. (2001). Intake of Garlic

Aronowski, J.; Strong, R. & Grotta, J.C. (1997). Reperfusion Injury: Demonstration of Brain

Bloom, D.A. & Jaiswal, A.K. (2003). Phosphorylation of Nrf2 at Ser40 by Protein Kinase C in

Calabrese, E.J.; Bachmann, K.A.; Bailer, A.J.; Bolger, P.M.; Borak, J.; Cai, L.; Cedergreen, N.;

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of Antioxidant Treatments in Cerebral Ischemia, In: *New Perspectives on Brain Cell Damage, Neurodegeneration and Neuroprotective Strategies*, A. Santamaría & M.E. Jiménez-Capdeville, (Ed.), 145–159, Research Signpost, ISBN 81-308-0164-7, Kerala,

Ezpinoza-Rojo, M.; Villeda-Hernández, J.; Sánchez-García, A. & Maldonado P.D. (2010). Aged Garlic Extract Delays the Appearance of Infarct Area in Cerebral Ischemia Model, an Effect Likely Conditioned by the Celular Antioxidant System.

the Nrf2-Keap1 Antioxidant Defence Pathway for Neurovascular Protection in Stroke. *Journal of Physiologie*, Vol.589, No.Pt17, (September), pp. 4125-4136, ISSN

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Response to Antioxidants Leads to the Release of Nrf2 From INrf2, but is not Required for Nrf2 Stabilization/Accumulation in the Nucleus and Transcriptional Activation of Antioxidant Response Element-Mediated NAD(P)H:Quinone Oxidoreductase-1 Gene Expression. *The Journal of Biological Chemistry*, Vol.278,

Cherian, M.G.; Chiueh, C.C.; Clarkson, T.W.; Cook, R.R.; Diamond, D.M.; Doolittle, D.J.; Dorato, M.A.; Duke, S.O.; Feinendegen, L.; Gardner, D.E.; Hart, R.W.; Hastings, K.L.; Hayes, A.W.; Hoffmann, G.R.; Ives, J.A.; Jaworowski, Z.; Johnson, T.E.; Jonas, W.B.; Kaminski, N.E.; Keller, J.G.; Klaunig, J.E.; Knudsen, T.B.; Kozumbo, W.J.; Lettieri, T.; Liu, S.Z.; Maisseu, A.; Maynard, K.I.; Masoro, E.J.; McClellan, R.O.; Mehendale, H.M.; Mothersill, C.; Newlin, D.B.; Nigg, H.N.; Oehme, F.W.; Phalen, R.F.; Philbert, M.A.; Rattan, S.I.; Riviere, J.E.; Rodricks, J., Sapolsky, R.M.; Scott, B.R.; Seymour, C.; Sinclair, D.A.; Smith-Sonneborn, J.; Snow,

ROS Reactive Oxygen Species SOD Superoxide Dismutase tBHQ tert-butylhydroquinone TXNRD1 Thioredoxine Reductase-1

**14. References** 

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Recently, it has been reported that some garlic compounds (diallyl trisulfide, dialyl disulfide, dialyl sulfide and S-ally-L-cysteine) are able to activate Nrf2 factor in liver, kidney, intestine and lung. (Chen et al., 2004; Fisher et al., 2007; Fukao et al., 2004; Gong et al., 2004; Guyonnet et al., 1999; Kalayarasan et al., 2008; 2009; Wu et al., 2002). However, there is no information on Nrf2 induction by these garlic compounds in the brain.

Altogether, these data indicate that S-ally-L-cysteine, diallyl trisulfide, dialyl disulfide, and dialyl sulfide may be alternative treatments for cerebral ischemia through Nrf2 upregulation.

### **11. Conclusion**

Nowadays is widely recognized that up-regulation of phase 2 response is a powerful, highly efficient and promising antioxidant strategy for protection against several diseases, including ischemic stroke. A wide range of dietary phytochemicals with medicinal properties have been reported to activate adaptive stress responses related with the induction of cytoprotective genes through Nrf2/ARE pathway. Unfortunately, few of these compounds (sulforaphane, curcumin, ter-butilhydroquinone) have been tested in cerebral ischemia experimental models. Moreover, these compounds have characteristics that limit their use as therapeutic agents in ischemic stroke. For example, sulforaphane is expensive, while curcumin poorly crosses the blood-brain barrier. Due to this, new agents should be evaluated. In this context, some garlic compounds (diallyl sulfide, diallyl disulfide, diallyl trisulfide and S-allylcysteine) could be promising agents for treatment of ischemic stroke because their physicochemical properties are promising, their absorption is high and most of them can easily cross the blood-brain barrier. Moreover, they have the ability to active Nrf2 factor and induce a phase 2 response in several models of hepatic and renal damage.

### **12. Acknowledgements**

This study was supported by CONACYT (Grant No. 103527 to PDM).

### **13. Abbreviation list**



### **14. References**

366 Advances in the Preclinical Study of Ischemic Stroke

Recently, it has been reported that some garlic compounds (diallyl trisulfide, dialyl disulfide, dialyl sulfide and S-ally-L-cysteine) are able to activate Nrf2 factor in liver, kidney, intestine and lung. (Chen et al., 2004; Fisher et al., 2007; Fukao et al., 2004; Gong et al., 2004; Guyonnet et al., 1999; Kalayarasan et al., 2008; 2009; Wu et al., 2002). However,

Altogether, these data indicate that S-ally-L-cysteine, diallyl trisulfide, dialyl disulfide, and dialyl sulfide may be alternative treatments for cerebral ischemia through Nrf2

Nowadays is widely recognized that up-regulation of phase 2 response is a powerful, highly efficient and promising antioxidant strategy for protection against several diseases, including ischemic stroke. A wide range of dietary phytochemicals with medicinal properties have been reported to activate adaptive stress responses related with the induction of cytoprotective genes through Nrf2/ARE pathway. Unfortunately, few of these compounds (sulforaphane, curcumin, ter-butilhydroquinone) have been tested in cerebral ischemia experimental models. Moreover, these compounds have characteristics that limit their use as therapeutic agents in ischemic stroke. For example, sulforaphane is expensive, while curcumin poorly crosses the blood-brain barrier. Due to this, new agents should be evaluated. In this context, some garlic compounds (diallyl sulfide, diallyl disulfide, diallyl trisulfide and S-allylcysteine) could be promising agents for treatment of ischemic stroke because their physicochemical properties are promising, their absorption is high and most of them can easily cross the blood-brain barrier. Moreover, they have the ability to active Nrf2 factor and induce a phase 2 response in several models of hepatic and renal damage.

there is no information on Nrf2 induction by these garlic compounds in the brain.

This study was supported by CONACYT (Grant No. 103527 to PDM).

ARE Antioxidant Response Element

G6PD Glucose-6phosphate dehydrogenase GCLC Glutamate cysteine ligase catalitic subunit GCLM Glutamate cysteine ligase modifier subunit

NQO1 NADPH:quinone oxidoreductase-1 Keap1 Kelch-like ECH-associated protein 1 Nrf2 Nuclear Factor-E2-related Factor 2

BH2 Dihydrobiopterin BH4 Tetrahydrobiopterin

GPx Glutathione Peroxidase GSH Reduced Glutathione GSSG Oxidized Glutathione HO-1 Heme oxygenase-1

RNS Reactive Nitrogen Species

CAT Catalase

upregulation.

**11. Conclusion** 

**12. Acknowledgements** 

**13. Abbreviation list** 


Nrf2 Activation, an Innovative Therapeutic Alternative in Cerebral Ischemia 369

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**16** 

*USA* 

**Preconditioning and Postconditioning** 

*1Department of Anatomy, Physiology & Genetics, and The Center for Neuroscience & Regenerative Medicine,* 

*AMEDD Center and School, Academy of Health Sciences,* 

*Fort Sam Houston, San Antonio, Texas* 

Joseph T. McCabe1, Michael W. Bentley2 and Joseph C. O'Sullivan2

Cerebral ischemic events from trauma, stroke, hemorrhagic shock, or other cerebral perfusion deficit, initiate a cascade of detrimental processes leading to long lasting tissue injury and poor neurological outcome. Correction of the perfusion deficit is vital. However, no interventions have been identified that protect compromised cerebral tissue during the

This chapter reviews two emerging concepts: *preconditioning*, which may have therapeutic utility for the protection of patients for *planned* treatments such as surgical intervention, and *postconditioning*, which may have benefits for amelioration of deficits from ischemic events, vascular injury and accidents. Preconditioning (described below) was first described from maneuvers that induced cytoprotection by temporarily occluding vessels serving the tissue or region of interest. There are inherent risks in performing such a maneuver, so that pharmacological agents—particularly for *delayed preconditioning* (described later)*—*affecting the aforementioned signal transduction and genomic pathways, are a safer, more realistic area of study. Conditioning is still primarily an experimental phenomenon. However, investigators have made considerable strides in uncovering the multiple, albeit complex signal transduction pathways that mediate conditioning effects. This knowledge may help

The preconditioning concept has roots in cardiovascular research. Ischemic preconditioning was originally described in a landmark 1986 study by Murry and associates (Murry, et al., 1986). Using a cardiac dog model, these investigators found that multiple, brief ischemic episodes protected the heart from a subsequent sustained ischemic insult. An experimental group of dogs experienced circumflex artery occlusion for 5-minute intervals with 5-minute reperfusions. This cycle was repeated a total of four times—clamping for 5 minutes and unclamping for 5 minutes. The circumflex artery was then clamped for 40 minutes. The control group of dogs had their circumflex artery occluded for 40 minutes. Surprisingly,

**1. Introduction** 

resolution of the ischemic event.

clinicians one day develop schemes for neuroprotection.

**2. Ischemic preconditioning: Origins in cardiology** 

*Uniformed Services University of the Health Sciences, Bethesda, Maryland 2U.S. Army Graduate School of Anesthesia Nursing, Graduate School,* 


## **Preconditioning and Postconditioning**

Joseph T. McCabe1, Michael W. Bentley2 and Joseph C. O'Sullivan2

*1Department of Anatomy, Physiology & Genetics, and The Center for Neuroscience & Regenerative Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 2U.S. Army Graduate School of Anesthesia Nursing, Graduate School, AMEDD Center and School, Academy of Health Sciences, Fort Sam Houston, San Antonio, Texas USA* 

### **1. Introduction**

378 Advances in the Preclinical Study of Ischemic Stroke

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Cerebral ischemic events from trauma, stroke, hemorrhagic shock, or other cerebral perfusion deficit, initiate a cascade of detrimental processes leading to long lasting tissue injury and poor neurological outcome. Correction of the perfusion deficit is vital. However, no interventions have been identified that protect compromised cerebral tissue during the resolution of the ischemic event.

This chapter reviews two emerging concepts: *preconditioning*, which may have therapeutic utility for the protection of patients for *planned* treatments such as surgical intervention, and *postconditioning*, which may have benefits for amelioration of deficits from ischemic events, vascular injury and accidents. Preconditioning (described below) was first described from maneuvers that induced cytoprotection by temporarily occluding vessels serving the tissue or region of interest. There are inherent risks in performing such a maneuver, so that pharmacological agents—particularly for *delayed preconditioning* (described later)*—*affecting the aforementioned signal transduction and genomic pathways, are a safer, more realistic area of study. Conditioning is still primarily an experimental phenomenon. However, investigators have made considerable strides in uncovering the multiple, albeit complex signal transduction pathways that mediate conditioning effects. This knowledge may help clinicians one day develop schemes for neuroprotection.

### **2. Ischemic preconditioning: Origins in cardiology**

The preconditioning concept has roots in cardiovascular research. Ischemic preconditioning was originally described in a landmark 1986 study by Murry and associates (Murry, et al., 1986). Using a cardiac dog model, these investigators found that multiple, brief ischemic episodes protected the heart from a subsequent sustained ischemic insult. An experimental group of dogs experienced circumflex artery occlusion for 5-minute intervals with 5-minute reperfusions. This cycle was repeated a total of four times—clamping for 5 minutes and unclamping for 5 minutes. The circumflex artery was then clamped for 40 minutes. The control group of dogs had their circumflex artery occluded for 40 minutes. Surprisingly,

Preconditioning and Postconditioning 381

Although not as effective as classical preconditioning, *delayed* or *late* preconditioning becomes apparent approximately 24 hours after initial preconditioning and it can persist for up to 72 hours (Ishida, et al., 1997). The most significant difference between classical and delayed preconditioning is the latter's requirement of the manufacture of new proteins needed to obtain protection; inhibition of new protein synthesis attenuates the protection

The triggers for delayed preconditioning are similar to classical preconditioning. These include cellular stress factors (sub-lethal ischemia, heat stress, cardiac pacing), which release factors such as reactive oxygen species, adenosine, and endogenous nitric oxide (NO). NO has been determined to have no effect on classical preconditioning and is a trigger specific only for delayed preconditioning (Bolli, 2000). Endogenous preconditioning agents also initiate delayed preconditioning and include adenosine agonists, bradykinin, opioids, NO donors, acetylcholine and norepinephrine (Bolli, 2000). Exogenous agents that activate the preconditioning pathways include, diazoxide, nicorandil, some hypercholesterolemic

Kinase intracellular pathways play a central role in delayed preconditioning, with activation of the PKC-ε isoform being particularly essential. Downstream to PKC-ε, tyrosine kinases and other kinases then activate the important transcription factor, NF-κB, which leads to the upregulation of many protective proteins in the nucleus. Some key proteins identified thus far include: inducible NO synthase (iNOS) (Takano, et al., 1998, cyclooxygenase (COX-2) (Shinmura, et al., 2000), the antioxidant enzyme superoxide dismutase (SOD) (Hoshida, et al., 2002), and heat shock proteins, HSP70, HSP25, and HSP32 (Bolli, 2000). Delayed preconditioning also reduces apoptosis by upregulating the anti-apoptotic protein, Bcl-2, which has been shown to inhibit opening of the mPTP, leading to cell survival (Maulik, et al., 1999). Mitochondrial KATP (mKATP) channel opening seems to be the final common pathway for these signaling pathways but it is not yet clear how the opening of these channels affords protection. Delayed preconditioning requires the opening of the mKATP channels during the ischemic event; its role after 24 hours, when delayed preconditioning

By 1993, an appreciable number of studies had confirmed that the direct application of various stimuli (hypoxia, ischemia, triggering agents) resulted in tissue and organ protection. In that year, however, Przyklenk and colleagues made a startling discovery. Using a canine model in which the circumflex artery was occluded (as in Murry's 1986 study), Przyklenk and colleagues observed that cardiac muscle supplied by the descending left coronary artery was also protected from ischemic insult, indicating that the preconditioning stimulus offered protection that was not confined to one area of an organ (Przyklenk, et al., 1993). Further experiments have shown *remote* classical ischemic preconditioning also is effective in other organs. A preconditioning trigger in one area of an organ offers protection to a different region of that same organ or to a different organ. For example, one group demonstrated that intermittent tourniquet application to a hind limb (ischemic preconditioning) implemented protection in other skeletal muscles (Addison, et al., 2003). Remote classical preconditioning of the heart was also obtained via transient ischemia of the small intestine (Liem, et al., 2005; Patel, et al., 2002) or the kidney (Gho, et al., 1996). Other studies have demonstrated remote delayed preconditioning. Induction of small intestine ischemia, for example, engenders myocardial protection 24-72 hours later (Wang,

derived from delayed preconditioning (Rizvi, et al., 1999).

agents, and volatile anesthetics (Gross, 2005).

occurs, is less clear (Takano, et al., 2000).

et al., 2001; Xiao, et al., 2001).

despite the additional 20 minutes of ischemia in the preconditioned animals, cardiac damage, measured by infarct size, was significantly reduced to just ¼th of the infarct observed in the hearts of dogs in the control group. This paper was the first to demonstrate and coin the term, *ischemic preconditioning*.

### **2.1 Preconditioning**

Preconditioning has been demonstrated in all species studied to date (rat, mouse, rabbit, dog, human, chicken, sheep, pig and including cell lines), and in all organs studied thus far, including skeletal muscle, brain, kidney, small intestine, heart, and liver (Peralta, et al., 1996; Glazier, et al., 1994; Pang, et al., 1995). Other preconditioning manipulations, such as global hypoxia (Emerson, et al., 1999) and thermal injury (Marber, et al., 1993), are also effective in protection from lasting insult. Prior to this 1986 discovery, the best pharmacological treatments (with varying reproducible results) for the protection of cardiac muscle from infarction only preserved 10-20% of tissue compared to the 75% protection afforded by preconditioning (Yellon & Downey, 2003).

Research has determined that ischemic preconditioning can be subdivided into two distinctive types; *classical* and *delayed*. In *classical* preconditioning, the protective effects of ischemia/perfusion cycles are evident within minutes after the insult and persist for 2-3 hours (Ishida, et al., 1997). Classical preconditioning is independent of protein synthesis, and is therefore dependent upon existing cellular pathways. It involves the direct modulation of energy supplies, pH regulation, Na+ and Ca2+ homeostasis and caspase inactivation (Carini, & Albano, 2003). Investigations have shown many triggers can activate classical ischemic preconditioning, including agonists of G protein-coupled receptors [bradykinin (Goto, et al., 1995), opioids (Schultz, et al., 1998), norepinephrine (Hu & Nattel, 1995), adenosine (Liu, et al., 1991), potassium ATP channel (KATP) openers, such as diazoxide, pinacidil (Legtenberg, et al., 2002), succinate dehydrogenase inhibitors, such as 3 nitropropionic acid (Ockaili, et al., 2001), and volatile anesthetics, such as sevoflurane and isoflurane (Zaugg, et al., 2002)].

With classic preconditioning, a trigger event, such as brief ischemia, activates a number of intracellular pathways that lead to the protected cell phenotype. The actual sequence of these pathways has not been determined, but some components of the cascade have been identified. G-coupled receptors, for example, activate the epsilon (ε) isoform of protein kinase C (PKC-ε; Mitchell, et al., 1995; Kilts, et al., 2005), which has been implicated as the key PKC subtype involved in preconditioning. Also important is the upstream signaling molecule, phosphatidylinositol-3-kinase (PI3K; Tong, et al., 2000), and mitogen-activated protein kinases (MAPKs; Armstrong, 2004). PI3K activates the serine/threonine kinase, Akt, which inactivates the pro-apoptotic kinase glycogen synthase kinase-3 (GSK-3) via phosphorylation (Tong, et al., 2002). Phosphorylation of GSK-3, in turn, inhibits the opening of the mitochondrial permeability transition pore (mPTP). Cell apoptosis or necrosis often occurs during reperfusion due to opening of the mPTP, a large nonselective pore traversing both inner and outer membranes of mitochondria. Cytochrome c and apoptotic-inducing factor (AIF) are both released through the mPTP during ischemic reperfusion, leading to the activation of caspase and caspase-independent apoptotic pathways (Kadenbach, et al., 2011; Penninger, & Kroemer, 2003). KATP channels are key intracellular triggers of early ischemic preconditioning. This channel will be described in more detail later in this chapter.

despite the additional 20 minutes of ischemia in the preconditioned animals, cardiac damage, measured by infarct size, was significantly reduced to just ¼th of the infarct observed in the hearts of dogs in the control group. This paper was the first to demonstrate

Preconditioning has been demonstrated in all species studied to date (rat, mouse, rabbit, dog, human, chicken, sheep, pig and including cell lines), and in all organs studied thus far, including skeletal muscle, brain, kidney, small intestine, heart, and liver (Peralta, et al., 1996; Glazier, et al., 1994; Pang, et al., 1995). Other preconditioning manipulations, such as global hypoxia (Emerson, et al., 1999) and thermal injury (Marber, et al., 1993), are also effective in protection from lasting insult. Prior to this 1986 discovery, the best pharmacological treatments (with varying reproducible results) for the protection of cardiac muscle from infarction only preserved 10-20% of tissue compared to the 75% protection afforded by

Research has determined that ischemic preconditioning can be subdivided into two distinctive types; *classical* and *delayed*. In *classical* preconditioning, the protective effects of ischemia/perfusion cycles are evident within minutes after the insult and persist for 2-3 hours (Ishida, et al., 1997). Classical preconditioning is independent of protein synthesis, and is therefore dependent upon existing cellular pathways. It involves the direct modulation of energy supplies, pH regulation, Na+ and Ca2+ homeostasis and caspase inactivation (Carini, & Albano, 2003). Investigations have shown many triggers can activate classical ischemic preconditioning, including agonists of G protein-coupled receptors [bradykinin (Goto, et al., 1995), opioids (Schultz, et al., 1998), norepinephrine (Hu & Nattel, 1995), adenosine (Liu, et al., 1991), potassium ATP channel (KATP) openers, such as diazoxide, pinacidil (Legtenberg, et al., 2002), succinate dehydrogenase inhibitors, such as 3 nitropropionic acid (Ockaili, et al., 2001), and volatile anesthetics, such as sevoflurane and

With classic preconditioning, a trigger event, such as brief ischemia, activates a number of intracellular pathways that lead to the protected cell phenotype. The actual sequence of these pathways has not been determined, but some components of the cascade have been identified. G-coupled receptors, for example, activate the epsilon (ε) isoform of protein kinase C (PKC-ε; Mitchell, et al., 1995; Kilts, et al., 2005), which has been implicated as the key PKC subtype involved in preconditioning. Also important is the upstream signaling molecule, phosphatidylinositol-3-kinase (PI3K; Tong, et al., 2000), and mitogen-activated protein kinases (MAPKs; Armstrong, 2004). PI3K activates the serine/threonine kinase, Akt, which inactivates the pro-apoptotic kinase glycogen synthase kinase-3 (GSK-3) via phosphorylation (Tong, et al., 2002). Phosphorylation of GSK-3, in turn, inhibits the opening of the mitochondrial permeability transition pore (mPTP). Cell apoptosis or necrosis often occurs during reperfusion due to opening of the mPTP, a large nonselective pore traversing both inner and outer membranes of mitochondria. Cytochrome c and apoptotic-inducing factor (AIF) are both released through the mPTP during ischemic reperfusion, leading to the activation of caspase and caspase-independent apoptotic pathways (Kadenbach, et al., 2011; Penninger, & Kroemer, 2003). KATP channels are key intracellular triggers of early ischemic

preconditioning. This channel will be described in more detail later in this chapter.

and coin the term, *ischemic preconditioning*.

preconditioning (Yellon & Downey, 2003).

isoflurane (Zaugg, et al., 2002)].

**2.1 Preconditioning** 

Although not as effective as classical preconditioning, *delayed* or *late* preconditioning becomes apparent approximately 24 hours after initial preconditioning and it can persist for up to 72 hours (Ishida, et al., 1997). The most significant difference between classical and delayed preconditioning is the latter's requirement of the manufacture of new proteins needed to obtain protection; inhibition of new protein synthesis attenuates the protection derived from delayed preconditioning (Rizvi, et al., 1999).

The triggers for delayed preconditioning are similar to classical preconditioning. These include cellular stress factors (sub-lethal ischemia, heat stress, cardiac pacing), which release factors such as reactive oxygen species, adenosine, and endogenous nitric oxide (NO). NO has been determined to have no effect on classical preconditioning and is a trigger specific only for delayed preconditioning (Bolli, 2000). Endogenous preconditioning agents also initiate delayed preconditioning and include adenosine agonists, bradykinin, opioids, NO donors, acetylcholine and norepinephrine (Bolli, 2000). Exogenous agents that activate the preconditioning pathways include, diazoxide, nicorandil, some hypercholesterolemic agents, and volatile anesthetics (Gross, 2005).

Kinase intracellular pathways play a central role in delayed preconditioning, with activation of the PKC-ε isoform being particularly essential. Downstream to PKC-ε, tyrosine kinases and other kinases then activate the important transcription factor, NF-κB, which leads to the upregulation of many protective proteins in the nucleus. Some key proteins identified thus far include: inducible NO synthase (iNOS) (Takano, et al., 1998, cyclooxygenase (COX-2) (Shinmura, et al., 2000), the antioxidant enzyme superoxide dismutase (SOD) (Hoshida, et al., 2002), and heat shock proteins, HSP70, HSP25, and HSP32 (Bolli, 2000). Delayed preconditioning also reduces apoptosis by upregulating the anti-apoptotic protein, Bcl-2, which has been shown to inhibit opening of the mPTP, leading to cell survival (Maulik, et al., 1999). Mitochondrial KATP (mKATP) channel opening seems to be the final common pathway for these signaling pathways but it is not yet clear how the opening of these channels affords protection. Delayed preconditioning requires the opening of the mKATP channels during the ischemic event; its role after 24 hours, when delayed preconditioning occurs, is less clear (Takano, et al., 2000).

By 1993, an appreciable number of studies had confirmed that the direct application of various stimuli (hypoxia, ischemia, triggering agents) resulted in tissue and organ protection. In that year, however, Przyklenk and colleagues made a startling discovery. Using a canine model in which the circumflex artery was occluded (as in Murry's 1986 study), Przyklenk and colleagues observed that cardiac muscle supplied by the descending left coronary artery was also protected from ischemic insult, indicating that the preconditioning stimulus offered protection that was not confined to one area of an organ (Przyklenk, et al., 1993). Further experiments have shown *remote* classical ischemic preconditioning also is effective in other organs. A preconditioning trigger in one area of an organ offers protection to a different region of that same organ or to a different organ. For example, one group demonstrated that intermittent tourniquet application to a hind limb (ischemic preconditioning) implemented protection in other skeletal muscles (Addison, et al., 2003). Remote classical preconditioning of the heart was also obtained via transient ischemia of the small intestine (Liem, et al., 2005; Patel, et al., 2002) or the kidney (Gho, et al., 1996). Other studies have demonstrated remote delayed preconditioning. Induction of small intestine ischemia, for example, engenders myocardial protection 24-72 hours later (Wang, et al., 2001; Xiao, et al., 2001).

Preconditioning and Postconditioning 383

employment of mechanical and pharmacological postconditioning suggest the activation of mitochondrial KATP (mKATP) channels initiates a series of events that close the mitochondrial permeability transition pore (mPTP) and converge onto the Reperfusion Injury Survival

KATP channels were first discovered in 1983 by A. Noma in a patch clamp study using cardiac muscle membrane preparations (Noma, 1983). There are cell surface KATP (sKATP) and mitochondrial KATP (mKATP) forms. Pharmacologically, these are different channels, but their opening (via PKC or pharmacological agents) leads to increased cell survival. One hypothesis is that activation of KATP channels hyperpolarizes the cell membrane thereby protecting the cell from detrimental depolarization (Kirino, 2002). In 1997, Garlid and coworkers presented evidence that mKATP channels have a cardioprotectve role in ischemia and reperfusion, and were a component in the mechanism for preconditioning (Garlid, et al., 1997). A prototypical mKATP channel, as reviewed by Aguilar-Bryan and Bryan (1999), is an octameric structure consisting of four sulfonylurea receptor (SUR1 or SUR2) subunits and four K+ inward-rectifying (Kir6.1 or Kir6.2) subunits. Attached to the SUR subunits are two nucleotide binding domains (NBD). The mKATP channel is activated in low energy states by ADP, binding to NBDs, allowing the influx of K+ into the mitochondrial inner matrix. Conversely, the mKATP channel is inhibited in high energy states when ATP closes the Kir channel. In the brain, it appears the predominant subtypes are SUR2 and Kir6.2, although the SUR1 and Kir6.1 subunits are present in smaller amounts (Lacza, et al., 2003). The mKATP channel may trigger preconditioning or postconditioning via mechanisms dependent on matrix volume stabilization, respiratory inhibition, controlled production of reactive oxygen

The physiological functions of mKATP channels have been debated. The activities of the mKATP channel and the K+/H+ exchanger are believed to maintain K+ homeostasis within mitochondria by controlling mitochondrial volume and moderating the outer-to-inner pH gradient needed to drive ATP synthesis. In the presence of hypoxia, whole cell pH decreases and ATP production declines. This causes a switch to anaerobic metabolism. A decrease in pH combined with an increase in the AMP/ADP ratio secondary to ATP metabolism causes the mKATP channel to open allowing the influx of K+ into the inner matrix. This, in turn, activates the K+(out)/H+(in) exchanger decreasing the hydrogen gradient between the outer membrane and inner matrix (Szewczyk & Marbán, 1999). By doing so, the proton motive forces driving ATP production are attenuated and mitochondria energetics slow. During this time, the mPTP is closed as membrane stability and electrical potential are better maintained by the simultaneous activity of the mKATP channel and the K+/H+ exchanger. In addition, reactive oxygen species (ROS) generation is proportional to the availability of oxygen and activation of mKATP channel appears to moderate ROS production (Ferranti, et al., 2003; Saitoh, et al., 2006). However, this effect is only protective to a limited extent. For example, a moderate or controlled production of ROS signals promote prosurvival signaling

As anaerobic metabolism continues in response to an extended period of severe hypoxia, ATP hydrolysis exceeds ATP generation causing a dramatic rise in H+ within the cell and mitochondrial inner matrix. At some point H+ entry becomes lethal as it exceeds the outward pumping capacity of the mitochondrial electron transport chain already hindered

Kinase (RISK) Pathway.

species (ROS), and the closure of the mPTP.

while excessive ROS production promotes apoptotic signaling.

**3.1 KATP channels** 

The precise mechanism of remote ischemic preconditioning is unknown, but putative factors have been identified. Protection from kidney or intestinal preconditioning on cardiac muscle was eliminated with application of the ganglionic blocker, hexamethonium, suggesting involvement of a neuronal pathway (Gho, et al., 1996). However, stronger evidence exists that a humoral factor may play a more important role. Effluent from a preconditioned heart, transferred by whole blood transfusion, protected a non-conditioned heart from ischemic insult (Dickson, et al., 1999a; 1999b). Remote preconditioning was not activated by adenosine or bradykinin, but was found to be attenuated by the opioid antagonist, naloxone, suggesting opioid receptor involvement (Dickson, et al., 2001).

### **2.2 Postconditioning**

One promising approach for neuroprotective therapies may be derived from *postconditioning*, where supportive measures are employed following an injury. Postconditioning is very similar to preconditioning with the exception of the temporal relationship of the protective maneuver in respect to the prolonged period of ischemia. Preconditioning is an intervention that occurs *prior to* injury while postconditioning interventions occur *after* an injury has occurred and thus may be more clinically relevant. The origin of postconditioning stems from the work of Okamoto and colleagues (Okamoto, et al., 1986). Okamoto's group established that post-ischemic damage could be limited by the use of timely low-pressure reperfusion. Following a period of ischemia, dog hearts were reperfused either with the sudden release of a coronary occlusion, or by low-pressure (40 to 50 mm Hg) coronary reperfusion with normal blood for 20 minutes before completely removing the coronary occlusion. This maneuver focused on the initial stage of reperfusion and established the basis for novel postconditioning approaches to resuscitation. Years later, Mizumura et al. (1995) first demonstrated pharmacological postconditioning. Mizumura's group used the KATP channel opener, bimakalin. Bimakalin markedly reduced cardiac infarct size in dogs when given 10 minutes before and during the 60-minute coronary reperfusion period following a set time of occlusive hypoxia. Collectively, these initial findings firmly established the concept of postconditioning and emphasized a critical factor that altering the initial moments of reperfusion was beneficial.

In 2003, Zhao and colleagues (2003) first used the term *postconditioning*. They found in a model of occlusive hypoxia in dogs that short, repeated (or stuttered) periods of arterial occlusion and release of previously occluded coronary arteries (three occlusions of 30 seconds each) prior to restoration of perfusion reduced infarct area by 44% as compared to controls. This was an example of a mechanical postconditioning intervention and implied that the first minute of reperfusion is critical in thwarting cellular demise. These findings were further validated by Kin et al. (2004). Kin and colleagues stated that the first minute of reperfusion in the rat was crucial for postconditioning. Three cycles of 10 seconds of coronary occlusion and 10 seconds of coronary release, preceding a full coronary occlusion release, decreased cardiac infarct size by 23%. In the broadest sense, the cellular processes activated by postconditioning are analogous to those activated by preconditioning, and the sole difference between the two interventions is the timing related to the prolonged period of ischemia.

### **3. Signaling processes linked to preconditioning and postconditioning**

Many studies have investigated the mechanism of postconditioning with the aspiration to utilize this powerful protective system in the clinical setting. Recent observations from the employment of mechanical and pharmacological postconditioning suggest the activation of mitochondrial KATP (mKATP) channels initiates a series of events that close the mitochondrial permeability transition pore (mPTP) and converge onto the Reperfusion Injury Survival Kinase (RISK) Pathway.

### **3.1 KATP channels**

382 Advances in the Preclinical Study of Ischemic Stroke

The precise mechanism of remote ischemic preconditioning is unknown, but putative factors have been identified. Protection from kidney or intestinal preconditioning on cardiac muscle was eliminated with application of the ganglionic blocker, hexamethonium, suggesting involvement of a neuronal pathway (Gho, et al., 1996). However, stronger evidence exists that a humoral factor may play a more important role. Effluent from a preconditioned heart, transferred by whole blood transfusion, protected a non-conditioned heart from ischemic insult (Dickson, et al., 1999a; 1999b). Remote preconditioning was not activated by adenosine or bradykinin, but was found to be attenuated by the opioid antagonist,

One promising approach for neuroprotective therapies may be derived from *postconditioning*, where supportive measures are employed following an injury. Postconditioning is very similar to preconditioning with the exception of the temporal relationship of the protective maneuver in respect to the prolonged period of ischemia. Preconditioning is an intervention that occurs *prior to* injury while postconditioning interventions occur *after* an injury has occurred and thus may be more clinically relevant. The origin of postconditioning stems from the work of Okamoto and colleagues (Okamoto, et al., 1986). Okamoto's group established that post-ischemic damage could be limited by the use of timely low-pressure reperfusion. Following a period of ischemia, dog hearts were reperfused either with the sudden release of a coronary occlusion, or by low-pressure (40 to 50 mm Hg) coronary reperfusion with normal blood for 20 minutes before completely removing the coronary occlusion. This maneuver focused on the initial stage of reperfusion and established the basis for novel postconditioning approaches to resuscitation. Years later, Mizumura et al. (1995) first demonstrated pharmacological postconditioning. Mizumura's group used the KATP channel opener, bimakalin. Bimakalin markedly reduced cardiac infarct size in dogs when given 10 minutes before and during the 60-minute coronary reperfusion period following a set time of occlusive hypoxia. Collectively, these initial findings firmly established the concept of postconditioning and emphasized a critical factor

In 2003, Zhao and colleagues (2003) first used the term *postconditioning*. They found in a model of occlusive hypoxia in dogs that short, repeated (or stuttered) periods of arterial occlusion and release of previously occluded coronary arteries (three occlusions of 30 seconds each) prior to restoration of perfusion reduced infarct area by 44% as compared to controls. This was an example of a mechanical postconditioning intervention and implied that the first minute of reperfusion is critical in thwarting cellular demise. These findings were further validated by Kin et al. (2004). Kin and colleagues stated that the first minute of reperfusion in the rat was crucial for postconditioning. Three cycles of 10 seconds of coronary occlusion and 10 seconds of coronary release, preceding a full coronary occlusion release, decreased cardiac infarct size by 23%. In the broadest sense, the cellular processes activated by postconditioning are analogous to those activated by preconditioning, and the sole difference between the two

naloxone, suggesting opioid receptor involvement (Dickson, et al., 2001).

that altering the initial moments of reperfusion was beneficial.

interventions is the timing related to the prolonged period of ischemia.

**3. Signaling processes linked to preconditioning and postconditioning** 

Many studies have investigated the mechanism of postconditioning with the aspiration to utilize this powerful protective system in the clinical setting. Recent observations from the

**2.2 Postconditioning** 

KATP channels were first discovered in 1983 by A. Noma in a patch clamp study using cardiac muscle membrane preparations (Noma, 1983). There are cell surface KATP (sKATP) and mitochondrial KATP (mKATP) forms. Pharmacologically, these are different channels, but their opening (via PKC or pharmacological agents) leads to increased cell survival. One hypothesis is that activation of KATP channels hyperpolarizes the cell membrane thereby protecting the cell from detrimental depolarization (Kirino, 2002). In 1997, Garlid and coworkers presented evidence that mKATP channels have a cardioprotectve role in ischemia and reperfusion, and were a component in the mechanism for preconditioning (Garlid, et al., 1997). A prototypical mKATP channel, as reviewed by Aguilar-Bryan and Bryan (1999), is an octameric structure consisting of four sulfonylurea receptor (SUR1 or SUR2) subunits and four K+ inward-rectifying (Kir6.1 or Kir6.2) subunits. Attached to the SUR subunits are two nucleotide binding domains (NBD). The mKATP channel is activated in low energy states by ADP, binding to NBDs, allowing the influx of K+ into the mitochondrial inner matrix. Conversely, the mKATP channel is inhibited in high energy states when ATP closes the Kir channel. In the brain, it appears the predominant subtypes are SUR2 and Kir6.2, although the SUR1 and Kir6.1 subunits are present in smaller amounts (Lacza, et al., 2003). The mKATP channel may trigger preconditioning or postconditioning via mechanisms dependent on matrix volume stabilization, respiratory inhibition, controlled production of reactive oxygen species (ROS), and the closure of the mPTP.

The physiological functions of mKATP channels have been debated. The activities of the mKATP channel and the K+/H+ exchanger are believed to maintain K+ homeostasis within mitochondria by controlling mitochondrial volume and moderating the outer-to-inner pH gradient needed to drive ATP synthesis. In the presence of hypoxia, whole cell pH decreases and ATP production declines. This causes a switch to anaerobic metabolism. A decrease in pH combined with an increase in the AMP/ADP ratio secondary to ATP metabolism causes the mKATP channel to open allowing the influx of K+ into the inner matrix. This, in turn, activates the K+(out)/H+(in) exchanger decreasing the hydrogen gradient between the outer membrane and inner matrix (Szewczyk & Marbán, 1999). By doing so, the proton motive forces driving ATP production are attenuated and mitochondria energetics slow. During this time, the mPTP is closed as membrane stability and electrical potential are better maintained by the simultaneous activity of the mKATP channel and the K+/H+ exchanger. In addition, reactive oxygen species (ROS) generation is proportional to the availability of oxygen and activation of mKATP channel appears to moderate ROS production (Ferranti, et al., 2003; Saitoh, et al., 2006). However, this effect is only protective to a limited extent. For example, a moderate or controlled production of ROS signals promote prosurvival signaling while excessive ROS production promotes apoptotic signaling.

As anaerobic metabolism continues in response to an extended period of severe hypoxia, ATP hydrolysis exceeds ATP generation causing a dramatic rise in H+ within the cell and mitochondrial inner matrix. At some point H+ entry becomes lethal as it exceeds the outward pumping capacity of the mitochondrial electron transport chain already hindered

Preconditioning and Postconditioning 385

forces within the mitochondria inner matrix and leads to degradation of the matrix membrane, causing the release of apoptotic factors, especially cytochrome C (Honda, et al., 2005). Also, as the mitochondrial membrane potential is perturbed, ATP synthase reverses its primary function and serves as an ATPase; further depleting cellular ATP concentrations

Feng and colleagues (2005) determined that volatile anesthesia-induced postconditioning prevented the opening of the mPTP by inhibiting glycogen synthase kinase 3β (GSKβ). This inactivation was a result of PI3K-AKT signaling pathway inactivation with the resulting phosphorylation and inactivation of GSKβ, which protected against reperfusion damage. Argaud et al. (2005) found that mechanical postconditioning decreased cellular Ca2+ and protected *in vivo* rabbit hearts, suggesting that the mPTP could be inhibited by the PI3K-AKT-eNOS cascade. Bopassa et al. (2006), using a rat heart preparation undergoing postconditioning, concluded that PI3K signaling regulates the closure of mPTP. In addition, Cohen, Yan, and Downey (2007) observed that postconditioning prevented mPTP opening as a result of inhaled CO2-induced acidosis during the first minutes of reperfusion. They suggested that low cellular pH inhibits the opening of mPTP in heart tissue, but as the cellular pH normalizes, the inhibition of mPTP is lost. They hypothesized that by maintaining the cellular pH at a lower level while introducing oxygen during reperfusion, it was possible to keep the mPTP closed allowing the redox signaling necessary to trigger preconditioning-like protection. Cohen, Yan, and Downey further suggest that moderate acidosis during reperfusion might be protective. This hypothesis was addressed through the use of sodium bicarbonate (NaHCO3) during postconditioning. In isolated rabbit hearts, acidic CO2 perfusate at the time of reperfusion mimicked postconditioning while an alkaline NaHCO3 perfusate blocked that effect. They hypothesized that an acidic environment inhibited mPTP opening while an alkaline environment favored mPTP opening. Fujita and colleagues (2007) also hypothesized that NaHCO3 would blunt the protective properties of postconditioning. Using *in vivo* dog hearts that underwent ischemia, the administration of NaHCO3 during four intermittent cycles of one-minute reperfusion with one-minute reocclusion of a coronary vessel completely abolished the postconditioning effects. Their results suggested that postconditioning leads to the opening of mKATP channels as a result of

The Reperfusion Injury Survival Kinase (RISK) pathway begins with the activation of PI3K and ERK to promote cell survival. RISK can be activated by insulin, urocortin, atorvastatin, adenosine, bradykinin, opioid agonists, volatile anesthetics, or diazoxide (Bell & Yellon, 2003a,b; Chiari, et al., 2005; Gross, et al., 2004; Jonassen, et al., 2001; Schulman, et al., 2002; Wang, et al., 2004; Yang, et al., 2004). The RISK pathway promotes pro-survival signaling while inhibiting pathways associated with apoptosis. In 2004, Tsang and colleagues (2004) reported that in isolated rat hearts, which had undergone mechanical postconditioning following a period of ischemia, postconditioning is mediated by the PI3K-AKTeNOS/p70s6K pathway. They also suggested MEK 1/2-ERK 1/2 pathways were indirectly involved. Zhu and coworkers (2006) followed by finding that cardioprotection from postconditioning in the remodeled rat myocardium is regulated through PI3K-AKT signaling. The role of ERK 1/2 was addressed by Darling et al. (2005) and Krolikowski et al. (2006). Darling and colleagues utilized mechanical postconditioning in isolated rabbit hearts

decreased pH, leading to the attenuation of cardiac infarct size.

**3.3 Reperfusion Survival Kinase Pathway** 

and increasing H+ levels.

by anaerobic metabolism. This results in a total loss of proton motive force driving ATP production. If prolonged, this loss results in osmotic matrix swelling, mitochondrial degradation, and release of apoptotic proteins such as cytochrome C.

Upon resolution of a perfusion defect, abrupt reperfusion following prolonged ischemia results in a substantial amount of ROS generation. Following restoration of flow to intact but vulnerable cells, ATP levels begin to rise, mKATP channels close and K+ transport into the mitochondrial matrix declines. This indirectly decreases the activity of the K+/H+ exchanger. As H+ ions are rapidly removed from the matrix during mitochondrial respiration, the inner matrix quickly alkalinizes, causing the mPTP to open (Vinten-Johansen, et al., 2007). Opening the mPTP rapidly elevates inner matrix osmotic pressure leading to matrix distension and if allowed to remain open, mitochondrial rupture.

In total, mKATP channel closure associated with abrupt reperfusion cand result in the significant elevation of ROS and increase the mitochondrial inner matrix osmotic pressure, causing the mitochondria to quickly swell or rupture, releasing apoptotic factors such as cytochrome C (Armstrong, 2004). It is reasonable to suggest that maintaining the patency of the mKATP channel would be beneficial during reperfusion. Allowing the mKATP channel to remain open during reperfusion could: 1) moderate the generation of ROS, 2) reduce osmotic force within the matrix by promoting ion exchange, and 3) reduce the activity of the mPTP thereby providing a protective effect.

Activation of mKATP channels have been shown to be protective during reperfusion in cardiac and brain tissue (Obal, et al., 2005; O'Sullivan, et al., 2007; Penna, et al., 2007; Wu, et al., 2006). Obal and colleagues (2005), for example, demonstrated the utility of inhaling volatile anesthetics as a postconditioning trigger through mKATP channel activation. In rats subjected to cardiac ischemia, postconditioning was invoked by administering 1 minimum alveolar concentration (MAC) of sevoflurane for 2 minutes with the onset of reperfusion. This resulted in a significant decrease in cardiac infarct size. Penna et al. (2007, isolated rat hearts and exposed them to an ischemic period followed by reperfusion. Their results suggested that postconditioning mechanisms are activated by a bradykinin or a diazoxide mechanism resulting in the upregulation of protein kinase G (PKG). This upregulation was dependent on early ROS generation triggered by mKATP channel activation. They emphasized that their results were different from mechanical manipulations by showing that pharmacological agents, such as bradykinin or diazoxide, administered during the reperfusion period could induce protection. ROS also regulate the activity of heat shock proteins (HSPs). Using an *in vitro* vascular smooth muscle preparation, Madamanchi and colleagues (2001) discovered that the application of H2O2 significantly upregulated HSP70.

### **3.2 mPTPs**

As previously mentioned, the mPTP is inhibited with the activation of mKATP. The existence of the mPTP was confirmed in 1992 in rat liver mitoblast membranes (Szabó & Zoratti, 1992). The primary components of the mPTP are the voltage-dependent anion channel in the outer membrane, the adenine nucleotide translocator, and the cyclophilin D protein within the matrix (Lin & Lechleiter, 2002). In general, it is thought that the opening of the mPTP occurs with a decrease in the inner matrix potential, decreased AMP and ADP levels, increased matrix Ca2+, with alkalinization, or during oxidative stress (Gateau-Roesch, et al., 2006). mPTP opening blocks ATP formation and allows for the equilibration of small molecules (Gateau-Roesch, et al., 2006; Halestrap, 2004). mPTP opening increases osmotic

by anaerobic metabolism. This results in a total loss of proton motive force driving ATP production. If prolonged, this loss results in osmotic matrix swelling, mitochondrial

Upon resolution of a perfusion defect, abrupt reperfusion following prolonged ischemia results in a substantial amount of ROS generation. Following restoration of flow to intact but vulnerable cells, ATP levels begin to rise, mKATP channels close and K+ transport into the mitochondrial matrix declines. This indirectly decreases the activity of the K+/H+ exchanger. As H+ ions are rapidly removed from the matrix during mitochondrial respiration, the inner matrix quickly alkalinizes, causing the mPTP to open (Vinten-Johansen, et al., 2007). Opening the mPTP rapidly elevates inner matrix osmotic pressure leading to matrix

In total, mKATP channel closure associated with abrupt reperfusion cand result in the significant elevation of ROS and increase the mitochondrial inner matrix osmotic pressure, causing the mitochondria to quickly swell or rupture, releasing apoptotic factors such as cytochrome C (Armstrong, 2004). It is reasonable to suggest that maintaining the patency of the mKATP channel would be beneficial during reperfusion. Allowing the mKATP channel to remain open during reperfusion could: 1) moderate the generation of ROS, 2) reduce osmotic force within the matrix by promoting ion exchange, and 3) reduce the activity of the

Activation of mKATP channels have been shown to be protective during reperfusion in cardiac and brain tissue (Obal, et al., 2005; O'Sullivan, et al., 2007; Penna, et al., 2007; Wu, et al., 2006). Obal and colleagues (2005), for example, demonstrated the utility of inhaling volatile anesthetics as a postconditioning trigger through mKATP channel activation. In rats subjected to cardiac ischemia, postconditioning was invoked by administering 1 minimum alveolar concentration (MAC) of sevoflurane for 2 minutes with the onset of reperfusion. This resulted in a significant decrease in cardiac infarct size. Penna et al. (2007, isolated rat hearts and exposed them to an ischemic period followed by reperfusion. Their results suggested that postconditioning mechanisms are activated by a bradykinin or a diazoxide mechanism resulting in the upregulation of protein kinase G (PKG). This upregulation was dependent on early ROS generation triggered by mKATP channel activation. They emphasized that their results were different from mechanical manipulations by showing that pharmacological agents, such as bradykinin or diazoxide, administered during the reperfusion period could induce protection. ROS also regulate the activity of heat shock proteins (HSPs). Using an *in vitro* vascular smooth muscle preparation, Madamanchi and colleagues (2001) discovered that the application of H2O2 significantly upregulated HSP70.

As previously mentioned, the mPTP is inhibited with the activation of mKATP. The existence of the mPTP was confirmed in 1992 in rat liver mitoblast membranes (Szabó & Zoratti, 1992). The primary components of the mPTP are the voltage-dependent anion channel in the outer membrane, the adenine nucleotide translocator, and the cyclophilin D protein within the matrix (Lin & Lechleiter, 2002). In general, it is thought that the opening of the mPTP occurs with a decrease in the inner matrix potential, decreased AMP and ADP levels, increased matrix Ca2+, with alkalinization, or during oxidative stress (Gateau-Roesch, et al., 2006). mPTP opening blocks ATP formation and allows for the equilibration of small molecules (Gateau-Roesch, et al., 2006; Halestrap, 2004). mPTP opening increases osmotic

degradation, and release of apoptotic proteins such as cytochrome C.

distension and if allowed to remain open, mitochondrial rupture.

mPTP thereby providing a protective effect.

**3.2 mPTPs** 

forces within the mitochondria inner matrix and leads to degradation of the matrix membrane, causing the release of apoptotic factors, especially cytochrome C (Honda, et al., 2005). Also, as the mitochondrial membrane potential is perturbed, ATP synthase reverses its primary function and serves as an ATPase; further depleting cellular ATP concentrations and increasing H+ levels.

Feng and colleagues (2005) determined that volatile anesthesia-induced postconditioning prevented the opening of the mPTP by inhibiting glycogen synthase kinase 3β (GSKβ). This inactivation was a result of PI3K-AKT signaling pathway inactivation with the resulting phosphorylation and inactivation of GSKβ, which protected against reperfusion damage. Argaud et al. (2005) found that mechanical postconditioning decreased cellular Ca2+ and protected *in vivo* rabbit hearts, suggesting that the mPTP could be inhibited by the PI3K-AKT-eNOS cascade. Bopassa et al. (2006), using a rat heart preparation undergoing postconditioning, concluded that PI3K signaling regulates the closure of mPTP. In addition, Cohen, Yan, and Downey (2007) observed that postconditioning prevented mPTP opening as a result of inhaled CO2-induced acidosis during the first minutes of reperfusion. They suggested that low cellular pH inhibits the opening of mPTP in heart tissue, but as the cellular pH normalizes, the inhibition of mPTP is lost. They hypothesized that by maintaining the cellular pH at a lower level while introducing oxygen during reperfusion, it was possible to keep the mPTP closed allowing the redox signaling necessary to trigger preconditioning-like protection. Cohen, Yan, and Downey further suggest that moderate acidosis during reperfusion might be protective. This hypothesis was addressed through the use of sodium bicarbonate (NaHCO3) during postconditioning. In isolated rabbit hearts, acidic CO2 perfusate at the time of reperfusion mimicked postconditioning while an alkaline NaHCO3 perfusate blocked that effect. They hypothesized that an acidic environment inhibited mPTP opening while an alkaline environment favored mPTP opening. Fujita and colleagues (2007) also hypothesized that NaHCO3 would blunt the protective properties of postconditioning. Using *in vivo* dog hearts that underwent ischemia, the administration of NaHCO3 during four intermittent cycles of one-minute reperfusion with one-minute reocclusion of a coronary vessel completely abolished the postconditioning effects. Their results suggested that postconditioning leads to the opening of mKATP channels as a result of decreased pH, leading to the attenuation of cardiac infarct size.

### **3.3 Reperfusion Survival Kinase Pathway**

The Reperfusion Injury Survival Kinase (RISK) pathway begins with the activation of PI3K and ERK to promote cell survival. RISK can be activated by insulin, urocortin, atorvastatin, adenosine, bradykinin, opioid agonists, volatile anesthetics, or diazoxide (Bell & Yellon, 2003a,b; Chiari, et al., 2005; Gross, et al., 2004; Jonassen, et al., 2001; Schulman, et al., 2002; Wang, et al., 2004; Yang, et al., 2004). The RISK pathway promotes pro-survival signaling while inhibiting pathways associated with apoptosis. In 2004, Tsang and colleagues (2004) reported that in isolated rat hearts, which had undergone mechanical postconditioning following a period of ischemia, postconditioning is mediated by the PI3K-AKTeNOS/p70s6K pathway. They also suggested MEK 1/2-ERK 1/2 pathways were indirectly involved. Zhu and coworkers (2006) followed by finding that cardioprotection from postconditioning in the remodeled rat myocardium is regulated through PI3K-AKT signaling. The role of ERK 1/2 was addressed by Darling et al. (2005) and Krolikowski et al. (2006). Darling and colleagues utilized mechanical postconditioning in isolated rabbit hearts

Preconditioning and Postconditioning 387

interfering with the formation of the apoptotic protease activating factor-1 (APAF-1) cytochrome c multimeric apoptosome and the activation of procaspase 9 (Bruey, et al., 2000; Concannon, et al., 2001; Garrido, et al., 1999). HSP27 also directly interacts with procaspase-3, decreasing the activity of activated caspase-3 (Concannon, et al., 2001). HSP27 serves as a signaling messenger by causing the activation of serine/threonine kinase Akt thereby inhibiting Bcl-2 and caspase-9 (Cardone, et al., 1998). HSP25/27 has other actions. Phosphorylated HSP27 can stabilize F-actin and increase the number of cells retaining microfilament organization thus stabilizing membrane structure (Lavoie, et al., 1995). Additionally, HSP27 is able to increase glutathione levels, thereby reducing levels of ROS

Over the last three decades, HSP70 has become the most thoroughly investigated protein of the HSP family of proteins. Like HSP25, HSP70 can inhibit cell death at various sites within the cell. However, unlike HSP25, HSP70 function is "ATP-dependent." HSP70 is typically found *in vivo* bound by ATP and HSP70 function is typically based upon the hydrolysis of the attached ATP molecule. HSP70 serves as a chaperone protein, inhibits stress signaling, prevents mitochondrial membrane permeabilization, and inhibits apoptotic pathways. HSP70 may chaperone kinases by binding to an unfolded carboxyl terminus, preventing aggregation, and allowing re-autophosphorylation of the kinase enzyme; thus stabilizing the enzyme and restoring function (Gao & Newton, 2002). HSP70 also binds the death receptors, DR4 and DR5, inhibiting Apo-2L/TRAIL-induced cell death (Guo, et al., (2005), and HSP70 blocks Bax translocation into the mitochondrial outer membrane. The latter effect prevents the permeabilization of the mitochondrial membrane and subsequent release of apoptosisinducing factor (AIF) and cytochrome C (Stankiewicz , et al., 2005). HSP70 binds AIF within the cytosol; inhibiting its nuclear translocation and limiting nuclear condensation (Ruchalski, et al., 2006). Similar to HSP25, HSP70 prevents cell death by binding to Apaf-1 and interfering in the formation of the apoptosome complex and subsequent recruitment of procaspase-9 (Beere, et al., 2000). Lastly, HSP70 suppresses apoptotic signaling by binding precursor forms of caspase-3 and caspase-7; preventing their cleavage and activation

Both HSP25 and HSP70 inhibit the cleavage of caspase-3 (Concannon, et al., 2001; Komarova, et al., 2004). Cleaved caspase-3 (CC3) is a primary executioner of apoptosis as it is responsible for the total or partial proteolytic cleavage of numerous key cellular survival proteins (Fernandes-Alnemri , et al., 1994). One of those proteins being the abundant nuclear enzyme polymerase, which functions in DNA repair and protein modification during oxidative stress (Smith, 2001). Thus, induction of HSP25 and HSP70 may alleviate cerebral ischemic injury and resuscitation injury that results from the mitochondrial release of

Wu and colleagues (Wu, et al., 2006) directly examined the roles of mPTP and the mKATP channel in preconditioning and postconditioning in a rat model of cerebral stroke. These

**4. Evidence of preconditioning and postconditioning in the brain** 

(Kretz-Remy, et al., 1996).

(Komarova, et al., 2004).

**3.5 Cleaved caspase 3** 

cytochrome C with subsequent cleavage of caspase-3.

**3.4.2 Heat Shock Protein 70** 

and found ERK1/2 but not PI3K activity provided cardiac protection. Krolikowski and colleagues exposed rabbits to isoflurane before and during early reperfusion and suggested a central role of ERK1/2, p70s6k, and eNOS in anesthetic-induced postconditioning.

Downstream in the RISK pathway, phosphorylation of AKT occurs with the subsequent phosphorylation of protein kinase C (PKC) and GSKβ. When PKC is phosphorylated it is stimulated while the phosphorylation of GSKβ inhibits its activity. In a rabbit model, Philipp and colleagues (2006) demonstrated through inhibitor studies that adenosine, PKC, and PI3K mediated the effects of mechanical postconditioning. In their investigation, they concluded that protection was conferred through the activation of adenosine receptors by endogenous adenosine, a cellular metabolite. This, in turn, activated the PI3K component of the RISK pathway resulting in activation of PKC. In regards to GSKβ, Feng and colleagues (2005), using isoflurane as a postconditioning trigger along with an AKT inhibitor, showed that when inhaled early in reperfusion, isoflurane phosphorylated AKT and GSKβ. Phosphorylated GSKβ was inhibited and could not promote the opening of mPTP. They also determined that while the PI3K-AKT signal was strong, the ERK1/2-p38 MAPK was not altered. This suggests a primary role of PI3K-AKT in the RISK pathway and in mPTP closure. Recently, in human tissue, it has been found that the cytoprotective proteins, HSP25 and HSP70, are upregulated by the PI3K-AKT pathway (Dickson, et al., 2001).

### **3.4 The heat shock response**

Both preconditioning and postconditioning upregulate proteins identified as Heat Shock Proteins (HSPs), specifically HSP25 and HSP70. The heat shock response was discovered in 1962. *Drosophilia* larvae, when heated, developed puffing patterns in certain chromosomal regions. This suggested a change in the synthetic activity of the chromosomal bands concerned (Ritossa, 1962). Sixteen years later, the RNA for *Drosophilia* exposed to a thermal stimulus was coded using hybrid-arrested translation and indicated that proteins of 83, 72, 70, 68, 28, 26, 23 and 22 kilodaltons were upregulated (Livak, et al., 1978). Over the following decades, the investigation of the heat shock response has confirmed that a family of highly conserved HSPs is upregulated following a variety of sublethal stressors, possibly as a result of non-native proteins accumulating in a stressed cell (Voellmy & Boellmann, 2007). These proteins are subcategorized by their molecular weight and are either inherently present or can be induced following sublethal stress (O'Sullivan, et al., 2008). In particular, HSP25 and HSP70 have been thoroughly investigated with the consensus being they are protective when upregulated following stress (Beere, et al., 2000; Garrido, et al., 2006; Takayama, et al., 2003).

### **3.4.1 Heat Shock Protein 25/27**

HSP25 is the rodent equivalent of the primate HSP27 and often the terms are used interchangeably. HSP27 confers protection at different levels as it can interact with several proteins implicated in cell death based upon its phosphorylation and oligomerization condition and not upon ATP. The main mechanisms for how HSP27 confers cytoprotection appear to be: molecular chaperoning, interference with cell death pathways, signaling of antiapoptotic pathways, stabilization of the cytoskeleton, and antioxidant activities. Serving as a chaperone, HSP27 can bind folded intermediate non-native proteins, inhibiting their aggregation, and in the presence of HSP70 these HSP27-bound proteins can be reactivated (Ehrnsperger, et al., 1997). Within the cytosol, HSP27 can sequester cytochrome C;

and found ERK1/2 but not PI3K activity provided cardiac protection. Krolikowski and colleagues exposed rabbits to isoflurane before and during early reperfusion and suggested

Downstream in the RISK pathway, phosphorylation of AKT occurs with the subsequent phosphorylation of protein kinase C (PKC) and GSKβ. When PKC is phosphorylated it is stimulated while the phosphorylation of GSKβ inhibits its activity. In a rabbit model, Philipp and colleagues (2006) demonstrated through inhibitor studies that adenosine, PKC, and PI3K mediated the effects of mechanical postconditioning. In their investigation, they concluded that protection was conferred through the activation of adenosine receptors by endogenous adenosine, a cellular metabolite. This, in turn, activated the PI3K component of the RISK pathway resulting in activation of PKC. In regards to GSKβ, Feng and colleagues (2005), using isoflurane as a postconditioning trigger along with an AKT inhibitor, showed that when inhaled early in reperfusion, isoflurane phosphorylated AKT and GSKβ. Phosphorylated GSKβ was inhibited and could not promote the opening of mPTP. They also determined that while the PI3K-AKT signal was strong, the ERK1/2-p38 MAPK was not altered. This suggests a primary role of PI3K-AKT in the RISK pathway and in mPTP closure. Recently, in human tissue, it has been found that the cytoprotective proteins, HSP25

a central role of ERK1/2, p70s6k, and eNOS in anesthetic-induced postconditioning.

and HSP70, are upregulated by the PI3K-AKT pathway (Dickson, et al., 2001).

Both preconditioning and postconditioning upregulate proteins identified as Heat Shock Proteins (HSPs), specifically HSP25 and HSP70. The heat shock response was discovered in 1962. *Drosophilia* larvae, when heated, developed puffing patterns in certain chromosomal regions. This suggested a change in the synthetic activity of the chromosomal bands concerned (Ritossa, 1962). Sixteen years later, the RNA for *Drosophilia* exposed to a thermal stimulus was coded using hybrid-arrested translation and indicated that proteins of 83, 72, 70, 68, 28, 26, 23 and 22 kilodaltons were upregulated (Livak, et al., 1978). Over the following decades, the investigation of the heat shock response has confirmed that a family of highly conserved HSPs is upregulated following a variety of sublethal stressors, possibly as a result of non-native proteins accumulating in a stressed cell (Voellmy & Boellmann, 2007). These proteins are subcategorized by their molecular weight and are either inherently present or can be induced following sublethal stress (O'Sullivan, et al., 2008). In particular, HSP25 and HSP70 have been thoroughly investigated with the consensus being they are protective when upregulated following stress (Beere, et al., 2000; Garrido, et al., 2006;

HSP25 is the rodent equivalent of the primate HSP27 and often the terms are used interchangeably. HSP27 confers protection at different levels as it can interact with several proteins implicated in cell death based upon its phosphorylation and oligomerization condition and not upon ATP. The main mechanisms for how HSP27 confers cytoprotection appear to be: molecular chaperoning, interference with cell death pathways, signaling of antiapoptotic pathways, stabilization of the cytoskeleton, and antioxidant activities. Serving as a chaperone, HSP27 can bind folded intermediate non-native proteins, inhibiting their aggregation, and in the presence of HSP70 these HSP27-bound proteins can be reactivated (Ehrnsperger, et al., 1997). Within the cytosol, HSP27 can sequester cytochrome C;

**3.4 The heat shock response** 

Takayama, et al., 2003).

**3.4.1 Heat Shock Protein 25/27** 

interfering with the formation of the apoptotic protease activating factor-1 (APAF-1) cytochrome c multimeric apoptosome and the activation of procaspase 9 (Bruey, et al., 2000; Concannon, et al., 2001; Garrido, et al., 1999). HSP27 also directly interacts with procaspase-3, decreasing the activity of activated caspase-3 (Concannon, et al., 2001). HSP27 serves as a signaling messenger by causing the activation of serine/threonine kinase Akt thereby inhibiting Bcl-2 and caspase-9 (Cardone, et al., 1998). HSP25/27 has other actions. Phosphorylated HSP27 can stabilize F-actin and increase the number of cells retaining microfilament organization thus stabilizing membrane structure (Lavoie, et al., 1995). Additionally, HSP27 is able to increase glutathione levels, thereby reducing levels of ROS (Kretz-Remy, et al., 1996).

### **3.4.2 Heat Shock Protein 70**

Over the last three decades, HSP70 has become the most thoroughly investigated protein of the HSP family of proteins. Like HSP25, HSP70 can inhibit cell death at various sites within the cell. However, unlike HSP25, HSP70 function is "ATP-dependent." HSP70 is typically found *in vivo* bound by ATP and HSP70 function is typically based upon the hydrolysis of the attached ATP molecule. HSP70 serves as a chaperone protein, inhibits stress signaling, prevents mitochondrial membrane permeabilization, and inhibits apoptotic pathways. HSP70 may chaperone kinases by binding to an unfolded carboxyl terminus, preventing aggregation, and allowing re-autophosphorylation of the kinase enzyme; thus stabilizing the enzyme and restoring function (Gao & Newton, 2002). HSP70 also binds the death receptors, DR4 and DR5, inhibiting Apo-2L/TRAIL-induced cell death (Guo, et al., (2005), and HSP70 blocks Bax translocation into the mitochondrial outer membrane. The latter effect prevents the permeabilization of the mitochondrial membrane and subsequent release of apoptosisinducing factor (AIF) and cytochrome C (Stankiewicz , et al., 2005). HSP70 binds AIF within the cytosol; inhibiting its nuclear translocation and limiting nuclear condensation (Ruchalski, et al., 2006). Similar to HSP25, HSP70 prevents cell death by binding to Apaf-1 and interfering in the formation of the apoptosome complex and subsequent recruitment of procaspase-9 (Beere, et al., 2000). Lastly, HSP70 suppresses apoptotic signaling by binding precursor forms of caspase-3 and caspase-7; preventing their cleavage and activation (Komarova, et al., 2004).

### **3.5 Cleaved caspase 3**

Both HSP25 and HSP70 inhibit the cleavage of caspase-3 (Concannon, et al., 2001; Komarova, et al., 2004). Cleaved caspase-3 (CC3) is a primary executioner of apoptosis as it is responsible for the total or partial proteolytic cleavage of numerous key cellular survival proteins (Fernandes-Alnemri , et al., 1994). One of those proteins being the abundant nuclear enzyme polymerase, which functions in DNA repair and protein modification during oxidative stress (Smith, 2001). Thus, induction of HSP25 and HSP70 may alleviate cerebral ischemic injury and resuscitation injury that results from the mitochondrial release of cytochrome C with subsequent cleavage of caspase-3.

### **4. Evidence of preconditioning and postconditioning in the brain**

Wu and colleagues (Wu, et al., 2006) directly examined the roles of mPTP and the mKATP channel in preconditioning and postconditioning in a rat model of cerebral stroke. These

Preconditioning and Postconditioning 389

postconditioning neuroprotection for hippocampal CA1 neurons and improved spatial learning and memory in rats. This protection was dependent upon DADLE-induced

As reviewed, the majority of research related to pre- and post-conditioning has not been performed in studies related to cerebral ischemia. As recently stated by Keep and colleagues (2010), the question remains—"Is there a place for cerebral preconditioning in the clinic?" The clinical utility of cerebral conditioning is potentially limited by issues of safety, the relatively narrow therapeutic window, and the need to present the stimulus before the

Brief periods of ischemia can enact classical and delayed conditioning. These momentary periods of ischemia have been shown to protect neuronal cells *in vitro* and to reduce injury *in vivo* in several experiment models and species (Koch, 2010). Since safety issues prevent deliberately inducing conditioning by cerebrovascular occlusion, research has focused on pharmacological agents, including volatile anesthetics, inhibitors of cellular metabolism, KATP channel activators, and inflammatory mediators (Keep, et al., 2010). Other agents that have been effective in producing conditioning are hyperbaric oxygen, cooling and hyperthermia, and acupuncture (Keep, et al., 2010). Recent research has also given credence to remote conditioning where ischemia to a hindlimb (e.g., by application of a tourniquet) protects the brain from later middle cerebral artery occlusion. Remote preconditioning or ischemia probably has the most practical use for clinical utilization. However, currently there are no clinical data to strongly support the use of any type of conditioning for brain

From a clinical standpoint, a major problem with the application of conditioning is timing. With the exception of a planned neurosurgical intervention, classically employed technique such as vascular clamping is impractible as a pretreatment. Pharmacological agents, then. Given at the time of reperfusion may hold promise. Agents such as MgSO4, erythropoietin, anti-hypertension drugs, anticoagulants, and statins all given to patients at risk for stroke

In addition, there is still little *clinical* evidence from basic research regarding the use of preconditioning for neuroprotection. Research models currently in use have at least four important limitations. First, experiments are routinely conducted on young, disease-free animals (Koch, 2010). The majority of patients who suffer cerebral ischemic events are older, and may have artherosclerosis, cardiac or kidney disease, or other co-morbidities, such as obesity, hypertension, diabetes, as well as additional risk factors such as sedentary lifestyle, and tobacco, alcohol, or illicit drug abuse. The 'chronic' ischemic state of these patients, with a chronic conditioning compensatory state, may not allow further conditioning protection with interventions. Secondly, both Keep, et al. (2010) and Koch (2010) noted that the effect of medications used by patients has a potential to interfere with preconditioning effects. Do certain prescribed medications or self-administered substances such as herbal products interfere with the conditioning signaling pathways? Third, the neuroprotective cascade might be very specific to gender, diet, genetic background, and age (Dirnagl, et al., 2009). Lastly, major issues to be resolved include determination of doses of preconditioning drugs that are safe and whether premorbid conditions, for example intermittent transient ischemic

have shown limited damage from a stroke should it occur (Keep, et al., 2010).

activation of the PI3K/Akt signaling.

**5. Conclusion** 

injury.

protection.

investigators activated the mKATP channel with diazoxide 20 minutes before middle cerebral artery occlusion followed by reperfusion, or inhibited the mPTP by infusion of cyclosporin A 15 minutes before reperfusion. It was discovered that both measures significantly increased functional performance scores and reduced infarction volumes. Importantly, both of these effects were abolished by blocking the adenine nucleotide port located on the mPTP. Their results strongly suggested that the mKATP channel and mPTP activity during reperfusion share a common protective pathway; the Reperfusion Survival Kinase Pathway (RISK). More recently, Feng, Rhodes, and Bhatt (2010) discovered that hypoxic preconditioning could invoke neuroprotection through the activation of AKT, a kinase that is part of the aforementioned RISK pathway. These investigators subjected newborn rats to 3 hours of 8% oxygen followed by 24 hours of reoxygenation. Following reoxygenation, the right carotid artery was permanently ligated and again the rats were subjected to 8% oxygen but for 140 minutes instead of 3 hours. Compared to rats subjected to normoxia prior to carotid ligation, preconditioned rats had a significant reduction in cerebral injury. It was found that preconditioning preserved RISK pathway signaling and attenuated caspase-3 activity.

Acute models of postconditioning have emphasized the benefit of cerebral reperfusion under controlled conditions. For example, several groups have shown that carefully controlled periods of reperfusion, before the full return of cerebral circulation, results in reduced injury. Zhao and colleagues (2006) employed permanent middle cerebral artery occlusion in combination with transient common carotid artery occlusions. Shorter periods of repeated common carotid occlusion resulted in a reduction in infarct size. Pignataro, et al. (2008) also employed middle cerebral artery occlusion for 100 minutes. Reperfusion of the artery that included a 10-minute period of occlusion was found to be the most effective, although intermittent occlusions were also beneficial. Gao, Ren and Zhao (2008) found that three cycles of reperfusion of the common carotid artery, in conjunction with permanent middle artery occlusion, reduced infarct size, while ten cycles was not effective. These publications, as well as numerous reports with cardiac models, emphasis the criticality of the duration of cerebral ischemia (longer periods of ischemia result in more cerebral damage, including irreversibility), as well as the essential specifics of the timing, duration, number of cycles, and inter-reperfusion intervals for effective postconditioning.

As well, recent work has shown the benefit of pharmacological postconditioning in cerebral ischemia. O'Sullivan and colleagues (2007) employed a rat model of combined hemorrhagic shock and permanent unilateral common carotid artery occlusion. The administration of diazoxide at the time of hemorrhagic resuscitation significantly increased the expression of heat shock proteins in the cerebral cortex and hippocampus. Robin and colleagues (2011), using a middle cerebral artery occlusion model, found that in Wistar strain rats ischemic postconditioning decreased infarct size by 40% and improved neurological outcomes. Specifically, pharmacological postconditioning by diazoxide administration decreased cerebral infarct by 60%. In addition, these beneficial effects in both ischemic postconditioning and diazoxide postconditioning were blocked through the use of the KATP blocker, 5-hydroxydecanoate (5-HD), which blocked the inhibition of the mPTP opening caused by ischemic postconditioning and diazoxide.

In 2011, Wang and colleagues discovered that selective delta opioid peptide [D-Ala2, D-Leu5] enkephalin (DADLE) provided a postconditioning effect by protecting hippocampal CA1 neurons in a model of forebrain ischemia. In this investigation, DADLE triggered postconditioning neuroprotection for hippocampal CA1 neurons and improved spatial learning and memory in rats. This protection was dependent upon DADLE-induced activation of the PI3K/Akt signaling.

### **5. Conclusion**

388 Advances in the Preclinical Study of Ischemic Stroke

investigators activated the mKATP channel with diazoxide 20 minutes before middle cerebral artery occlusion followed by reperfusion, or inhibited the mPTP by infusion of cyclosporin A 15 minutes before reperfusion. It was discovered that both measures significantly increased functional performance scores and reduced infarction volumes. Importantly, both of these effects were abolished by blocking the adenine nucleotide port located on the mPTP. Their results strongly suggested that the mKATP channel and mPTP activity during reperfusion share a common protective pathway; the Reperfusion Survival Kinase Pathway (RISK). More recently, Feng, Rhodes, and Bhatt (2010) discovered that hypoxic preconditioning could invoke neuroprotection through the activation of AKT, a kinase that is part of the aforementioned RISK pathway. These investigators subjected newborn rats to 3 hours of 8% oxygen followed by 24 hours of reoxygenation. Following reoxygenation, the right carotid artery was permanently ligated and again the rats were subjected to 8% oxygen but for 140 minutes instead of 3 hours. Compared to rats subjected to normoxia prior to carotid ligation, preconditioned rats had a significant reduction in cerebral injury. It was found that preconditioning preserved RISK pathway signaling and attenuated caspase-3

Acute models of postconditioning have emphasized the benefit of cerebral reperfusion under controlled conditions. For example, several groups have shown that carefully controlled periods of reperfusion, before the full return of cerebral circulation, results in reduced injury. Zhao and colleagues (2006) employed permanent middle cerebral artery occlusion in combination with transient common carotid artery occlusions. Shorter periods of repeated common carotid occlusion resulted in a reduction in infarct size. Pignataro, et al. (2008) also employed middle cerebral artery occlusion for 100 minutes. Reperfusion of the artery that included a 10-minute period of occlusion was found to be the most effective, although intermittent occlusions were also beneficial. Gao, Ren and Zhao (2008) found that three cycles of reperfusion of the common carotid artery, in conjunction with permanent middle artery occlusion, reduced infarct size, while ten cycles was not effective. These publications, as well as numerous reports with cardiac models, emphasis the criticality of the duration of cerebral ischemia (longer periods of ischemia result in more cerebral damage, including irreversibility), as well as the essential specifics of the timing, duration,

number of cycles, and inter-reperfusion intervals for effective postconditioning.

caused by ischemic postconditioning and diazoxide.

As well, recent work has shown the benefit of pharmacological postconditioning in cerebral ischemia. O'Sullivan and colleagues (2007) employed a rat model of combined hemorrhagic shock and permanent unilateral common carotid artery occlusion. The administration of diazoxide at the time of hemorrhagic resuscitation significantly increased the expression of heat shock proteins in the cerebral cortex and hippocampus. Robin and colleagues (2011), using a middle cerebral artery occlusion model, found that in Wistar strain rats ischemic postconditioning decreased infarct size by 40% and improved neurological outcomes. Specifically, pharmacological postconditioning by diazoxide administration decreased cerebral infarct by 60%. In addition, these beneficial effects in both ischemic postconditioning and diazoxide postconditioning were blocked through the use of the KATP blocker, 5-hydroxydecanoate (5-HD), which blocked the inhibition of the mPTP opening

In 2011, Wang and colleagues discovered that selective delta opioid peptide [D-Ala2, D-Leu5] enkephalin (DADLE) provided a postconditioning effect by protecting hippocampal CA1 neurons in a model of forebrain ischemia. In this investigation, DADLE triggered

activity.

As reviewed, the majority of research related to pre- and post-conditioning has not been performed in studies related to cerebral ischemia. As recently stated by Keep and colleagues (2010), the question remains—"Is there a place for cerebral preconditioning in the clinic?" The clinical utility of cerebral conditioning is potentially limited by issues of safety, the relatively narrow therapeutic window, and the need to present the stimulus before the injury.

Brief periods of ischemia can enact classical and delayed conditioning. These momentary periods of ischemia have been shown to protect neuronal cells *in vitro* and to reduce injury *in vivo* in several experiment models and species (Koch, 2010). Since safety issues prevent deliberately inducing conditioning by cerebrovascular occlusion, research has focused on pharmacological agents, including volatile anesthetics, inhibitors of cellular metabolism, KATP channel activators, and inflammatory mediators (Keep, et al., 2010). Other agents that have been effective in producing conditioning are hyperbaric oxygen, cooling and hyperthermia, and acupuncture (Keep, et al., 2010). Recent research has also given credence to remote conditioning where ischemia to a hindlimb (e.g., by application of a tourniquet) protects the brain from later middle cerebral artery occlusion. Remote preconditioning or ischemia probably has the most practical use for clinical utilization. However, currently there are no clinical data to strongly support the use of any type of conditioning for brain protection.

From a clinical standpoint, a major problem with the application of conditioning is timing. With the exception of a planned neurosurgical intervention, classically employed technique such as vascular clamping is impractible as a pretreatment. Pharmacological agents, then. Given at the time of reperfusion may hold promise. Agents such as MgSO4, erythropoietin, anti-hypertension drugs, anticoagulants, and statins all given to patients at risk for stroke have shown limited damage from a stroke should it occur (Keep, et al., 2010).

In addition, there is still little *clinical* evidence from basic research regarding the use of preconditioning for neuroprotection. Research models currently in use have at least four important limitations. First, experiments are routinely conducted on young, disease-free animals (Koch, 2010). The majority of patients who suffer cerebral ischemic events are older, and may have artherosclerosis, cardiac or kidney disease, or other co-morbidities, such as obesity, hypertension, diabetes, as well as additional risk factors such as sedentary lifestyle, and tobacco, alcohol, or illicit drug abuse. The 'chronic' ischemic state of these patients, with a chronic conditioning compensatory state, may not allow further conditioning protection with interventions. Secondly, both Keep, et al. (2010) and Koch (2010) noted that the effect of medications used by patients has a potential to interfere with preconditioning effects. Do certain prescribed medications or self-administered substances such as herbal products interfere with the conditioning signaling pathways? Third, the neuroprotective cascade might be very specific to gender, diet, genetic background, and age (Dirnagl, et al., 2009). Lastly, major issues to be resolved include determination of doses of preconditioning drugs that are safe and whether premorbid conditions, for example intermittent transient ischemic

Preconditioning and Postconditioning 391

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Finally, optimal neuroprotection may be a combination of physiological manipulations (e.g., body temperature regulation) and pharmacological treatment(s). Gidday (2010) provides an excellent overview of the current state of pharmacological approaches for neuroprotection. Related to the present review, the translational possibilities require continued bench science to characterize the signal transduction pathways mediating neuroprotection, and whether or not they have potential clinical applicability. There are many "gaps" in understanding the mechanisms of action of the >20 drugs presently known to be beneficial (Gidday, 2010), and we must determine how best to employ these agents.

The landmark study by Murry and colleagues on cardiac tissue heralded new and exciting research regarding classic and delayed and remote preconditioning as well as the more clinically important postconditioning effect. Research continues with pharmacological or physical manipulations that can mimic pre- or post- conditioning and this could eventually have significant clinical ramifications. Further work is needed that considers the aforementioned limitations. Reducing the long-term effect of stroke or traumatic brain injury by preserving ischemic tissue can vastly improve the quality of life for patients. Likewise, billions of dollars saved from long-term care requirements, lost wages, family care-giver issues, and the reduced burden on our health care system will all stand to benefit from progress in this critical field of study.

### **6. Acknowledgments**

The views expressed in this work are those of the authors and do not reflect those of the United States Army, Department of Defense, or the United States Governmental Institution.

### **7. References**


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Finally, optimal neuroprotection may be a combination of physiological manipulations (e.g., body temperature regulation) and pharmacological treatment(s). Gidday (2010) provides an excellent overview of the current state of pharmacological approaches for neuroprotection. Related to the present review, the translational possibilities require continued bench science to characterize the signal transduction pathways mediating neuroprotection, and whether or not they have potential clinical applicability. There are many "gaps" in understanding the mechanisms of action of the >20 drugs presently known to be beneficial (Gidday, 2010), and

The landmark study by Murry and colleagues on cardiac tissue heralded new and exciting research regarding classic and delayed and remote preconditioning as well as the more clinically important postconditioning effect. Research continues with pharmacological or physical manipulations that can mimic pre- or post- conditioning and this could eventually have significant clinical ramifications. Further work is needed that considers the aforementioned limitations. Reducing the long-term effect of stroke or traumatic brain injury by preserving ischemic tissue can vastly improve the quality of life for patients. Likewise, billions of dollars saved from long-term care requirements, lost wages, family care-giver issues, and the reduced burden on our health care system will all stand to benefit

The views expressed in this work are those of the authors and do not reflect those of the United States Army, Department of Defense, or the United States Governmental Institution.

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**17** 

*Italy* 

**Could Mannitol-Induced Delay of Anoxic** 

Maurizio Balestrino\*, Enrico Adriano and Patrizia Garbati

**Depolarization be Relevant in Stroke Patients?** 

The use of hyperosmotic agents in stroke is still a matter of debate, since their usefulness has repeatedely been suggested but not conclusively demonstrated (Righetti E et al., 2002). Better understanding of the possible mechanism of protection by hyperosmotic agents may help identifying clinical situations where they may be more useful. It is generally assumed that their effect in stroke is due to their capacity to reduce brain edema. However, increasing extracellular osmolarity has direct effects on neuronal electrical function (Osehobo and Andrew, 1993; Rudehill et al., 1993), and one of us has previously reported that adding mannitol to the perfusing medium of brain slices delays anoxic depolarization (AD) (Balestrino, 1995a; Balestrino, 1995b). Since the latter is a factor in causing neuronal damage in anoxia and ischemia (Balestrino and Somjen, 1986; Jarvis et al., 2001; Kaminogo et al., 1998; Somjen et al., 1990), this may be another mechanism of brain protection by hyperosmotic agents in stroke. This study investigates whether or not this delay occurs at values of hyperosmolarity that may be obtained in clinical practice. We first carried out a survey of the literature on osmolarity changes after administration of hyperosmotic agents in vivo, under both clinical and experimental conditions. Then, we did a dose-response study of mannitol-induced delay of AD. Finally we compared the two sets of data to gauge whether or not mannitol-induced delay of AD occurs in the range of hyperosmolarity that

Sprague-Dawley female rats (155-190g.) were anaesthetised with ether and decapitated. The left hippocampus was dissected free and cut in 600 µm thick transversal slices. Slices were immediately transferred into an "interface" recording chamber (Fine Science Tools, Vancouver B. C. Canada) and incubated at 35±1°C. They were bathed by Artificial CerebroSpinal Fluid (ACSF) flowing at 2 ml/min and having the following composition: NaCl 130 mM, KCl 3.5 mM, NaH2PO4 1.25 mM, NaHCO3 24 mM, CaCl2 2.4 mM, MgSO4 1.2 mM, glucose 10 mM. This medium was continuously bubbled with 95% O2 / 5% CO2, resulting in a pH of 7.35-7.40. The same warmed, humidified 95% O2 / 5% CO2 mixture aerated the slices representing the gas phase. Anoxia was induced by replacing oxygen with

**1. Introduction** 

might be obtained in clinical practice.

**2. Materials and methods** 

Corresponding Author

 \*

*Department of Neuroscience, Ophtalmology and Genetics, University of Genova,* 


## **Could Mannitol-Induced Delay of Anoxic Depolarization be Relevant in Stroke Patients?**

Maurizio Balestrino\*, Enrico Adriano and Patrizia Garbati *Department of Neuroscience, Ophtalmology and Genetics, University of Genova, Italy* 

### **1. Introduction**

398 Advances in the Preclinical Study of Ischemic Stroke

Zhao, H., Sapolsky, R.M. & Steinberg, G.K. (2006). Interrupting reperfusion as a stroke

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Zhu, M., Feng, J., Lucchinetti, E., Fischer, G., Xu, L., Pedrazzini, T., Schaub, M. C. & Zaugg,

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No. 1, pp. 152-162

therapy: ischemic postconditioning reduces infarct size after focal ischemia in rats. *J* 

Johansen, J. (2003). Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. *Am J Physiol Heart* 

M. (2006). Ischemic postconditioning protects remodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvage kinase pathway. *Cardiovasc Res*, Vol. 72,

> The use of hyperosmotic agents in stroke is still a matter of debate, since their usefulness has repeatedely been suggested but not conclusively demonstrated (Righetti E et al., 2002). Better understanding of the possible mechanism of protection by hyperosmotic agents may help identifying clinical situations where they may be more useful. It is generally assumed that their effect in stroke is due to their capacity to reduce brain edema. However, increasing extracellular osmolarity has direct effects on neuronal electrical function (Osehobo and Andrew, 1993; Rudehill et al., 1993), and one of us has previously reported that adding mannitol to the perfusing medium of brain slices delays anoxic depolarization (AD) (Balestrino, 1995a; Balestrino, 1995b). Since the latter is a factor in causing neuronal damage in anoxia and ischemia (Balestrino and Somjen, 1986; Jarvis et al., 2001; Kaminogo et al., 1998; Somjen et al., 1990), this may be another mechanism of brain protection by hyperosmotic agents in stroke. This study investigates whether or not this delay occurs at values of hyperosmolarity that may be obtained in clinical practice. We first carried out a survey of the literature on osmolarity changes after administration of hyperosmotic agents in vivo, under both clinical and experimental conditions. Then, we did a dose-response study of mannitol-induced delay of AD. Finally we compared the two sets of data to gauge whether or not mannitol-induced delay of AD occurs in the range of hyperosmolarity that might be obtained in clinical practice.

### **2. Materials and methods**

Sprague-Dawley female rats (155-190g.) were anaesthetised with ether and decapitated. The left hippocampus was dissected free and cut in 600 µm thick transversal slices. Slices were immediately transferred into an "interface" recording chamber (Fine Science Tools, Vancouver B. C. Canada) and incubated at 35±1°C. They were bathed by Artificial CerebroSpinal Fluid (ACSF) flowing at 2 ml/min and having the following composition: NaCl 130 mM, KCl 3.5 mM, NaH2PO4 1.25 mM, NaHCO3 24 mM, CaCl2 2.4 mM, MgSO4 1.2 mM, glucose 10 mM. This medium was continuously bubbled with 95% O2 / 5% CO2, resulting in a pH of 7.35-7.40. The same warmed, humidified 95% O2 / 5% CO2 mixture aerated the slices representing the gas phase. Anoxia was induced by replacing oxygen with

<sup>\*</sup> Corresponding Author

Could Mannitol-Induced Delay of Anoxic Depolarization be Relevant in Stroke Patients? 401

Osmotic agent

(Cloyd et al., 1986) Humans Mannitol 0.5, 0.7 g/Kg 10 - 18 mOsm (Cloyd et al., 1986) Dog Mannitol 0.5, 1, 1.5 g/Kg 43-66 mOsm

(Manninen et al., 1987) Humans Mannitol 1, 2 g/Kg 32 mOsm (Newman, 1979) Humans Mannitol 2 g/Kg Not reported

(Ostensen et al., 1987) Dog Mannitol 40 mOsm (Rudehill et al., 1993) Humans Mannitol 1 g/Kg 12 mOsm.

Table 1. **Literature data on changes in serum osmolarity after i.v. infusion of osmotic agents.** The table summarizes available literature data on changes in serum osmolarity after i.v. infusion of osmotic agents. When different changes in osmolarity are reported following different doses of osmotic agent, in the table the range of increases is given. The values given in the table are either the numbers provided by the Authors or those obtained by measuring graphs in their papers. In the latter case, the value is obviously less precise. When the Authors reported mean±SD for pre- and post-infusion osmolarity, in the table the

lower increase of 10 mOsm or less afterwards (Rudehill et al., 1993). Higher doses of mannitol (4 g/Kg and 8 g/Kg, with or without the addition of furosemide) lead, in rats, to a rather high serum osmolarity increase, reaching an average as high as 67 mOsm (Thenuwara et al., 2002). Under experimental conditions in vivo, glycerol infusion leads to average increases in serum osmolarity of 6 mOsm in guinea pigs (Noi and Makimoto, 1998) and of up to 119 mOsm in dogs (Jansson and Rask-Anderson, 1993). Using Urografin® infusion, a 25 mOsm increase in serum osmolarity was obtained in guinea

The previously shown robust effect of mannitol in delaying AD was first confirmed in double wash-out experiments, where the same slice was subjected to transient anoxia in the presence of mannitol, then in control ACSF, then again in the presence of mannitol. These experiments are illustrated in figure 2. Mannitol concentrations of 100 and 500 mM were used in these experiments, as they were those that had been previously shown to most

The effects of mannitol where then investigated at different concentrations. Fig. 3 summarizes these results. As it can be seen, 1 and 10 mM were not effective in delaying AD. Twenty-five mM significantly delayed AD, while 50 mM did not show a statistically

infused Dose Serum osmolarity

5.2 g /kg 12-119 mOsm

of 50% glycerol 6 mOsm

30-min infusion

1, 4, 8 g/Kg, with or without furosemide

of 76% Urographin® increase

25 mOsm

4-67 mOsm

Paper Animal

(Jansson and

species

(Noi and Makimoto, 1998) Guinea pig Urographin®

(Thenuwara et al., 2002) Rat Mannitol

corresponding difference between means is given.

pigs (Noi and Makimoto, 1998).

reliably delay AD (Balestrino, 1995).

**3.2 Effects of increasing mannitol in vitro** 

Rask-Anderson, 1993) Mice Glycerol 1.3, 2.6 and

(Noi and Makimoto, 1998) Guinea pig Glycerol 30-min infusion

nitrogen in the gas phase. The DC-coupled, ground-referenced extracellular potential of the tissue was constantly monitored in the cell body layer of CA1. As soon as the sudden fall in this potential that is the hallmark of AD was observed, oxygen flow was restored. A crossover study design was observed, with the same slice being subjected to anoxia, at 30' intervals, both in the presence and in the absence of mannitol. Each slice was subjected to two anoxic episodes. The sequence of treatments (mannitol first, or control ACSF first) was alternated in consecutive experiments, to minimize the bias due to possible effects of repeated anoxia *per se* on AD latency. In two experiments, the same slice was subjected to three anoxic episodes the first one in mannitol, the second in control ACSF, the third one in mannitol again. For statistical analysis, in each experiment the difference in latency between AD in mannitol and AD in control ACSF was computed, and used as a gauge of mannitol efficacy in that experiment.

### **3. Results**

**3.1 Literature search on serum osmolarity changes in vivo** 

Results are summarized in figure 1 and in Table 1.

Fig. 1. **Increases in serum osmolarity reported in the literature:** This figure depicts the highest increase in serum osmolarity reported in each of the papers listed in Table 1. It refers to papers quoted in the Reference List. This figure is meant to graphically visualize the highest reported increases. Refer to Table I and to text for further information.

In human patients, use of mannitol at the dose of 0.5-2 g/Kg body weight is reported (Cloyd et al., 1986; Newman, 1979; Rudehill et al., 1993). Such a dose leads, still in human patients, to a maximum increase in serum osmolarity of about 10-32 mOsm (Cloyd et al., 1986; Manninen et al., 1987; Rudehill et al., 1993). When experimental animals are considered, administration of 1 g/Kg body weight to rats yelded a serum osmolarity increase of 4 mOsm (Thenuwara et al., 2002). In dogs, mannitol administration of 0.5, 1 or 1.5 g/Kg lead to a peak increase (mean±SD) of 43±18, 66±18 and 52±23 mOsm, respectively, during the brief time of the infusion, and to the

nitrogen in the gas phase. The DC-coupled, ground-referenced extracellular potential of the tissue was constantly monitored in the cell body layer of CA1. As soon as the sudden fall in this potential that is the hallmark of AD was observed, oxygen flow was restored. A crossover study design was observed, with the same slice being subjected to anoxia, at 30' intervals, both in the presence and in the absence of mannitol. Each slice was subjected to two anoxic episodes. The sequence of treatments (mannitol first, or control ACSF first) was alternated in consecutive experiments, to minimize the bias due to possible effects of repeated anoxia *per se* on AD latency. In two experiments, the same slice was subjected to three anoxic episodes the first one in mannitol, the second in control ACSF, the third one in mannitol again. For statistical analysis, in each experiment the difference in latency between AD in mannitol and AD in control ACSF was computed, and used as a gauge of mannitol

Fig. 1. **Increases in serum osmolarity reported in the literature:** This figure depicts the highest increase in serum osmolarity reported in each of the papers listed in Table 1. It refers to papers quoted in the Reference List. This figure is meant to graphically visualize the

In human patients, use of mannitol at the dose of 0.5-2 g/Kg body weight is reported (Cloyd et al., 1986; Newman, 1979; Rudehill et al., 1993). Such a dose leads, still in human patients, to a maximum increase in serum osmolarity of about 10-32 mOsm (Cloyd et al., 1986; Manninen et al., 1987; Rudehill et al., 1993). When experimental animals are considered, administration of 1 g/Kg body weight to rats yelded a serum osmolarity increase of 4 mOsm (Thenuwara et al., 2002). In dogs, mannitol administration of 0.5, 1 or 1.5 g/Kg lead to a peak increase (mean±SD) of 43±18, 66±18 and 52±23 mOsm, respectively, during the brief time of the infusion, and to the

highest reported increases. Refer to Table I and to text for further information.

efficacy in that experiment.

**3.1 Literature search on serum osmolarity changes in vivo** 

Results are summarized in figure 1 and in Table 1.

**3. Results** 


Table 1. **Literature data on changes in serum osmolarity after i.v. infusion of osmotic agents.** The table summarizes available literature data on changes in serum osmolarity after i.v. infusion of osmotic agents. When different changes in osmolarity are reported following different doses of osmotic agent, in the table the range of increases is given. The values given in the table are either the numbers provided by the Authors or those obtained by measuring graphs in their papers. In the latter case, the value is obviously less precise. When the Authors reported mean±SD for pre- and post-infusion osmolarity, in the table the corresponding difference between means is given.

lower increase of 10 mOsm or less afterwards (Rudehill et al., 1993). Higher doses of mannitol (4 g/Kg and 8 g/Kg, with or without the addition of furosemide) lead, in rats, to a rather high serum osmolarity increase, reaching an average as high as 67 mOsm (Thenuwara et al., 2002). Under experimental conditions in vivo, glycerol infusion leads to average increases in serum osmolarity of 6 mOsm in guinea pigs (Noi and Makimoto, 1998) and of up to 119 mOsm in dogs (Jansson and Rask-Anderson, 1993). Using Urografin® infusion, a 25 mOsm increase in serum osmolarity was obtained in guinea pigs (Noi and Makimoto, 1998).

### **3.2 Effects of increasing mannitol in vitro**

The previously shown robust effect of mannitol in delaying AD was first confirmed in double wash-out experiments, where the same slice was subjected to transient anoxia in the presence of mannitol, then in control ACSF, then again in the presence of mannitol. These experiments are illustrated in figure 2. Mannitol concentrations of 100 and 500 mM were used in these experiments, as they were those that had been previously shown to most reliably delay AD (Balestrino, 1995).

The effects of mannitol where then investigated at different concentrations. Fig. 3 summarizes these results. As it can be seen, 1 and 10 mM were not effective in delaying AD. Twenty-five mM significantly delayed AD, while 50 mM did not show a statistically

Could Mannitol-Induced Delay of Anoxic Depolarization be Relevant in Stroke Patients? 403

300 CONTROLS MANNITOL

**(\*) (\*) (\*)**

1 10 25 50 100 500 <sup>0</sup>

Fig. 3. **Effects of different mannitol concentrations in delaying anoxic depolarization.** The bars show latency of AD (mean± SD)in both control and mannitol-fortified ACSF for different mannitol concentrations. Asterisks mark the groups in which the difference is statistically significant (p<0.03, t-test for paired data). N=3 for 1mM, N=4 for 10 mM, N=8 for 25 mM, N=7 for 50 mM, N=6 for 100 mM, N=6 for 500 mM. See text for experiment

In a further analysis, we calculated for each slice the difference between the latency of AD in mannitol and the latency of AD in control ACSF. Such a difference was used as a gauge of mannitol effectiveness in that particular slice. If the difference had been positive, it would have indicated that latency in mannitol was longer that in control (i.e., AD occurred later), thus showing protection by mannitol and quantifying its degree. The opposite would have been true for a negative difference. Results are shown in Figure 5. As it can be seen, above 10 mM all concentrations of mannitol delay AD to a comparable extent. Such a finding has already been reported, from our laboratory, for AD delay by

Figure 4 shows an example of AD delay by mannitol.

MANNITOL CONCENTRATION, mM

50

100

150

AD LATENCY, SECONDS

design.

creatine (Balestrino, 1995).

200

250

significant effect. The quite high concentrations of 100 and 500 mM significantly increased the latency of AD.

Fig. 2. **Double wash-out experiments showing mannitol effectiveness in delaying anoxic depolarization.** In two different slices, anoxia was induced in mannitol-fortified Artificial CerebroSpinal Fluid (ACSF), then in control ACSF, then again in ACSF with added mannitol. In one experiment (set of bars at left) 100 mM mannitol were used, in the other (set of bars at right) 500 mM mannitol were used. Bars represent latency of AD in each anoxia episode. Control ACSF reversibly decreased AD latency, thus confirming the previously published efficacy of mannitol in increasing AD latency.

significant effect. The quite high concentrations of 100 and 500 mM significantly increased

1st ANOXIA (MANNITOL)

3rd ANOXIA (MANNITOL)

2nd ANOXIA

(CONTROL ACSF)

<sup>100</sup> <sup>500</sup> <sup>50</sup>

previously published efficacy of mannitol in increasing AD latency.

MANNITOL CONCENTRATION (mM)

Fig. 2. **Double wash-out experiments showing mannitol effectiveness in delaying anoxic depolarization.** In two different slices, anoxia was induced in mannitol-fortified Artificial CerebroSpinal Fluid (ACSF), then in control ACSF, then again in ACSF with added mannitol. In one experiment (set of bars at left) 100 mM mannitol were used, in the other (set of bars at right) 500 mM mannitol were used. Bars represent latency of AD in each anoxia episode. Control ACSF reversibly decreased AD latency, thus confirming the

the latency of AD.

60

70

AD LATENCY (SECONDS)

80

Fig. 3. **Effects of different mannitol concentrations in delaying anoxic depolarization.** The bars show latency of AD (mean± SD)in both control and mannitol-fortified ACSF for different mannitol concentrations. Asterisks mark the groups in which the difference is statistically significant (p<0.03, t-test for paired data). N=3 for 1mM, N=4 for 10 mM, N=8 for 25 mM, N=7 for 50 mM, N=6 for 100 mM, N=6 for 500 mM. See text for experiment design.

Figure 4 shows an example of AD delay by mannitol.

In a further analysis, we calculated for each slice the difference between the latency of AD in mannitol and the latency of AD in control ACSF. Such a difference was used as a gauge of mannitol effectiveness in that particular slice. If the difference had been positive, it would have indicated that latency in mannitol was longer that in control (i.e., AD occurred later), thus showing protection by mannitol and quantifying its degree. The opposite would have been true for a negative difference. Results are shown in Figure 5. As it can be seen, above 10 mM all concentrations of mannitol delay AD to a comparable extent. Such a finding has already been reported, from our laboratory, for AD delay by creatine (Balestrino, 1995).

Could Mannitol-Induced Delay of Anoxic Depolarization be Relevant in Stroke Patients? 405

1 10 25 50

Fig. 5. **Measure of AD delay in different mannitol concentrations.** Same experiments as in figure 3. This figure depicts more precisely the increase in AD latency determined by mannitol at each concentration. For each slice, the difference (AD latency in ACSF with mannitol) – (AD latency in control ACSF) was computed. A positive difference means that AD latency was longer in mannitol (i.e., AD occurred later), the opposite is true for a negative difference. Data were grouped for mannitol concentration. For each concentration, mean±SD is provided. Number of experiments as in figure 3. Concentrations of 25 mM

The effectiveness of hyperosmolarity in delaying AD was confirmed by these findings. Delay of AD may be relevant to neuroprotection in stroke, because AD is a factor in the generation of anoxic damage, and its delay has been associated with better outcome under experimental conditions (Balestrino and Somjen, 1986; Jarvis et al., 2001; Kaminogo et al., 1998; Somjen et al., 1990). The present study indicates that significant delay of AD is obtained at mannitol concentrations greater than 10 mM, 25 mM being the lowest effective dose among those tested. An overview of the literature showed that in human patients serum osmolarity increases, under common clinical settings, by 10-32 mOsm after administration of 1 g/Kg mannitol (Table 1 and fig. 1). This is equivalent to adding 10-32 mM mannitol to in vitro†slices1. At the lower end of this range, such an

1 Since the molecule of mannitol does not split in acqueous solutions, the molarity of mannitol in solution (here expressed in mM) corresponds to the consequent increase in osmolarity (1mM=1mOsm).

MANNITOL CONCENTRATION, mM

100 500


mannitol and higher all delay AD to the same extent.




0

10

AD LATENCY, SECONDS,

DIFFERENCE FROM

**4. Discussion** 

PAIRED CONTROL

20

30

40

Fig. 4. **Sample anoxic depolarization in control and mannitol-treated ACSF.** Two different anoxic episodes in the same slice. The dotted line represents DC tracing (showing anoxic depolarization) during anoxia in control ACSF, the solid line represents the same tracing during anoxia in ACSF with added mannitol. AD occurs later in mannitol-fortified ACSF.

Fig. 5. **Measure of AD delay in different mannitol concentrations.** Same experiments as in figure 3. This figure depicts more precisely the increase in AD latency determined by mannitol at each concentration. For each slice, the difference (AD latency in ACSF with mannitol) – (AD latency in control ACSF) was computed. A positive difference means that AD latency was longer in mannitol (i.e., AD occurred later), the opposite is true for a negative difference. Data were grouped for mannitol concentration. For each concentration, mean±SD is provided. Number of experiments as in figure 3. Concentrations of 25 mM mannitol and higher all delay AD to the same extent.

### **4. Discussion**

404 Advances in the Preclinical Study of Ischemic Stroke

Fig. 4. **Sample anoxic depolarization in control and mannitol-treated ACSF.** Two different anoxic episodes in the same slice. The dotted line represents DC tracing (showing anoxic depolarization) during anoxia in control ACSF, the solid line represents the same tracing during anoxia in ACSF with added mannitol. AD occurs later in mannitol-fortified ACSF.

The effectiveness of hyperosmolarity in delaying AD was confirmed by these findings. Delay of AD may be relevant to neuroprotection in stroke, because AD is a factor in the generation of anoxic damage, and its delay has been associated with better outcome under experimental conditions (Balestrino and Somjen, 1986; Jarvis et al., 2001; Kaminogo et al., 1998; Somjen et al., 1990). The present study indicates that significant delay of AD is obtained at mannitol concentrations greater than 10 mM, 25 mM being the lowest effective dose among those tested. An overview of the literature showed that in human patients serum osmolarity increases, under common clinical settings, by 10-32 mOsm after administration of 1 g/Kg mannitol (Table 1 and fig. 1). This is equivalent to adding 10-32 mM mannitol to in vitro†slices1. At the lower end of this range, such an

<sup>1</sup> Since the molecule of mannitol does not split in acqueous solutions, the molarity of mannitol in solution (here expressed in mM) corresponds to the consequent increase in osmolarity (1mM=1mOsm).

Could Mannitol-Induced Delay of Anoxic Depolarization be Relevant in Stroke Patients? 407

Balestrino M. (1995) Studies on anoxic depolarization. In: Brain Slices in Basic and Clinical Research (A.Schurr, B.M.Rigor, eds), pp 273-293 Boca Raton, Florida: CRC Press. Balestrino M (Pathophysiology of anoxic depolarization: new findings and a working

Balestrino M, Somjen GG (Chlorpromazine protects brain tissue in hypoxia by delaying spreading depression-mediated calcium influx. Brain Res 385:219-226.1986). Chen Q, Chopp M, Bodzin G, Chen H (Temperature modulation of cerebral depolarization

Cloyd JC, Snyder BD, Cleeremans B, Bundlie SR, Blomquist CH, Lakatua DJ (Mannitol

Jansson B, Rask-Anderson H (Correlations between serum osmolality and endolymphatic

Jarvis CR, Anderson TR, Andrew RD (Anoxic Depolarization Mediates Acute Damage

Kaminogo M, Suyama K, Ichikura A, Onizuka M, Shibata S (Anoxic depolarization

Manninen P, Lam A, Gelb A, Brown S (The effect of high-dose mannitol on serum and urine

Newman SL (Monitoring serum osmolality in mannitol treatment of Reye's syndrome. N

Noi O, Makimoto K (Comparative effects of glycerol and Urografin on cochlear blood flow

Ohta K, Graf R, Rosner G, Heiss WD (Profiles of cortical tissue depolarization in cat focal

Osehobo EP, Andrew RD (Osmotic effects upon the theta rhythm, a natural brain oscillation

Ostensen J, Bugge JF, Stokke ES, Langberg H, Kiil F (Mechanism of osmotic diuresis studied

Righetti E, Celani MG, Cantisani T, Sterzi R, Boysen G, Ricci S (2002) Glycerol for acute

Rudehill A, Gordon E, Ohman G, Lindqvist C, Andersson P (Pharmacokinetics and effects

osmolality during intracranial surgery. J Neurosurg Anesthesiol 5:12.1993). Somjen GG, Aitken PG, Balestrino M, Herreras O, Kawasaki K (Spreading depression-like

determines ischemic brain injury. Neurol Res 20:343-348.1998).

Anesthesia / Journal canadien d'anesthésie 34:442-446.1987).

production. J Cereb Blood Flow Metab 17:1170-1181.1997).

in the hippocampal slice. Exp Neurol 124:192-199.1993).

and serum osmolarity. Hear Res 123:55-60.1998).

during focal cerebral ischemia in rats: correlation with ischemic injury. J Cereb

pharmacokinetics and serum osmolality in dogs and humans. J Pharmacol Exp

sac response using hypertonic glycerol. ORL J Otorhinolaryngol Relat Spec 55:185-

Independent of Glutamate in Neocortical Brain Slices. Cerebral Cortex 11:249-

electrolytes and osmolality in neurosurgical patients. Canadian Journal of

cerebral ischemia in relation to calcium ion homeostasis and nitric oxide

by infusion of NaHCO3 and mannitol in dogs. Acta Physiol Scand 131:397-

stroke (Cochrane Review). In: The Cochrane Library, Issue 2 2002 Oxford: Update

of mannitol on hemodynamics, blood and cerebrospinal fluid electrolytes, and

depolarization and selective vulnerability of neurons. A brief review. Stroke

hypothesis. J Neurosci Methods 59:99-103.1995).

Blood Flow Metab 13:389-394.1993).

Ther 236:301-306.1986).

Engl J Med 301:945-946.1979).

192.1993).

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Software.

21:III179-III183.1990).

**6. References** 

increase would be insufficient (10 mM (Cloyd et al., 1986; Rudehill et al., 1993) ) or perhaps barely sufficient (18 mM (Cloyd et al., 1986)) to afford delay of AD. In the upper end, a 32 mM increase (Manninen et al., 1987) would probably be somehow effective in delaying AD. In fact, we showed that in vitro the addition of 25 mM mannitol (increasing osmolarity by 25 mOsm) significantly delays AD. The delay in AD was not significant after addition of 50 mM (increasing osmolarity by 50 mOsm), indicating that these osmolarity increases (25-50 mOsm) are of borderline efficacy. However, much higher increases, up to 100 mOsm and more, were reliably effective in vitro, and have been reported under experimental conditions in laboratory animals, apparently without severe adverse effects (Table 1 and figure 1). The latter increases would be in a range that does cause AD delay (compare figure 5 with figure 1). If further studies suggested that a comparable increase in serum osmolarity can be safely obtained in human patients, it might be useful not only by decreasing brain edema, but also by having a direct effect on tissue depolarization.

Two more considerations are in order.

First, in human stroke mannitol or other hyperosmotic agents would be adiminstered when AD has already occurred. In fact, AD is an event that occurs in the core of an infarction soon after ischemia. Nevertheless, under experimental conditions continuous or repeated depolarizations have been demonstrated in the hours following stroke (Chen et al., 1993; Ohta et al., 1997). Their reduction has been associated with better outcome (Chen et al., 1993). Given the striking similarity of these events with "classical" AD, it is very likely that hyperosmolarity can delay or suppress these waveforms as well, thus providing protection.

Second, the changes reported in the literature are in serum, not in the interstitial space of the brain. To the best of our knowledge, no study has yet measured increases in osmolarity in the interstitial space of the brain, probably due to the technical difficulty of this investigation. However, it is reasonable to assume that an increase in osmotic pressure in the serum draws water from the brain interstitial space, thus increasing its osmolarity to a comparable degree. Therefore, increase in serum osmolarity should be comparable, at least to an extent, to increase in osmolarity of the brain interstitial space.

Finally, it should be noted that future clinical studies on hyperosmotic agents in stroke should take into account the increase in serum osmolarity that was obtained in the single patients. In fact, our data indicate that the latter is a critical variable in determining whether the hyperosmotic therapy will be effective or not.

Summing up, we conclude that the increase in serum osmolarity that is commonly obtained in clinical practice is not sufficient to delay AD. Larger increases in serum osmolarity have been, however, reported in animal experiments. If further studies indicated that such increases were safe in humans as well as in animals, they might provide brain protection by decreasing AD and AD-like depolarizations. Future clinical studies on hyperosmotic agents in stroke should measure and take into account the degree of changes that were obtained in serum osmolarity.

### **5. Acknowledgment**

We thank Prof. Aroldo Cupello for his useful comments on the manuscript.

### **6. References**

406 Advances in the Preclinical Study of Ischemic Stroke

increase would be insufficient (10 mM (Cloyd et al., 1986; Rudehill et al., 1993) ) or perhaps barely sufficient (18 mM (Cloyd et al., 1986)) to afford delay of AD. In the upper end, a 32 mM increase (Manninen et al., 1987) would probably be somehow effective in delaying AD. In fact, we showed that in vitro the addition of 25 mM mannitol (increasing osmolarity by 25 mOsm) significantly delays AD. The delay in AD was not significant after addition of 50 mM (increasing osmolarity by 50 mOsm), indicating that these osmolarity increases (25-50 mOsm) are of borderline efficacy. However, much higher increases, up to 100 mOsm and more, were reliably effective in vitro, and have been reported under experimental conditions in laboratory animals, apparently without severe adverse effects (Table 1 and figure 1). The latter increases would be in a range that does cause AD delay (compare figure 5 with figure 1). If further studies suggested that a comparable increase in serum osmolarity can be safely obtained in human patients, it might be useful not only by decreasing brain edema, but also by having a direct effect on

First, in human stroke mannitol or other hyperosmotic agents would be adiminstered when AD has already occurred. In fact, AD is an event that occurs in the core of an infarction soon after ischemia. Nevertheless, under experimental conditions continuous or repeated depolarizations have been demonstrated in the hours following stroke (Chen et al., 1993; Ohta et al., 1997). Their reduction has been associated with better outcome (Chen et al., 1993). Given the striking similarity of these events with "classical" AD, it is very likely that hyperosmolarity can delay or suppress these waveforms as well, thus

Second, the changes reported in the literature are in serum, not in the interstitial space of the brain. To the best of our knowledge, no study has yet measured increases in osmolarity in the interstitial space of the brain, probably due to the technical difficulty of this investigation. However, it is reasonable to assume that an increase in osmotic pressure in the serum draws water from the brain interstitial space, thus increasing its osmolarity to a comparable degree. Therefore, increase in serum osmolarity should be comparable, at least

Finally, it should be noted that future clinical studies on hyperosmotic agents in stroke should take into account the increase in serum osmolarity that was obtained in the single patients. In fact, our data indicate that the latter is a critical variable in determining whether

Summing up, we conclude that the increase in serum osmolarity that is commonly obtained in clinical practice is not sufficient to delay AD. Larger increases in serum osmolarity have been, however, reported in animal experiments. If further studies indicated that such increases were safe in humans as well as in animals, they might provide brain protection by decreasing AD and AD-like depolarizations. Future clinical studies on hyperosmotic agents in stroke should measure and take into account the degree of changes that were obtained in

to an extent, to increase in osmolarity of the brain interstitial space.

We thank Prof. Aroldo Cupello for his useful comments on the manuscript.

the hyperosmotic therapy will be effective or not.

tissue depolarization.

providing protection.

serum osmolarity.

**5. Acknowledgment** 

Two more considerations are in order.


**Fasudil (a Rho Kinase Inhibitor) Specifically** 

**Vasospasm After Subarachnoid Hemorrhage** 

Subarachnoid hemorrhage due to a rupture of cerebral aneurysm is a severe disease with morbidity and mortality. Although, if patients' conditions are fair before surgery, they are rather safely operated by either clipping or coiling, vasospasm remains as a major complication of this disease. There are still many patients who suffer from vasospasm causing neurological deficits. Both strong vasoconstriction and inflammation are involved in the pathphysiological mechanism of vasospasm. In 1992 we had reported specific effects of a vasodilating drug "fasudil" in the treatment of vasospasm, but mechanisms how fasudil

RhoA/Rho kinase had been found in 1996 and was revealed to act as molecular on-off switches that control multiple signaling pathways. Upregulated Rho kinase is known to be involved in various diseases from vascular disease to cancer. In cerebral vasospasm, upregulated Rho kinase was found to be involved in many aspects, such as increased calcium sensitivity, reduced production of nitric oxide, migration of inflammatory cells and their production of superoxide anions and increased blood viscosity. Interestingly, fasudil was found to specifically increase cerebral blood flow in the area with vasospasm. In the present paper pathophysiological mechanism of vasospasm and effects of fasudil are reviewed and mechanisms why fasudil increases cerebral blood flow in the area with

Cerebral infarction due to delayed vasospasm is still the leading cause of a poor postoperative outcome of patients with a ruptured cerebral aneurysm especially if we consider deficits in higher neurological functions such as cognitive functions. Several days after a subarachnoid hemorrhage (SAH), blood vessels begin to be contracted by substances eluted from the blood clot such as oxyhemoglobin, endothelin, amines and many other chemical substances. Most of the patients show contraction of the blood vessels (angiographic spasm). In about one third of the patients, signs of neurological deficits appear (symptomatic spasm) on an average of day 7 after the hemorrhage (Bederson et al., 2009, Shibuya et al., 1992). Patients may even die of severe spasm, especially due to vasospasm of arteries supplying basal part of the brain: hypothalamus and brainstem. A

vasospasm without so much changing that of normal flow area will be discussed.

**2. Cerebral vasospasm following subarachnoid hemorrhage** 

representative case of a patient with severe vasospasm is shown in Fig. 1.

ameliorated vasospasm had not been clearly understood as it is today.

**1. Introduction** 

**Increases Cerebral Blood Flow in Area of** 

Masato Shibuya, Kenko Meda and Akira Ikeda *Department of Neurosurgery, Chukyo Hospital, Nagoya,* 

*Japan* 

Thenuwara K, Todd MM, Brian JE, Jr. (Effect of mannitol and furosemide on plasma osmolality and brain water. Anesthesiology 96:416-421.2002). **18** 

### **Fasudil (a Rho Kinase Inhibitor) Specifically Increases Cerebral Blood Flow in Area of Vasospasm After Subarachnoid Hemorrhage**

Masato Shibuya, Kenko Meda and Akira Ikeda *Department of Neurosurgery, Chukyo Hospital, Nagoya, Japan* 

### **1. Introduction**

408 Advances in the Preclinical Study of Ischemic Stroke

Thenuwara K, Todd MM, Brian JE, Jr. (Effect of mannitol and furosemide on plasma

Subarachnoid hemorrhage due to a rupture of cerebral aneurysm is a severe disease with morbidity and mortality. Although, if patients' conditions are fair before surgery, they are rather safely operated by either clipping or coiling, vasospasm remains as a major complication of this disease. There are still many patients who suffer from vasospasm causing neurological deficits. Both strong vasoconstriction and inflammation are involved in the pathphysiological mechanism of vasospasm. In 1992 we had reported specific effects of a vasodilating drug "fasudil" in the treatment of vasospasm, but mechanisms how fasudil ameliorated vasospasm had not been clearly understood as it is today.

RhoA/Rho kinase had been found in 1996 and was revealed to act as molecular on-off switches that control multiple signaling pathways. Upregulated Rho kinase is known to be involved in various diseases from vascular disease to cancer. In cerebral vasospasm, upregulated Rho kinase was found to be involved in many aspects, such as increased calcium sensitivity, reduced production of nitric oxide, migration of inflammatory cells and their production of superoxide anions and increased blood viscosity. Interestingly, fasudil was found to specifically increase cerebral blood flow in the area with vasospasm. In the present paper pathophysiological mechanism of vasospasm and effects of fasudil are reviewed and mechanisms why fasudil increases cerebral blood flow in the area with vasospasm without so much changing that of normal flow area will be discussed.

### **2. Cerebral vasospasm following subarachnoid hemorrhage**

Cerebral infarction due to delayed vasospasm is still the leading cause of a poor postoperative outcome of patients with a ruptured cerebral aneurysm especially if we consider deficits in higher neurological functions such as cognitive functions. Several days after a subarachnoid hemorrhage (SAH), blood vessels begin to be contracted by substances eluted from the blood clot such as oxyhemoglobin, endothelin, amines and many other chemical substances. Most of the patients show contraction of the blood vessels (angiographic spasm). In about one third of the patients, signs of neurological deficits appear (symptomatic spasm) on an average of day 7 after the hemorrhage (Bederson et al., 2009, Shibuya et al., 1992). Patients may even die of severe spasm, especially due to vasospasm of arteries supplying basal part of the brain: hypothalamus and brainstem. A representative case of a patient with severe vasospasm is shown in Fig. 1.

Fasudil (a Rho Kinase Inhibitor) Specifically Increases

prevent development of further neurological deficits.

hemiparesis.

Cerebral Blood Flow in Area of Vasospasm After Subarachnoid Hemorrhage 411

home. He was slightly drowsy and disoriented (Hunt & Hess grade III) with mild weakness in the left arm and leg. A head computed tomography (CT) (upper right) showed a diffuse subarachnoid hemorrhage and a large hematoma in the right Sylvian fissure. A 3-D CT angiogram (upper left) showed a 10mm long aneurysm at the bifurcation of the right middle cerebral artery (MCA) (arrow). The aneurysm was clipped and subarachnoid space was washed with urokinase on the same day. He smoothly recovered from surgery and he was treated routinely postoperatively to prevent vasospasm with careful management of blood pressure, water and electrolytes balance. Fasudil 30mg (i.v./30min, t.i.d.) was started on day

1. His postoperative course was smooth with clear consciousness and a mild left

A routine checkup, on day 9, by a magnetic resonance angiography (MRA, middle left) showed a moderately severe vasospasm in the right MCA, a segmental vasospasm in the left MCA and proximal portion of the right anterior cerebral artery (ACA). Diffusion weighted magnetic resonance image (DWI) showed no abnormality (middle right). His blood pressure was elevated with dopamine and daily dose of fasudil was increased to 60mg (i.v., t.i.d.) to

However, the next day (day 10), his left hemiparesis deteriorated and he became drowsy. MRA on day 15 showed that vasospasm in bilateral MCAs progressed. Especially, distal branches of the right MCA were hardly seen. Segmental vasospasm appeared in the proximal portion of the right MCA, left ACA and distal portion of the vertebral arteries (lower left). However, vasospasm in the proximal portion of the right ACA improved. DWI on the same day showed an infarction in the right MCA territory (lower right). In spite of

consciousness the same day. Although he was communicable and could eat by himself, his left hemiplegia did not improve and he was discharged to a rehabilitation hospital. Now,

Vasospasm is not a simple contraction of blood vessels but it is complex pathological phenomena consisting of abnormal contraction of blood vessels which is not easily relaxed by usual calcium antagonists and inflammation. Tissue damage is seen in vascular endothelium and smooth muscle cells in the medial wall caused by free radicals released from inflammatory cells. Decreased production of nitric oxide (NO) is also contributing to both contraction and tissue damage. Rho kinase has been found to be deeply implicated in the pathophysiology of vasospasm (Miyagi et al., 2000; Sato et al., 2000) and use of a Rho kinase inhibitor: fasudil dramatically improved patients' outcome (Shibuya et al., 1992)

Fasudil HCl: (hexahydro-1-5-isoquinolinesulfonyl)-1H-1.4-diazepine HCl, (also called HA1077, AT877, or Eril@) is originally considered to be an intracellular calcium antagonist. By experimental studies in dogs we had found that fasudil dilated spastic arteries without causing systemic hypotension, which could not been shown by any of the previously presented drugs (Takayasu et al., 1986). The effectiveness was also confirmed in patients by a double blind trial (Shibuya et al., 1992). Fasudil showed stronger brain protection from ischemic damage than dilatation of the spastic artery itself, suggesting its possible effects in patients with cerebral infarction as well. Fasudil is now routinely used in Japan for patients with SAH. Zhao et al. (2007) in China showed by a randomized trial that fasudil was

deterioration of vasospasm on MRI and MRA on day 15, he began to recover his

**3. Effects of Fasudil, a Rho kinase inhibitor on cerebral vasospasm** 

two years after the onset, he is bed ridden and taken care at his home.

Fig. 1. Representative case of a patient with severe vasospasm.

Patient is a 70y/o male with a past history of prostatic cancer, hypertension and diabetes mellitus. He had a sudden onset of severe headache and lost consciousness two times at

Fig. 1. Representative case of a patient with severe vasospasm.

Patient is a 70y/o male with a past history of prostatic cancer, hypertension and diabetes mellitus. He had a sudden onset of severe headache and lost consciousness two times at

home. He was slightly drowsy and disoriented (Hunt & Hess grade III) with mild weakness in the left arm and leg. A head computed tomography (CT) (upper right) showed a diffuse subarachnoid hemorrhage and a large hematoma in the right Sylvian fissure. A 3-D CT angiogram (upper left) showed a 10mm long aneurysm at the bifurcation of the right middle cerebral artery (MCA) (arrow). The aneurysm was clipped and subarachnoid space was washed with urokinase on the same day. He smoothly recovered from surgery and he was treated routinely postoperatively to prevent vasospasm with careful management of blood pressure, water and electrolytes balance. Fasudil 30mg (i.v./30min, t.i.d.) was started on day 1. His postoperative course was smooth with clear consciousness and a mild left hemiparesis.

A routine checkup, on day 9, by a magnetic resonance angiography (MRA, middle left) showed a moderately severe vasospasm in the right MCA, a segmental vasospasm in the left MCA and proximal portion of the right anterior cerebral artery (ACA). Diffusion weighted magnetic resonance image (DWI) showed no abnormality (middle right). His blood pressure was elevated with dopamine and daily dose of fasudil was increased to 60mg (i.v., t.i.d.) to prevent development of further neurological deficits.

However, the next day (day 10), his left hemiparesis deteriorated and he became drowsy. MRA on day 15 showed that vasospasm in bilateral MCAs progressed. Especially, distal branches of the right MCA were hardly seen. Segmental vasospasm appeared in the proximal portion of the right MCA, left ACA and distal portion of the vertebral arteries (lower left). However, vasospasm in the proximal portion of the right ACA improved. DWI on the same day showed an infarction in the right MCA territory (lower right). In spite of deterioration of vasospasm on MRI and MRA on day 15, he began to recover his consciousness the same day. Although he was communicable and could eat by himself, his left hemiplegia did not improve and he was discharged to a rehabilitation hospital. Now, two years after the onset, he is bed ridden and taken care at his home.

Vasospasm is not a simple contraction of blood vessels but it is complex pathological phenomena consisting of abnormal contraction of blood vessels which is not easily relaxed by usual calcium antagonists and inflammation. Tissue damage is seen in vascular endothelium and smooth muscle cells in the medial wall caused by free radicals released from inflammatory cells. Decreased production of nitric oxide (NO) is also contributing to both contraction and tissue damage. Rho kinase has been found to be deeply implicated in the pathophysiology of vasospasm (Miyagi et al., 2000; Sato et al., 2000) and use of a Rho kinase inhibitor: fasudil dramatically improved patients' outcome (Shibuya et al., 1992)

### **3. Effects of Fasudil, a Rho kinase inhibitor on cerebral vasospasm**

Fasudil HCl: (hexahydro-1-5-isoquinolinesulfonyl)-1H-1.4-diazepine HCl, (also called HA1077, AT877, or Eril@) is originally considered to be an intracellular calcium antagonist. By experimental studies in dogs we had found that fasudil dilated spastic arteries without causing systemic hypotension, which could not been shown by any of the previously presented drugs (Takayasu et al., 1986). The effectiveness was also confirmed in patients by a double blind trial (Shibuya et al., 1992). Fasudil showed stronger brain protection from ischemic damage than dilatation of the spastic artery itself, suggesting its possible effects in patients with cerebral infarction as well. Fasudil is now routinely used in Japan for patients with SAH. Zhao et al. (2007) in China showed by a randomized trial that fasudil was

Fasudil (a Rho Kinase Inhibitor) Specifically Increases

**5. Effects of fasudil on cerebral infarction** 

vasospasm (Shibuya et al., 2008).

region to a normal region.

**6. Discussion 6.1 Rho kinase** 

Cerebral Blood Flow in Area of Vasospasm After Subarachnoid Hemorrhage 413

(50.4±8.4ml, n=125). We also have shown by using 99mTc-HMPAO that fasudil (30- 60mg/i.v./30 min) significantly increased rCBF in the operated side of the brain in patients showing ischemic signs of vasospasm. Such difference was not found in patients without

These data suggest that upregulated Rho kinase is involved in the decrease of rCBF in patients with vasospasm which was specifically improved by a Rho kinase inhibitor fasudil. On the other hand calcium antagonist dilated normal arteries more than spastic arteries leading to a systemic hypotension and a steal phenomenon, a steal of blood from a spastic

Rho kinase is also up-regulated in patients with cerebral infarction, both in ischemic brain and in migrated WBCs. It is involved in many aspects of ischemic brain damage caused by migration of inflammatory cells to the ischemic site and their production of free radicals by activated NADPH oxidase. Rho kinase elevates blood viscosity by producing the tissue factor (also called factor III, thrombokinase, or CD142) which triggers the coagulation cascade. Blood viscosity is also elevated by reduced plasticity of RBCs due to polymerization of actin fibers which is induced by activated Rho kinase and protein kinase C (Arai et al., 1993; Brabeck et al., 2003; Feske et al., 2009; Satoh et al., 2010). Effectiveness of fasudil on cerebral infarction has been shown both by experimental (Tsuchiya et al., 1993) and clinical studies (Shibuya et al., 2005). After specific effects of fasudil on cerebral vasospasm and infarction had been shown, it has been tried and showed effectiveness in various kinds of vascular diseases such as coronary ischemia, glaucoma, pulmonary hypertension, chronic kidney disease and so on (Dong et al., 2010, Schmandke et al., 2007).

Rho kinase is the immediate downstream target of RhoA, a small GTP binding protein belonging to Ras, Rho, Rab and Ran subfamilies and acts as molecular on-off switches that control multiple signaling pathways. Inactive form of Rho-GDP is activated by guanine nucleotide exchange factors (GEFs) and Guanine dissociation inhibitors (GDIs) through stimulation by lysophosphatidic acid (LPA) and sphyngosine-1-phosphate (S1P). Active form (GTP and membrane-bound) RhoA is inactivated by GTPase activating proteins (GAPs) to GDP bound form in cytosole. Rho kinase is a serine-threonine protein kinase that are involved in diverse cellular functions including vascular smooth muscle cell (SMC) contraction such as cerebral and coronary vasospasm, atherosclerosis, actin cytoskelton

arrangement, cell adhesion, motility and gene expression (Noma et al., 2006).

**6.2 Upregulated Rho kinase and increased sensitivity to calcium in vasospasm** 

Miyagi et al. (2000) showed that RhoA and mRNA of Rho kinase was increased in the basilar artery of SAH rats. Sato et al. (2000) clearly showed, in a two hemorrhage dog model, that Rho kinase was up-regulated with the decrease in basilar artery diameter and with the increase of phosphorylation of myosin binding subunit (MBS) of myosin phosphatase of the basilar artery, all of which were inhibited by a Rho kinase inhibitor Y27632. Activated Rho kinase inhibits MLC phosphatase by phosphorylating its component MBS at Thr697 (Feng et

significantly better for vasospasm than nimodipine which was most commonly used in the western countries.

After Rho kinase was found (Kimura et al., 1996), it became clear that upregulated Rho kinase worked unfavorably to the host in many vascular diseases and effects of fasudil on vasospasm mainly depended on its inhibition of Rho kinase. Fasudil was found to inhibit Rho kinase most strongly than any other protein kinases such as protein kinases C, A, and G (Hidaka et al., 2005). Fasudil is metabolized in human to hydroxyfasudil. Both fasudil and hydroxyfasudil are strong inhibitors of Rho kinase, however biological half-life of fasudil and hydroxyfasudil after an intravenous infusion of fasudil in human are 18 min and 6 hours, respectively. Thus major effect is considered to depend on hydroxyfasudil rather than fasudil itself.

Upregulated Rho kinase inhibits relaxation of the contracted blood vessels by inhibiting dephosphorylation of phosphorylated myosin light chain (MLC) either directly or through inhibition of endothelial NO synthase (eNOS). In an experimental model of vasospasm induced by PGF2α, double phosphorylation of MLC, at Thr18 in addition to Ser19, was found. This is considered to be the underlying mechanism of the strong contraction or increased sensitivity to Ca++. Furthermore, fasudil was found to inhibit the second (pathological) phosphorylation at Thr18 of MLC more strongly (IC50: 0.3uM) than the first phosphorylation at Ser19 (IC50: 3uM) (Seto et al., 1991).

### **4. Fasudil specifically increases rCBF in area with vasospasm**

Specific effect of fasudil on cerebral vasospasm has been suggested to depend on its inhibition of the abnormal phosphorylation of MLC. On the other hand, under normal situation, increased intracellular calcium phosphorylates MLC by activating calmodulin and myosin light chain kinase (MLCK) which is relaxed by dephosphorylation of MLC by phosphatase.

In a two hemorrhage canine model of SAH, basilar artery diameter is decreased to about 60% on day 7. Intravenous administration of a calcium antagonist nicardipine (0.1mg/kg, i.v./30min) did not dilate the spastic basilar artery but caused systemic hypotension. While fasudil (HA1077) (0.5~3mg/kg, i.v. /30min) significantly dilated the spastic artery without causing hypotension (Takayasu et al., 1986). It can be explained by specific inhibition of Rho kinase by fasudil. In other words, fasudil dilated spastic artery more specifically than normal or non-spastic arteries.

Specific vasodilating effect of fasudil has been shown by measuring regional cerebral blood flow (rCBF). In patients who had been operated on their ruptured aneurysms, Ueda (2000) compared the effects of fasudil on rCBF using 99mTc-HMPAO with that of nicardipine. Nicardipine (2mg, i.v.) decreased BP and increased pulse rate. It decreased rCBF in the low flow (spastic) area ( to -10%, P<0.05) without changing rCBF of the normal flow area, suggesting a loss of autoregulation in the spastic area. On the other hand, fasudil (15 mg, i.v.) increased rCBF in the low flow area by 16% (P<0.05) without changing that of normal flow area.

Using CT perfusion method in patients with SAH, Ono et al. (2005) have examined changes in the cerebral blood perfusion (CBP) by fasudil (30mg, i.v./30min) in both normal (>40ml/100g/min) and low flow (<40ml) regions due to vasospasm. The mean CBP in the low flow area (34.4±4.7ml) was significantly increased (to 41.0±8.2 ml, P<0.05, n=43), whereas the mean CBP of the normal flow region (51.8±7.6ml) did not change after fasudil (50.4±8.4ml, n=125). We also have shown by using 99mTc-HMPAO that fasudil (30- 60mg/i.v./30 min) significantly increased rCBF in the operated side of the brain in patients showing ischemic signs of vasospasm. Such difference was not found in patients without vasospasm (Shibuya et al., 2008).

These data suggest that upregulated Rho kinase is involved in the decrease of rCBF in patients with vasospasm which was specifically improved by a Rho kinase inhibitor fasudil. On the other hand calcium antagonist dilated normal arteries more than spastic arteries leading to a systemic hypotension and a steal phenomenon, a steal of blood from a spastic region to a normal region.

### **5. Effects of fasudil on cerebral infarction**

Rho kinase is also up-regulated in patients with cerebral infarction, both in ischemic brain and in migrated WBCs. It is involved in many aspects of ischemic brain damage caused by migration of inflammatory cells to the ischemic site and their production of free radicals by activated NADPH oxidase. Rho kinase elevates blood viscosity by producing the tissue factor (also called factor III, thrombokinase, or CD142) which triggers the coagulation cascade. Blood viscosity is also elevated by reduced plasticity of RBCs due to polymerization of actin fibers which is induced by activated Rho kinase and protein kinase C (Arai et al., 1993; Brabeck et al., 2003; Feske et al., 2009; Satoh et al., 2010). Effectiveness of fasudil on cerebral infarction has been shown both by experimental (Tsuchiya et al., 1993) and clinical studies (Shibuya et al., 2005). After specific effects of fasudil on cerebral vasospasm and infarction had been shown, it has been tried and showed effectiveness in various kinds of vascular diseases such as coronary ischemia, glaucoma, pulmonary hypertension, chronic kidney disease and so on (Dong et al., 2010, Schmandke et al., 2007).

### **6. Discussion**

412 Advances in the Preclinical Study of Ischemic Stroke

significantly better for vasospasm than nimodipine which was most commonly used in the

After Rho kinase was found (Kimura et al., 1996), it became clear that upregulated Rho kinase worked unfavorably to the host in many vascular diseases and effects of fasudil on vasospasm mainly depended on its inhibition of Rho kinase. Fasudil was found to inhibit Rho kinase most strongly than any other protein kinases such as protein kinases C, A, and G (Hidaka et al., 2005). Fasudil is metabolized in human to hydroxyfasudil. Both fasudil and hydroxyfasudil are strong inhibitors of Rho kinase, however biological half-life of fasudil and hydroxyfasudil after an intravenous infusion of fasudil in human are 18 min and 6 hours, respectively. Thus major effect is considered to depend on hydroxyfasudil rather

Upregulated Rho kinase inhibits relaxation of the contracted blood vessels by inhibiting dephosphorylation of phosphorylated myosin light chain (MLC) either directly or through inhibition of endothelial NO synthase (eNOS). In an experimental model of vasospasm induced by PGF2α, double phosphorylation of MLC, at Thr18 in addition to Ser19, was found. This is considered to be the underlying mechanism of the strong contraction or increased sensitivity to Ca++. Furthermore, fasudil was found to inhibit the second (pathological) phosphorylation at Thr18 of MLC more strongly (IC50: 0.3uM) than the first

Specific effect of fasudil on cerebral vasospasm has been suggested to depend on its inhibition of the abnormal phosphorylation of MLC. On the other hand, under normal situation, increased intracellular calcium phosphorylates MLC by activating calmodulin and myosin light chain kinase (MLCK) which is relaxed by dephosphorylation of MLC by

In a two hemorrhage canine model of SAH, basilar artery diameter is decreased to about 60% on day 7. Intravenous administration of a calcium antagonist nicardipine (0.1mg/kg, i.v./30min) did not dilate the spastic basilar artery but caused systemic hypotension. While fasudil (HA1077) (0.5~3mg/kg, i.v. /30min) significantly dilated the spastic artery without causing hypotension (Takayasu et al., 1986). It can be explained by specific inhibition of Rho kinase by fasudil. In other words, fasudil dilated spastic artery more specifically than

Specific vasodilating effect of fasudil has been shown by measuring regional cerebral blood flow (rCBF). In patients who had been operated on their ruptured aneurysms, Ueda (2000) compared the effects of fasudil on rCBF using 99mTc-HMPAO with that of nicardipine. Nicardipine (2mg, i.v.) decreased BP and increased pulse rate. It decreased rCBF in the low flow (spastic) area ( to -10%, P<0.05) without changing rCBF of the normal flow area, suggesting a loss of autoregulation in the spastic area. On the other hand, fasudil (15 mg, i.v.) increased rCBF in the low flow area by 16% (P<0.05) without changing that of normal

Using CT perfusion method in patients with SAH, Ono et al. (2005) have examined changes in the cerebral blood perfusion (CBP) by fasudil (30mg, i.v./30min) in both normal (>40ml/100g/min) and low flow (<40ml) regions due to vasospasm. The mean CBP in the low flow area (34.4±4.7ml) was significantly increased (to 41.0±8.2 ml, P<0.05, n=43), whereas the mean CBP of the normal flow region (51.8±7.6ml) did not change after fasudil

phosphorylation at Ser19 (IC50: 3uM) (Seto et al., 1991).

**4. Fasudil specifically increases rCBF in area with vasospasm** 

western countries.

than fasudil itself.

phosphatase.

flow area.

normal or non-spastic arteries.

### **6.1 Rho kinase**

Rho kinase is the immediate downstream target of RhoA, a small GTP binding protein belonging to Ras, Rho, Rab and Ran subfamilies and acts as molecular on-off switches that control multiple signaling pathways. Inactive form of Rho-GDP is activated by guanine nucleotide exchange factors (GEFs) and Guanine dissociation inhibitors (GDIs) through stimulation by lysophosphatidic acid (LPA) and sphyngosine-1-phosphate (S1P). Active form (GTP and membrane-bound) RhoA is inactivated by GTPase activating proteins (GAPs) to GDP bound form in cytosole. Rho kinase is a serine-threonine protein kinase that are involved in diverse cellular functions including vascular smooth muscle cell (SMC) contraction such as cerebral and coronary vasospasm, atherosclerosis, actin cytoskelton arrangement, cell adhesion, motility and gene expression (Noma et al., 2006).

### **6.2 Upregulated Rho kinase and increased sensitivity to calcium in vasospasm**

Miyagi et al. (2000) showed that RhoA and mRNA of Rho kinase was increased in the basilar artery of SAH rats. Sato et al. (2000) clearly showed, in a two hemorrhage dog model, that Rho kinase was up-regulated with the decrease in basilar artery diameter and with the increase of phosphorylation of myosin binding subunit (MBS) of myosin phosphatase of the basilar artery, all of which were inhibited by a Rho kinase inhibitor Y27632. Activated Rho kinase inhibits MLC phosphatase by phosphorylating its component MBS at Thr697 (Feng et

Fasudil (a Rho Kinase Inhibitor) Specifically Increases

al., 20022Satoh et al., 2010).

infarction are shown in Fig. 2.

infarction

Cerebral Blood Flow in Area of Vasospasm After Subarachnoid Hemorrhage 415

through the carotid artery for one hour and then removed, blood viscosity measured 24 hours later by a cone-plated discometer (at 37.5 rpm), was elevated from a control of 5.31 centipoise (cP) to 6.05 cP. Fasudil (1~10mg/kg) dose dependently inhibited the elevation of blood viscosity (Hitomi et al., 2000). Both production of the tissue factor (Zhang et al., 2007) and Rho kinase-activated polymerization of f-actin are considered to be involved in the increase of blood viscosity which were ameliorated by fasudil(Feske et al., 2009, Nagata et

RhoA/Rho kinase pathway has been shown to be involved in many other vascular diseases such as angiogenesis, atherosclerosis, cerebral and coronary spasm and infarction, glomerulosclerosis, hypertension, ischemia-reperfusion injury, neointimal proliferation, bronchial asthma, glaucoma and so on. Our current concepts about the Rho-kinase related mechanisms and effects of a Rho kinase inhibitor fasudil in cerebral vasospasm and

Fig. 2. Rho kinase related mechanisms and effects of fasudil in cerebral vasospasm and

Chemical ligands eluted from subarachnoid blood clot or from ischemic brain such as oxyhemoglobin, angiotensin II and endothelin increase intracellular lysophosphatidic acid (LPA) and sphyngosine-1-phosphate (S1-P) which activate RhoA through activation of guanine nucleotide exchange factors (GEFs) from an inactive GDP-Rho in the cytosole to an active and membrane bound GTP-Rho. Activated Rho kinase contracts blood vessels by inhibiting myosin light chain (MLC) phosphatase by phosphorylating its component myosin

binding subunit (MBS) at Thr696 through activation of protein kinase C-potentiated

al., 1999) either directly or through activation of protein kinase C (PKC). PKC activated protein kinase C-potentiated inhibitory protein-17 (CPI-17) by phosphorylating at Thr38 (Koyama et al., 2000). In vasospastic condition, contraction force is increased without changes in intracellular concentration of Ca++. Thus double (sometimes triple) phosphorylation of MLC by upregulated Rho kinase is considered to be the mechanism of so called increased sensitivity to Ca++.

### **6.3 Involvement of inflammation in vasospasm**

Inflammatory cells migrate to vasospasm or infarction sites and cause tissue injury by producing free radicals. When human WBCs were incubated in a Boyden chamber, WBCs migrated through a millipore filter by adding a chemoattractant such as formyl-methionylleucyl-phenylalanine (fMLP) to one side of the chamber. This migration was dose dependently inhibited by fasudil (Satoh et al., 1999). When WBCs were incubated with phorbol myristate acetate (PMA), a protein kinase C activator, they produced superoxide anion (O2-) by NADPH oxidase, which also was dose dependently inhibited by fasudil (Arai et al., 1993). Free radicals such as O2 - are known to cause structural damage in endothelial cells and SMCs, leading to a decreased production of nitric oxide (NO) by endothelial NO synthase (eNOS).

### **6.4 Inhibition of NO synthase (eNOS) by Rho kinase**

Nitric oxide (NO) plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, suppression of SMC proliferation and prevention of leukocyte recruitment to the vessel wall. Activity of eNOS is controlled by a variety of signals surrounding blood vessels. Laminar shear stress, O2 tension and transforming growth factor (TGF)β1 can regulate eNOS expression at the transcriptional level. Chronic hypoxia, tissue necrosis factor (TNF)α, thrombin, oxidized low density lipoprotein (LDL) and cellular proliferation are known to regulate eNOS expression at postscriptional level. Shear stress and vascular endothelial growth factor (VEGF) rapidly activated eNOS by phosphorylating at Ser1177. Hypoxia is known to upregulate Rho kinase which inhibits eNOS by phosphorylating at Thr495 (Flemming et al., 2001; Noma et al., 2006; Sugimoto et al., 2007). On the other hand, inhibition of Rho kinase by hydroxyfasudil increased phosphorylation of protein kinase Akt Ser473 and production of NO (Wolfrum et al., 2004). NO relaxes blood vessels by activating guanylate cyclase (which produced cyclic GMP) and protein kinase G, which activated MLC phosphatase by phosphorylating its component MBS at Ser695 (Nakamura & Ikebe, 2007, see also Fig. 2).

Pulmonary hypertension is a fatal disease in which eNOS activity is decreased. When human vascular endothelium was incuvated under hypoxic state of 3% O2, both expression of mRNA of eNOS and eNOS activity were suppressed. The suppression was ameliorated by Rho kinase inhibitors, botulinus C3 transferase and fasudil (Takemoto et al., 2002). Actually, fasudil showed good results in patients with pulmonary hypertension (Fukumoto et al., 2005).

### **6.5 Increased blood viscosity in cerebral vasospasm and infarction**

Blood viscosity is elevated in patients with acute cerebral infarction (Coull et al., 1991). However, it is not clear if this reflects a pre-existing risk factor or an acute phase response to the stroke itself or both. In rats model of temporary ischemia, by passing a nylon thread

al., 1999) either directly or through activation of protein kinase C (PKC). PKC activated protein kinase C-potentiated inhibitory protein-17 (CPI-17) by phosphorylating at Thr38 (Koyama et al., 2000). In vasospastic condition, contraction force is increased without changes in intracellular concentration of Ca++. Thus double (sometimes triple) phosphorylation of MLC by upregulated Rho kinase is considered to be the mechanism of

Inflammatory cells migrate to vasospasm or infarction sites and cause tissue injury by producing free radicals. When human WBCs were incubated in a Boyden chamber, WBCs migrated through a millipore filter by adding a chemoattractant such as formyl-methionylleucyl-phenylalanine (fMLP) to one side of the chamber. This migration was dose dependently inhibited by fasudil (Satoh et al., 1999). When WBCs were incubated with phorbol myristate acetate (PMA), a protein kinase C activator, they produced superoxide anion (O2-) by NADPH oxidase, which also was dose dependently inhibited by fasudil (Arai et al., 1993). Free radicals such as O2- are known to cause structural damage in endothelial cells and SMCs, leading to a decreased production of nitric oxide (NO) by endothelial NO

Nitric oxide (NO) plays an important role in the regulation of vascular tone, inhibition of platelet aggregation, suppression of SMC proliferation and prevention of leukocyte recruitment to the vessel wall. Activity of eNOS is controlled by a variety of signals surrounding blood vessels. Laminar shear stress, O2 tension and transforming growth factor (TGF)β1 can regulate eNOS expression at the transcriptional level. Chronic hypoxia, tissue necrosis factor (TNF)α, thrombin, oxidized low density lipoprotein (LDL) and cellular proliferation are known to regulate eNOS expression at postscriptional level. Shear stress and vascular endothelial growth factor (VEGF) rapidly activated eNOS by phosphorylating at Ser1177. Hypoxia is known to upregulate Rho kinase which inhibits eNOS by phosphorylating at Thr495 (Flemming et al., 2001; Noma et al., 2006; Sugimoto et al., 2007). On the other hand, inhibition of Rho kinase by hydroxyfasudil increased phosphorylation of protein kinase Akt Ser473 and production of NO (Wolfrum et al., 2004). NO relaxes blood vessels by activating guanylate cyclase (which produced cyclic GMP) and protein kinase G, which activated MLC phosphatase by phosphorylating its component MBS at Ser695

Pulmonary hypertension is a fatal disease in which eNOS activity is decreased. When human vascular endothelium was incuvated under hypoxic state of 3% O2, both expression of mRNA of eNOS and eNOS activity were suppressed. The suppression was ameliorated by Rho kinase inhibitors, botulinus C3 transferase and fasudil (Takemoto et al., 2002). Actually, fasudil showed good results in patients with pulmonary hypertension (Fukumoto

Blood viscosity is elevated in patients with acute cerebral infarction (Coull et al., 1991). However, it is not clear if this reflects a pre-existing risk factor or an acute phase response to the stroke itself or both. In rats model of temporary ischemia, by passing a nylon thread

**6.5 Increased blood viscosity in cerebral vasospasm and infarction** 

so called increased sensitivity to Ca++.

synthase (eNOS).

et al., 2005).

**6.3 Involvement of inflammation in vasospasm** 

**6.4 Inhibition of NO synthase (eNOS) by Rho kinase** 

(Nakamura & Ikebe, 2007, see also Fig. 2).

through the carotid artery for one hour and then removed, blood viscosity measured 24 hours later by a cone-plated discometer (at 37.5 rpm), was elevated from a control of 5.31 centipoise (cP) to 6.05 cP. Fasudil (1~10mg/kg) dose dependently inhibited the elevation of blood viscosity (Hitomi et al., 2000). Both production of the tissue factor (Zhang et al., 2007) and Rho kinase-activated polymerization of f-actin are considered to be involved in the increase of blood viscosity which were ameliorated by fasudil(Feske et al., 2009, Nagata et al., 20022Satoh et al., 2010).

RhoA/Rho kinase pathway has been shown to be involved in many other vascular diseases such as angiogenesis, atherosclerosis, cerebral and coronary spasm and infarction, glomerulosclerosis, hypertension, ischemia-reperfusion injury, neointimal proliferation, bronchial asthma, glaucoma and so on. Our current concepts about the Rho-kinase related mechanisms and effects of a Rho kinase inhibitor fasudil in cerebral vasospasm and infarction are shown in Fig. 2.

Fig. 2. Rho kinase related mechanisms and effects of fasudil in cerebral vasospasm and infarction

Chemical ligands eluted from subarachnoid blood clot or from ischemic brain such as oxyhemoglobin, angiotensin II and endothelin increase intracellular lysophosphatidic acid (LPA) and sphyngosine-1-phosphate (S1-P) which activate RhoA through activation of guanine nucleotide exchange factors (GEFs) from an inactive GDP-Rho in the cytosole to an active and membrane bound GTP-Rho. Activated Rho kinase contracts blood vessels by inhibiting myosin light chain (MLC) phosphatase by phosphorylating its component myosin binding subunit (MBS) at Thr696 through activation of protein kinase C-potentiated

Fasudil (a Rho Kinase Inhibitor) Specifically Increases

88:E68-E75.

167:411-432

67:1929-1939

475:197-200

93:471-476

*Res* 87:195-200

*Pharmacol* 80:41-48

469.

*272*

system. *Am J physiol* 290:C661-C668

722

patients after ischemic attack. *Brain Res* 1257:89-93

Rho-associated kinase (Rho-kinase). *Science* 273:245-258

Cerebral Blood Flow in Area of Vasospasm After Subarachnoid Hemorrhage 417

Feske, S.K., Sorond, F.A., Henderson, G.V. et al. (2009) Increased leukocyte ROCK activity in

Flemming, I., Fisslthaler, B., Dimmeler, S. et al. (2001) Phosphorylation of Thr 495 regulates

Fukumoto, Y., Matoba, T., Ito, A. et al. (2005) Acute vasodilator effects of a Rho-kinase inhibitor fasudil in patients with severe pulmonary hypertension. *Heart* 91:391-392 Hidaka, H., Shibuya, M., Suzuki, Y. et al. (2005) Isoquinolinesulfonamide: A specific

Hitomi, A., Satoh, S., Ikegaki, I. et al. (2000) Hemorrheological abnormalities in experimental

Kimura, K., Ito, M., Amano, M. et al. (1996) Regulation of myosin phosphatase by Rho and

Koyama, M., Ito, M., Feng, J. et al. (2000) Phosphorylation of CPI-17, an inhibitory

Miyagi, Y., Carpenter, R.C., Meguro, T. et al. (2000) Upregulation of RhoA and Rho kinase

Nagata, K., Ishibashi, T., Sakamoto, T. et al. (2002) Rho/Rho-kinase is involved in the synthesis of tissue factor in human monocytes. *Atherosclerosis* 163:39-47 Nakamura, K. & Ikebe, M. (2007) cGMP-dependent relaxation of smooth muscle is coupled

Noma, K., Oyama, N. & Liao, J.K. (2006) Physiological role of ROCKs in the cardiovascular

Ono, K., Shirotani, T., Yuba, K. et al. (2005) Cerebral circulation dynamics following fasudil intravenous infusion: a CT perfusion study. *Brain Nerve (Tokyo)* 57:779-783. Sato, M., Tani, E., Fujikawa, H. et al. (2000) Involvement of Rho kinase mediated

Satoh, S., Hitomi, A., Ikegaki, I. et al. (2010) Amelioration of endothelial damage

Satoh, S., Kobayashi, T., Hitomi, A. et al. (1999) Inhibition of neutrophil migration by a

Schmandke, A., Schmandke, A. & Strittmatter, S.M. (2007) ROCK and Rho: Biochemistry

Seto, M., Sasaki, Y., Sasaki, Y. et al. (1991) Effect of HA1077, a protein kinase inhibitor, on

inhibitors against ischemic brain damage. *Brain Res Bull* 81:191-195

Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. *Circ Res*

inhibitor of Rho-kinase and the clinical aspect of anti-Rho-kinase therapy. *HEP* 

cerebral ischemia and effects of protein kinase inhibitor on blood fluidity. *Life Sci*

phosphoprotein of smooth muscle myosin phosphatase, by rho-kinse. *FEBS Let* 

RNAs in the basilar artery of a rat model of subarachnoid hemorrhage. *J Neurosurg* 

with the change in the phosphorylation of myosin phosphatase. *Circ Res* 101: 712-

phosphorylation of myosin light chain in enhancement of cerebral vasospasm. *Circ* 

/dysfunction is a possible mechanism for neuroprotecive effects of Rho-kinase

protein kinase inhibitor for the treatment of ischemic brain infarction. *Jpn J* 

and neuronal functions of Rho-associated protein kinases. Neuroscientist 13:454-

myosin phosphorylation and a tension in smooth muscle. *Eur J Pharmacol 195:267-*

inhibitory protein-17 (CPI-17). Rho kinase also inhibits relaxation of contracted blood vessels by inhibiting endothelial nitric oxide synthase (eNOS) through inhibition of phosphatidylinositol-3kinase (PI3K)/protein kinase Akt. On the other hand, eNOS is activated by dephosphorylation at Thr495 or phosphorylation at Ser1177 when Rho kinase is inhibited. NO relaxes blood vessels by activating guanylate cyclase and protein kinase G (PKG). PKG activates MLC phosphatase by phosphorylating its component myosin binding subunit (MBS) at Ser695.

On the other hand, migration of inflammatory cells like WBCs and their production of free radicals by NADPH oxidase are stimulated by upregulated Rho kinase and protein kinase C. Rho kinase also increases blood viscosity by producing the tissue factor which triggers the coagulation cascade and also by decreasing plasticity of RBCs. Plasticity of RBCs is decreased when f-actin, consisting cytoskeleton, is polymerized by Rho kinase and protein kinase C.

These adverse phenomena: abnormal contraction of blood vessels, migration of inflammatory cells and their production of free radicals, increase of blood viscosity had all been ameliorated by a Rho kinase inhibitor fasudil which showed in turn that upregulated Rho kinase is involved in each of these sites (see text for references). Arrow head indicates acting points of fasudil.

### **7. Conclusion**

Upregulated Rho kinase is deeply implicated in the complex mechanisms of delayed cerebral vasospasm after a subarachnoid hemorrhage, in both vasoconstriction and inflammation. Double phosphorylation of myosin light chain leading to pathological contraction, suppression of eNOS, production of free radicals are all induced by upregulated Rho kinase. Fasudil improved these situations by mainly inhibiting upregulated Rho kinase, which can explain why fasudil specifically increased cerebral blood flow in the area with vasospasm.

### **8. References**


inhibitory protein-17 (CPI-17). Rho kinase also inhibits relaxation of contracted blood vessels

activated by dephosphorylation at Thr495 or phosphorylation at Ser1177 when Rho kinase is inhibited. NO relaxes blood vessels by activating guanylate cyclase and protein kinase G (PKG). PKG activates MLC phosphatase by phosphorylating its component myosin binding

On the other hand, migration of inflammatory cells like WBCs and their production of free radicals by NADPH oxidase are stimulated by upregulated Rho kinase and protein kinase C. Rho kinase also increases blood viscosity by producing the tissue factor which triggers the coagulation cascade and also by decreasing plasticity of RBCs. Plasticity of RBCs is decreased when f-actin, consisting cytoskeleton, is polymerized by Rho kinase and protein

inflammatory cells and their production of free radicals, increase of blood viscosity had all been ameliorated by a Rho kinase inhibitor fasudil which showed in turn that upregulated Rho kinase is involved in each of these sites (see text for references). Arrow head indicates

Upregulated Rho kinase is deeply implicated in the complex mechanisms of delayed cerebral vasospasm after a subarachnoid hemorrhage, in both vasoconstriction and inflammation. Double phosphorylation of myosin light chain leading to pathological contraction, suppression of eNOS, production of free radicals are all induced by upregulated Rho kinase. Fasudil improved these situations by mainly inhibiting upregulated Rho kinase, which can explain why fasudil specifically increased cerebral blood flow in the area with

Arai, M., Sasaki, Y. & Nozawa, R. (1993) Inhibition by the protein kinase inhibitor HA1077

Bederson, J.B., Connolly, E.S.Jr, Batjer, H.H. et al. (2009) American Heart Association.

Brabeck, C., Mittelbronn, M., Bekure, K. et al. (2003) Effect of cerebral infarctions on regional

Coull, B.M., Beamer, N., de Garmo, P. et al. (1991) Chronic blood hyperviscosity in subjects

Dong, M., Yan, B.P., Liao J.K. et al. (2010) Rho-kinase inhibition: a novel therapeutic target for the treatment of cardiovascular diseases. Drug Discovery Today 15:622-629. Feng, J., Ito, M., Ichikawa, K. et al. (1999) Inhibitory phosphorylation site for rho-associated kinase on smooth muscle myosin phosphatase. *J Biol Chem* 274:3744-3752

RhoA and RhoB expression. *Arch Neurol* 60:1245-1249

of the activation of NADPH oxidase in human neutrophils. *Biochem Pharmacol* 

Guidelines for the management of aneurysmal subarachnoid hemorrhage. Stroke

with acute stroke, transient ischemic attack, and risk factors for stroke. *Stroke*

phosphatidylinositol-3kinase (PI3K)/protein kinase Akt. On the other hand, eNOS is

by inhibiting endothelial nitric oxide synthase (eNOS) through inhibition of

These adverse phenomena: abnormal contraction of blood vessels, migration of

subunit (MBS) at Ser695.

acting points of fasudil.

**7. Conclusion** 

vasospasm.

**8. References** 

46:1487-1490

40:994-1025

22:162-168

kinase C.


**19** 

*Italy* 

**Endogenous Agents That Contribute** 

*1Anestesiologia e Rianimazione, Università Degli Studi di Salerno* 

Ornella Piazza1 and Giuliana Scarpati2

**to Generate or Prevent Ischemic Damage** 

*2Anestesiologia e Rianimazione, Università Degli Studi di Napoli Federico II* 

From single to multicellular organisms, protective mechanisms have evolved against endogenous and exogenous noxious stimuli. Over the past decades numerous signaling pathways by which the brain senses and reacts to such insults as neurotoxins, substrate deprivation and inflammation have been discovered. Research on preconditioning is aimed at understanding endogenous neuroprotection to boost it or to supplement its effectors therapeutically once damage to the brain has occurred, such as after stroke or brain trauma. Another goal of establishing preconditioning protocols is to induce endogenous neuroprotection in anticipation of incipient brain damage. Currently several endogenous neuroprotectants are being investigated in controlled clinical trials. There is consensus that many of the neuroprotectants, which were highly effective in animal models of stroke, but failed in clinical trials, were unsuccessful because of side effects, which in many cases led to premature termination of the trial. Nowadays research aims to overcome this problem by developing compounds which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. In the present review we will give a short overview on the signals, sensors, transducers, and effectors of endogenous neuroprotection. We will first focus on common mechanisms, on which pathways of endogenous neuroprotection converge. We will then discuss various applications of endogenous neuroprotectors and explore the prospects of endogenous neuroprotective

Development of stroke prophylaxis involves the understanding of the mechanisms of damage following cerebral ischemia and elucidation of the endogenous mechanisms that

The binding of glutamate to its receptors and the activation of voltage-gated Ca2+ channels (VGCC) causes calcium to influx into the cell. Calcium is among the mediators that initiate the genomic response to cerebral ischemia. The superoxide dismutase (SOD) gene is upregulated to neutralize the reactive oxygen species (ROS). The generation of nitrous oxide (NO) in the neuron is cytotoxic. The interaction between antiapoptotic genes, such as Bcl-2, and proapoptotic genes, such as Bax, determines whether cytochrome c will be translocated

**1. Introduction** 

therapeutic approaches.

**2. Phisiopatology of cerebral ischemia** 

combat further brain injury (FIGURE 1).


### **Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage**

Ornella Piazza1 and Giuliana Scarpati2

*1Anestesiologia e Rianimazione, Università Degli Studi di Salerno 2Anestesiologia e Rianimazione, Università Degli Studi di Napoli Federico II Italy* 

### **1. Introduction**

418 Advances in the Preclinical Study of Ischemic Stroke

Shibuya, M., Suzuki, Y., Sugita, K. et al. (1992) Effect of AT877 on cerebral vasospasm after

Shibuya, M., Hirai, S, Seto, M. et al. (2005) Effects of fasudil in ischemic stroke: results of a prospective placebo-controlled double blind trial. *J Neurol Sci* 238:31-39 Shibuya, M, Ikeda, A., Ohsuka, K. et al. (2008) Fasudil (a rho kinase inhibitor) may specifically increase rCBF in spastic area. *Acta Neurochir Suppl* 104:275-278 Sugimoto, M., Nakayama, M., Gotoh, T.M. et al. (2007) Rho-kinase phosphorylates eNOS at

Takayasu, M., Suzuki, Y., Shibuya, M. et al. (1986) The effects of HA compound calcium antagonists on delayed cerebral vasospasm in dogs. *J Neurosurg* 65:80-85 Takemoto, M., Sun, J., Hiroki, J. et al. (2002) Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. *Circulation* 106:57-62 Tsuchiya, T., Sako, K., Yonemasu, Y. et al. (1993) The effects of HA1077, a novel protein

Ueda, T. (2000) Effect on increase of cerebral blood flow with cerebral vasospasm by fasudil

Wolfrum, S., Dendorfer, A., Rikitake, Y. et al (2004) Inhibition of Rho-kinase leads to rapid

Zhao, J., Zhou, D., Guo, J. et al. (2006) Effect of fasudil hydrochloride, a protein kinase

Zhang, X.P., Hu, Y., Hong, M., et al., (2007) Plasma thrombomodulin, fibrinogen, and

kinase inhibitor, on reductions of cerebral blood flow and glucose metabolism following acute and/or chronic bilateral carotid ligation in Wistar rats. *Exp Brain* 

activation of phosphatidylinositol 3 kinase/protein kinase Akt and cardiovascular

inhibitor, on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Results of a randomized trial of fasudil hydrochloride versus nimodipine. *Neurol* 

activity of tissue factor as risk factors for acute cerebral infarction. *Am J Clin Pathol*

aneurysmal subarachnoid hemorrhage. *J Neurosurg* 76: 571-577

threonine 495 in endothelial cells. *BBRC* 361:462-467

hydrochloride. *Med Pharmacy (Tokyo)* 42:753-759.

protection. *Atheroscler thromb Vasc Biol* 24:1842-1847

*Res* 97:233-238

128:287-292

*Med Chir (Tokyo)* 46:421-428

From single to multicellular organisms, protective mechanisms have evolved against endogenous and exogenous noxious stimuli. Over the past decades numerous signaling pathways by which the brain senses and reacts to such insults as neurotoxins, substrate deprivation and inflammation have been discovered. Research on preconditioning is aimed at understanding endogenous neuroprotection to boost it or to supplement its effectors therapeutically once damage to the brain has occurred, such as after stroke or brain trauma. Another goal of establishing preconditioning protocols is to induce endogenous neuroprotection in anticipation of incipient brain damage. Currently several endogenous neuroprotectants are being investigated in controlled clinical trials. There is consensus that many of the neuroprotectants, which were highly effective in animal models of stroke, but failed in clinical trials, were unsuccessful because of side effects, which in many cases led to premature termination of the trial. Nowadays research aims to overcome this problem by developing compounds which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. In the present review we will give a short overview on the signals, sensors, transducers, and effectors of endogenous neuroprotection. We will first focus on common mechanisms, on which pathways of endogenous neuroprotection converge. We will then discuss various applications of endogenous neuroprotectors and explore the prospects of endogenous neuroprotective therapeutic approaches.

### **2. Phisiopatology of cerebral ischemia**

Development of stroke prophylaxis involves the understanding of the mechanisms of damage following cerebral ischemia and elucidation of the endogenous mechanisms that combat further brain injury (FIGURE 1).

The binding of glutamate to its receptors and the activation of voltage-gated Ca2+ channels (VGCC) causes calcium to influx into the cell. Calcium is among the mediators that initiate the genomic response to cerebral ischemia. The superoxide dismutase (SOD) gene is upregulated to neutralize the reactive oxygen species (ROS). The generation of nitrous oxide (NO) in the neuron is cytotoxic. The interaction between antiapoptotic genes, such as Bcl-2, and proapoptotic genes, such as Bax, determines whether cytochrome c will be translocated

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 421

believed to result from depletion of cellular energy stores from overexcited neurons. However, the influx of Ca2+ seems to be the major pathogenic event contributing to cell death. This translocation of Ca2+ is accomplished through glutamate, particularly through the NMDA receptor, as well as through voltage-gated Ca2+ channels that open after cell depolarization. Calcium channel antagonists have displayed neural protection in animal models but have not shown benefit in clinical trials partially because they were administered too late after stroke onset or in insufficient quantity. However, it appears that Ca2+ influx

Reactive oxygen species are produced after the induction of ischemia and upon reperfusion. The oxidative stress produced by the reactive oxygen species destroys the cell through lipid peroxidation, protein oxidation, and DNA damage. Certain endogenous antioxidants scavenge and neutralize the reactive oxygen species. In particular, the antioxidant

hydrogen peroxide (H2O2). Glutathione peroxidase can then convert H2O2 into oxygen and water. During times of oxidative stress, the superoxide dismutase gene is upregulated. Neural protection strategies have included both the administration of exogenous superoxide dismutase and manipulation of the superoxide dismutase gene family. Nitric oxide (NO) is another free radical that is increased during ischemia due to an increase in intracellular Ca2+. The formation of NO is catalyzed by the enzyme NO synthase (NOS). NOS has several isoforms—a neuronal type (nNOS) located in neurons and an endothelial type (eNOS) in the vascular endothelium. NO is also generated in microglia, astrocytes and invading macrophages after the induction of an inducible isoform (iNOS). Initially after ischemia, the formation of NO in the vascular endothelium by eNOS may improve CBF through vasodilatation offering neuroprotection. However, synthesis of NO by nNOS and iNOS is cytotoxic, leading to an inhibition of mitochondrial respiration, glycolysis, and DNA synthesis. Because of the dual role of NO in cerebral ischemia, neuronal protection strategies need to target the specific isoform of NOS. For instance, deletion of the nNOS or iNOS gene

After an ischemic event, cells in the penumbra may initiate a program of autodestruction known as apoptosis. Apoptosis occurs in the developing brain. More than half of progenitor neurons undergo this process of programmed cell death while forming neural circuits. During ischemia, cells in the ischemic core undergo necrosis while cells in the ischemic penumbra may actually self destruct through this process of apoptosis. The mitochondria is regarded as the apoptotic headquarters of the cell. One of the key events in apoptosis is the translocation of cytochrome c from the intermembrane of the mitochondria into the cytosol. In the cytosol, cytochrome c combines then with apoptotic activating factor (Apaf-1) to activate a set of proteases known as caspases. These caspases actually dismantle the cell during apoptosis. A family of death-promoting genes, known as the Bcl-2 family, determines whether a cell will undergo apoptosis. The Bcl-2 gene is antiapoptotic and prevents the translocation of cytochrome c and activation of caspases. However, the Bax gene (one of the members of the Bcl-2 family) is proapoptotic, facilitating the translocation of cytochrome c and apoptosis. During ischemia, proapoptotic genes such as Bax are

) free radical by converting it to

into the cell is only the initial step in a complex biochemical cascade.

superoxide dismutase detoxifies the superoxide (O2-

in animal models has provided neuronal protection.

**2.2 Free radicals** 

**2.3 Apoptosis** 

from the mitochondria to the cytosol. In the cytosol, cytochrome c combines with Apaf-1 to activate the caspases. Proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a), are generated. Survival pathways involving growth factors (GFs), immediate early genes (IEGs), and heat shock proteins (Hsps) are also stimulated. Ultimately, the activation of these genetic pathways determines the fate of the ischemic cell.

Fig. 1. Phisiopatology of cerebral ischemia

### **2.1 Excitotoxicity and calcium**

Although the brain represents only 2% of body weight, it uses an astonishing 20% of the body's oxygen in adults. The innumerable cells of the brain require an almost continuous flow of oxygen and glucose, making them exquisitely sensitive to any interruption in energy supply. Energy depletion and reduced levels of adenosine triphosphate initiate a series of events that cause cells to die. Glutamate, the main excitatory neurotransmitter of the central nervous system, is a trigger of neuronal loss during stroke. During ischemia, an excess of glutamate is released into the extracellular space. The mechanism to clear glutamate is energy dependent; glutamate quickly builds to toxic levels when energy is depleted. Glutamate causes ionic shifts; Na+ enters the cell and K+ exits. Water passively follows the influx of Na+ leading to cellular swelling and edema. The membrane potential is lost and the cell depolarizes. In the ischemic core, cells undergo anoxic depolarization and never repolarize. However, cells in the penumbra initially retain the ability to repolarize so that they may depolarize again. As cells in the penumbra undergo these peri-infarct depolarizations the energy supply and ionic homeostasis are further compromised,resulting in an increase in the size of the ischemic lesion. Glutamate activates three main families of receptors: N-methyl-d-aspartate (NMDA), a-amino-3-hydroxy-5 methylisoxazole/kainate, and metabotropic glutamate receptors. Activation of these receptors leads to a buildup of Ca2+ within the cell. Ischemia therefore triggers glutamate receptor-mediated excitotoxicity and Ca2+ overload within the cell. Originally, neuronal death from excitotoxicity was

believed to result from depletion of cellular energy stores from overexcited neurons. However, the influx of Ca2+ seems to be the major pathogenic event contributing to cell death. This translocation of Ca2+ is accomplished through glutamate, particularly through the NMDA receptor, as well as through voltage-gated Ca2+ channels that open after cell depolarization. Calcium channel antagonists have displayed neural protection in animal models but have not shown benefit in clinical trials partially because they were administered too late after stroke onset or in insufficient quantity. However, it appears that Ca2+ influx into the cell is only the initial step in a complex biochemical cascade.

### **2.2 Free radicals**

420 Advances in the Preclinical Study of Ischemic Stroke

from the mitochondria to the cytosol. In the cytosol, cytochrome c combines with Apaf-1 to activate the caspases. Proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a), are generated. Survival pathways involving growth factors (GFs), immediate early genes (IEGs), and heat shock proteins (Hsps) are also stimulated. Ultimately, the activation of these genetic pathways determines the fate of the ischemic cell.

Although the brain represents only 2% of body weight, it uses an astonishing 20% of the body's oxygen in adults. The innumerable cells of the brain require an almost continuous flow of oxygen and glucose, making them exquisitely sensitive to any interruption in energy supply. Energy depletion and reduced levels of adenosine triphosphate initiate a series of events that cause cells to die. Glutamate, the main excitatory neurotransmitter of the central nervous system, is a trigger of neuronal loss during stroke. During ischemia, an excess of glutamate is released into the extracellular space. The mechanism to clear glutamate is energy dependent; glutamate quickly builds to toxic levels when energy is depleted. Glutamate causes ionic shifts; Na+ enters the cell and K+ exits. Water passively follows the influx of Na+ leading to cellular swelling and edema. The membrane potential is lost and the cell depolarizes. In the ischemic core, cells undergo anoxic depolarization and never repolarize. However, cells in the penumbra initially retain the ability to repolarize so that they may depolarize again. As cells in the penumbra undergo these peri-infarct depolarizations the energy supply and ionic homeostasis are further compromised,resulting in an increase in the size of the ischemic lesion. Glutamate activates three main families of receptors: N-methyl-d-aspartate (NMDA), a-amino-3-hydroxy-5 methylisoxazole/kainate, and metabotropic glutamate receptors. Activation of these receptors leads to a buildup of Ca2+ within the cell. Ischemia therefore triggers glutamate receptor-mediated excitotoxicity and Ca2+ overload within the cell. Originally, neuronal death from excitotoxicity was

Fig. 1. Phisiopatology of cerebral ischemia

**2.1 Excitotoxicity and calcium** 

Reactive oxygen species are produced after the induction of ischemia and upon reperfusion. The oxidative stress produced by the reactive oxygen species destroys the cell through lipid peroxidation, protein oxidation, and DNA damage. Certain endogenous antioxidants scavenge and neutralize the reactive oxygen species. In particular, the antioxidant superoxide dismutase detoxifies the superoxide (O2- ) free radical by converting it to hydrogen peroxide (H2O2). Glutathione peroxidase can then convert H2O2 into oxygen and water. During times of oxidative stress, the superoxide dismutase gene is upregulated. Neural protection strategies have included both the administration of exogenous superoxide dismutase and manipulation of the superoxide dismutase gene family. Nitric oxide (NO) is another free radical that is increased during ischemia due to an increase in intracellular Ca2+. The formation of NO is catalyzed by the enzyme NO synthase (NOS). NOS has several isoforms—a neuronal type (nNOS) located in neurons and an endothelial type (eNOS) in the vascular endothelium. NO is also generated in microglia, astrocytes and invading macrophages after the induction of an inducible isoform (iNOS). Initially after ischemia, the formation of NO in the vascular endothelium by eNOS may improve CBF through vasodilatation offering neuroprotection. However, synthesis of NO by nNOS and iNOS is cytotoxic, leading to an inhibition of mitochondrial respiration, glycolysis, and DNA synthesis. Because of the dual role of NO in cerebral ischemia, neuronal protection strategies need to target the specific isoform of NOS. For instance, deletion of the nNOS or iNOS gene in animal models has provided neuronal protection.

### **2.3 Apoptosis**

After an ischemic event, cells in the penumbra may initiate a program of autodestruction known as apoptosis. Apoptosis occurs in the developing brain. More than half of progenitor neurons undergo this process of programmed cell death while forming neural circuits. During ischemia, cells in the ischemic core undergo necrosis while cells in the ischemic penumbra may actually self destruct through this process of apoptosis. The mitochondria is regarded as the apoptotic headquarters of the cell. One of the key events in apoptosis is the translocation of cytochrome c from the intermembrane of the mitochondria into the cytosol. In the cytosol, cytochrome c combines then with apoptotic activating factor (Apaf-1) to activate a set of proteases known as caspases. These caspases actually dismantle the cell during apoptosis. A family of death-promoting genes, known as the Bcl-2 family, determines whether a cell will undergo apoptosis. The Bcl-2 gene is antiapoptotic and prevents the translocation of cytochrome c and activation of caspases. However, the Bax gene (one of the members of the Bcl-2 family) is proapoptotic, facilitating the translocation of cytochrome c and apoptosis. During ischemia, proapoptotic genes such as Bax are

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 423

hypoxia induces the gene expression of Epo in the kidney, the main site for Epo production, and in the liver (Cotena et al, 2008) in a negative feedback system between the kidney and the bone marrow. Research performed in the last decade has shown that Epo and its receptor (EpoR) are expressed in tissues other than those involved in erythropoiesis. These include the brain, the reproductive tract (Kobayashi et al, 2002; Marti et al, 1996; Masuda et al, 2000), the lung, the spleen, and the heart (Fandrey and Bunn, 1993). Accordingly, a novel cytoprotective effect of Epo was established in several organs. For example, Epo reduced injury and dysfunction after ischemia-reperfusion in the mouse kidney (Patel et al, 2004), and it showed protection in various myocardial ischemia models (Bogoyevith,2004; Cai et al,

Epo is mainly produced in the interstitial fibroblasts in the adult kidney and the hepatocytes of the fetus, whereas EpoR is normally expressed in erythroid precursor cells in the bone marrow (Marti, 2004). However, recent data have shown that the expression of Epo and its receptor, EpoR (both mRNA and protein), coincides in the same organ and even within the same cell. Epo and EpoR expression are widely distributed in the mammalian brain (Genc *et al*, 2004; Marti, 2004), albeit at lower levels than in the kidney (Brines and Cerami, 2005). Epo thus has to be added to the growing list of hematopoietic growth factors found to be

Epo/EpoR mRNA and protein were detected is several regions of the murine and primate brain, including cortex, hippocampus and amygdale, cerebellum, hypothalamus, and caudate nucleus (Siren et al, 2001). With respect to the type of cells in the brain that express Epo, astrocytes are the main source of Epo in the brain (Masuda et al, 1994). Moreover, it has been shown in vitro and in vivo that neurons express Epo (Bernaudin et al, 1999, 2000). Similarly, EpoR is expressed on neurons and astrocytes. In addition, primary cultures of human neurons, astrocytes, and microglia express EpoR mRNA (Nagai et al, 2001), and EpoR expression was also detected in primary cultures of rat oligodendrocytes (Genc et al, 2006). In addition to neurons, oligodendrocytes, and glial cells, a strong immunoreactivity for EpoR was found to be associated with brain vascular endothelial cells, showing that these cells also express EpoR (Brines et al, 2000). These findings implicate a broad spectrum

As mentioned above, Epo is upregulated in response to hypoxia. As, for many of the hypoxic adaptation processes in the body, the regulation of Epo expression is based on the transcriptional regulation of two hypoxia-inducible factors HIF-1 and HIF-2 (Wenger, 2000). HIFs are heterodimers composed of an α- and a β -subunit. Two forms of the oxygen-labile α exist, 1α and 2α. The α-subunit is stabilized under hypoxic conditions leading to the binding of the heterodimer HIF-1 or HIF-2 to specific DNA sequences located in the hypoxia response elements of target genes such as Epo or vascular endothelial growth factor (VEGF) (Wenger, 2002). Although HIF-1α was originally identified as the transcription factor responsible for Epo expression (Semenza et al, 1991), more recent evidence suggests that Epo is a target of HIF-2 (Eckardt and Kurtz, 2005). The stability of HIF-α is regulated by

2003; Parsa et al, 2003).

**3.1 Epo/EpoR expression and regulation** 

**3.2 Expression of Epo/EpoR in the brain** 

of actions of Epo in the brain.

**3.3 Regulation of Epo/EpoR expression** 

expressed and act in the central nervous system (CNS).

activated, resulting in the autodestruction of the cell. Thus, neuronal protection may be gained through blocking these death-promoting genes. Other strategies include giving caspase antagonists or preventing the translocation of cytochrome c from the mitochondria. Preventing apoptosis in the penumbra is another effective technique in animal models for neuronal protection.

### **2.4 Inflammation**

The inflammatory response may be an important part of the ischemic cascade. Soon after the onset of stroke, leukocytes invade the ischemic zone. The mechanisms by which these inflammatory cells contribute to the evolution of ischemia include microvascular occlusion by adherence to the endothelium, producing cytotoxic enzymes and generating injurious free radicals. Cytokines are intracellular messengers that mediate the recruitment of the leukocytes and the induction of adhesion molecules. The two main proinflammatory cytokines are interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a). The adhesion molecules that facilitate the movement of leukocytes along the surface of the endothelium are the E and P selectins, whereas intracellular adhesion molecules attach the leukocytes to the endothelium so that they may leave the vascular space and enter the site of injury. Research has been focusing on the manipulation of these proinflammatory cytokines and adhesion molecules to provide neuronal protection.

### **2.4.1 Survival pathways**

The cytokines that are activated during ischemia also include growth factors that actually promote neuronal survival and, in some cases, neuronal outgrowth and synapse formation. Fibroblast growth factor is the most extensively studied growth factor. Although the exact mechanism of neuroprotection of fibroblast growth factor is not fully understood, it includes upregulation of free radical scavenging enzymes and Ca2+ binding proteins, downregulation of the NMDA receptor and vasodilatation. The administration of growth factors has provided cerebral protection in animal models. Because they exert both protective and trophic influences on neurons, growth factors remain an exciting prospect in drug development for stroke. Other gene families and proteins are activated during ischemia. Immediate early genes, such as those of the Fos and Jun families, are activated soon after ischemia. It is believed that Ca2+ and reactive oxygen species are involved in the expression of immediate early genes. Although the exact role of each of the immediate early genes in ischemia is not yet understood, they are known to participate in apoptosis. Some immediate early genes may even afford neuronal protection. Ischemia also induces the expression of molecular chaperones known as heat shock proteins, which maintain protein function and assist in protein transport in response to injury. Increasing the expression of heat shock proteins to combat ischemia has been attempted.

### **3. Erythropoietin**

The hormone erythropoietin (Epo) is a 165-amino acid (~30 kDa) glycoprotein that belongs to the cytokine type I superfamily. Originally, it was believed that the only role of Epo was the regulation of erythropoiesis. This role is attributed to the ability of Epo to inhibit programmed cell death (apoptosis) in erythroid cells and thus allow the maturation of erythrocytes. Since blood oxygen availability is the main regulator of erythropoiesis, hypoxia induces the gene expression of Epo in the kidney, the main site for Epo production, and in the liver (Cotena et al, 2008) in a negative feedback system between the kidney and the bone marrow. Research performed in the last decade has shown that Epo and its receptor (EpoR) are expressed in tissues other than those involved in erythropoiesis. These include the brain, the reproductive tract (Kobayashi et al, 2002; Marti et al, 1996; Masuda et al, 2000), the lung, the spleen, and the heart (Fandrey and Bunn, 1993). Accordingly, a novel cytoprotective effect of Epo was established in several organs. For example, Epo reduced injury and dysfunction after ischemia-reperfusion in the mouse kidney (Patel et al, 2004), and it showed protection in various myocardial ischemia models (Bogoyevith,2004; Cai et al, 2003; Parsa et al, 2003).

### **3.1 Epo/EpoR expression and regulation**

422 Advances in the Preclinical Study of Ischemic Stroke

activated, resulting in the autodestruction of the cell. Thus, neuronal protection may be gained through blocking these death-promoting genes. Other strategies include giving caspase antagonists or preventing the translocation of cytochrome c from the mitochondria. Preventing apoptosis in the penumbra is another effective technique in animal models for

The inflammatory response may be an important part of the ischemic cascade. Soon after the onset of stroke, leukocytes invade the ischemic zone. The mechanisms by which these inflammatory cells contribute to the evolution of ischemia include microvascular occlusion by adherence to the endothelium, producing cytotoxic enzymes and generating injurious free radicals. Cytokines are intracellular messengers that mediate the recruitment of the leukocytes and the induction of adhesion molecules. The two main proinflammatory cytokines are interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a). The adhesion molecules that facilitate the movement of leukocytes along the surface of the endothelium are the E and P selectins, whereas intracellular adhesion molecules attach the leukocytes to the endothelium so that they may leave the vascular space and enter the site of injury. Research has been focusing on the manipulation of these proinflammatory cytokines and

The cytokines that are activated during ischemia also include growth factors that actually promote neuronal survival and, in some cases, neuronal outgrowth and synapse formation. Fibroblast growth factor is the most extensively studied growth factor. Although the exact mechanism of neuroprotection of fibroblast growth factor is not fully understood, it includes upregulation of free radical scavenging enzymes and Ca2+ binding proteins, downregulation of the NMDA receptor and vasodilatation. The administration of growth factors has provided cerebral protection in animal models. Because they exert both protective and trophic influences on neurons, growth factors remain an exciting prospect in drug development for stroke. Other gene families and proteins are activated during ischemia. Immediate early genes, such as those of the Fos and Jun families, are activated soon after ischemia. It is believed that Ca2+ and reactive oxygen species are involved in the expression of immediate early genes. Although the exact role of each of the immediate early genes in ischemia is not yet understood, they are known to participate in apoptosis. Some immediate early genes may even afford neuronal protection. Ischemia also induces the expression of molecular chaperones known as heat shock proteins, which maintain protein function and assist in protein transport in response to injury. Increasing the expression of heat shock

The hormone erythropoietin (Epo) is a 165-amino acid (~30 kDa) glycoprotein that belongs to the cytokine type I superfamily. Originally, it was believed that the only role of Epo was the regulation of erythropoiesis. This role is attributed to the ability of Epo to inhibit programmed cell death (apoptosis) in erythroid cells and thus allow the maturation of erythrocytes. Since blood oxygen availability is the main regulator of erythropoiesis,

neuronal protection.

**2.4 Inflammation** 

**2.4.1 Survival pathways** 

**3. Erythropoietin** 

adhesion molecules to provide neuronal protection.

proteins to combat ischemia has been attempted.

Epo is mainly produced in the interstitial fibroblasts in the adult kidney and the hepatocytes of the fetus, whereas EpoR is normally expressed in erythroid precursor cells in the bone marrow (Marti, 2004). However, recent data have shown that the expression of Epo and its receptor, EpoR (both mRNA and protein), coincides in the same organ and even within the same cell. Epo and EpoR expression are widely distributed in the mammalian brain (Genc *et al*, 2004; Marti, 2004), albeit at lower levels than in the kidney (Brines and Cerami, 2005). Epo thus has to be added to the growing list of hematopoietic growth factors found to be expressed and act in the central nervous system (CNS).

### **3.2 Expression of Epo/EpoR in the brain**

Epo/EpoR mRNA and protein were detected is several regions of the murine and primate brain, including cortex, hippocampus and amygdale, cerebellum, hypothalamus, and caudate nucleus (Siren et al, 2001). With respect to the type of cells in the brain that express Epo, astrocytes are the main source of Epo in the brain (Masuda et al, 1994). Moreover, it has been shown in vitro and in vivo that neurons express Epo (Bernaudin et al, 1999, 2000). Similarly, EpoR is expressed on neurons and astrocytes. In addition, primary cultures of human neurons, astrocytes, and microglia express EpoR mRNA (Nagai et al, 2001), and EpoR expression was also detected in primary cultures of rat oligodendrocytes (Genc et al, 2006). In addition to neurons, oligodendrocytes, and glial cells, a strong immunoreactivity for EpoR was found to be associated with brain vascular endothelial cells, showing that these cells also express EpoR (Brines et al, 2000). These findings implicate a broad spectrum of actions of Epo in the brain.

### **3.3 Regulation of Epo/EpoR expression**

As mentioned above, Epo is upregulated in response to hypoxia. As, for many of the hypoxic adaptation processes in the body, the regulation of Epo expression is based on the transcriptional regulation of two hypoxia-inducible factors HIF-1 and HIF-2 (Wenger, 2000). HIFs are heterodimers composed of an α- and a β -subunit. Two forms of the oxygen-labile α exist, 1α and 2α. The α-subunit is stabilized under hypoxic conditions leading to the binding of the heterodimer HIF-1 or HIF-2 to specific DNA sequences located in the hypoxia response elements of target genes such as Epo or vascular endothelial growth factor (VEGF) (Wenger, 2002). Although HIF-1α was originally identified as the transcription factor responsible for Epo expression (Semenza et al, 1991), more recent evidence suggests that Epo is a target of HIF-2 (Eckardt and Kurtz, 2005). The stability of HIF-α is regulated by

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 425

This basic mechanism of regulation seems to be of relevance for brain-expressed Epo, since in several experimental systems Epo was upregulated under hypoxic conditions in the brain of several mammalian species including mouse, rat, monkey, and human (Marti et al, 1996, 2000; Siren et al, 2001). However, depending on the severity of hypoxia, Epo mRNA level can increase 3- to 20-fold in the brain in contrast to 200-fold in the kidney. Moreover, although the increase in Epo expression in the kidney seems to be transient with a decrease after 8 h of continuous hypoxia, the level of Epo in the brain remains high for at least 24 h (Chikuma et al, 2000). This indicates a tissue-specific degree of regulation. Indeed, although HIF-1α levels in the kidney under systemic hypoxia peak after 1 h and again reach basal levels 4 h thereafter, in the brain the HIF-1α peak level is reached after only 5 h and returns to the basal level not before 12 h (Stroka et al, 2001). A possible explanation for the different time course in the brain might be an altered composition of the various PHD forms. It has to be noted that hypoxia is not the only factor activating HIF. Several studies have shown that pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or lipopolysaccharide (LPS) induce the expression of HIF (Frede et al, 2007). With regard to the EpoR, it is regulated by pro-inflammatory cytokines (Nagai et al, 2001), such as TNF-α, IL-1β, and Epo itself (Chin et al, 2000). The role of hypoxia in the regulation of EpoR expression is controversial. Whereas we did not observe hypoxic induction of EpoR expression in neurons or astrocytes (Bernaudin et al, 2000), anemic stress induced EpoR expression in the brain of human EpoR transgenic mice (Chin et al, 2000). Moreover, in the same study, hypoxia increased EpoR expression in neuronal cells in vitro. The mechanism of hypoxic EpoR regulation remains to be established, since EpoR has not been identified as

HIF target gene so far.

Epo promotes cell survival through inhibiting apoptosis (FIGURE 3).

In erythroid cells, after binding of Epo to its receptor (EpoR), Janus tyrosine kinase 2 (JAK2) is phosphorylated and thus activated. This leads to engaging secondary signaling molecules such as signal transducer and activator of transcription 5 (STAT5), followed by the activation of Ras mitogen-activated protein kinase (MAPK), ERK-1/-2, and PI3K/Akt (22). Moreover, Epo induced the upregulation of the anti-apoptotic protein BCL-XL (Kilic et al, 2005). The functional significance of these signaling molecules in erythropoiesis is not absolutely clear though. For instance, whereas in one study STAT5 knockout adult mice were largely unaffected in their erythroid lineage (Teglund et al, 1998), in another study STAT5 knockout embryos suffered from severe anemia, showed a reduced number of erythroid progenitors cells, and had higher numbers of apoptotic cells (Socolovsky et al, 1999). Most of these pathways seem also to be functional in the brain (Brines and Cerami, 2005; Kilic et al, 2005). In vitro, inhibition of MAPK and PI3K blocked Epo-mediated protection of rat hippocampal neurons against hypoxia (Siren et al, 2001b). Moreover, using ERK-1/-2 and Akt inhibitors, Kilic et al. showed that activation of these proteins is essential for Epo-mediated neuroprotection in an animal model of focal cerebral ischemia. The role of STAT5 in Epo-induced neuroprotection is, however, controversial. STAT5 phosphorylation has been shown to occur in hippocampal CA1 neurons after transient global cerebral ischemia in rats (Zhang et al, 2007). Therefore, the authors concluded that STAT5 plays a role in Epo-mediated neuroprotection. However, in a very recent study, in an in vitro model of glutamate toxicity using hippocampal neuronal culture from STAT5 knockout mouse

**3.4 Epo signaling** 

enzymatic hydroxylation of specific amino acids on the α subunit by a group of oxygenases (FIGURE 2). Under normoxic conditions, a specific prolyl hydroxylation within the oxygendependent degradation domain of HIF-α takes place. This prolyl hydroxylation allows binding of the von Hippel-Lindau protein (pVHL), leading to ubiquitylation and proteasomal degradation of the HIF-α subunit (Ivan et al, 2001; Jaakkola et al, 2001). The enzymes responsible for this hydroxylation are termed prolyl hydroxylase domain enzymes (PHD1-3) (Bruick and McKnight, 2001; Epstein et al, 2001) and are widely expressed. Furthermore, in the presence of oxygen, another hydroxylation reaction takes place on an asparaginyl group in the COOH-terminal transactivation domain of HIF-α, blocking its binding to the transcriptional coactivators (Lando et al, 2002a). This process is governed by a specific asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) (Hewitson et al, 2002; Lando et al, 2002b ). So, under normoxia, FIH and PHD(s) are active, leading to transcriptional inactivation and degradation of HIF-α, whereas under hypoxic conditions both enzymes are inactive. HIF is then stabilized and able to induce the expression of target genes, including Epo.

Fig. 2. Under normoxic conditions, specific prolyl hydroxylation within the oxygendependent degradation domain of HIF- α takes place. By contrast, under hypoxic conditions, FIH and PHD(s) are both inactive, and HIF is stabilized and able to induce the expression of target genes including Epo.

This basic mechanism of regulation seems to be of relevance for brain-expressed Epo, since in several experimental systems Epo was upregulated under hypoxic conditions in the brain of several mammalian species including mouse, rat, monkey, and human (Marti et al, 1996, 2000; Siren et al, 2001). However, depending on the severity of hypoxia, Epo mRNA level can increase 3- to 20-fold in the brain in contrast to 200-fold in the kidney. Moreover, although the increase in Epo expression in the kidney seems to be transient with a decrease after 8 h of continuous hypoxia, the level of Epo in the brain remains high for at least 24 h (Chikuma et al, 2000). This indicates a tissue-specific degree of regulation. Indeed, although HIF-1α levels in the kidney under systemic hypoxia peak after 1 h and again reach basal levels 4 h thereafter, in the brain the HIF-1α peak level is reached after only 5 h and returns to the basal level not before 12 h (Stroka et al, 2001). A possible explanation for the different time course in the brain might be an altered composition of the various PHD forms. It has to be noted that hypoxia is not the only factor activating HIF. Several studies have shown that pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or lipopolysaccharide (LPS) induce the expression of HIF (Frede et al, 2007). With regard to the EpoR, it is regulated by pro-inflammatory cytokines (Nagai et al, 2001), such as TNF-α, IL-1β, and Epo itself (Chin et al, 2000). The role of hypoxia in the regulation of EpoR expression is controversial. Whereas we did not observe hypoxic induction of EpoR expression in neurons or astrocytes (Bernaudin et al, 2000), anemic stress induced EpoR expression in the brain of human EpoR transgenic mice (Chin et al, 2000). Moreover, in the same study, hypoxia increased EpoR expression in neuronal cells in vitro. The mechanism of hypoxic EpoR regulation remains to be established, since EpoR has not been identified as HIF target gene so far.

### **3.4 Epo signaling**

424 Advances in the Preclinical Study of Ischemic Stroke

enzymatic hydroxylation of specific amino acids on the α subunit by a group of oxygenases (FIGURE 2). Under normoxic conditions, a specific prolyl hydroxylation within the oxygendependent degradation domain of HIF-α takes place. This prolyl hydroxylation allows binding of the von Hippel-Lindau protein (pVHL), leading to ubiquitylation and proteasomal degradation of the HIF-α subunit (Ivan et al, 2001; Jaakkola et al, 2001). The enzymes responsible for this hydroxylation are termed prolyl hydroxylase domain enzymes (PHD1-3) (Bruick and McKnight, 2001; Epstein et al, 2001) and are widely expressed. Furthermore, in the presence of oxygen, another hydroxylation reaction takes place on an asparaginyl group in the COOH-terminal transactivation domain of HIF-α, blocking its binding to the transcriptional coactivators (Lando et al, 2002a). This process is governed by a specific asparaginyl hydroxylase termed factor-inhibiting HIF (FIH) (Hewitson et al, 2002; Lando et al, 2002b ). So, under normoxia, FIH and PHD(s) are active, leading to transcriptional inactivation and degradation of HIF-α, whereas under hypoxic conditions both enzymes are inactive. HIF is then stabilized and able to induce the expression of target

NORMOXIA HYPOXIA

**α**

**α**

TCA

**Transcriptional activation** (TCA: transcriptional coactivator)

**β**

TCA

**α**

**β**

**α**

**α**

*Proteosomal degradation*

Fig. 2. Under normoxic conditions, specific prolyl hydroxylation within the oxygendependent degradation domain of HIF- α takes place. By contrast, under hypoxic

conditions, FIH and PHD(s) are both inactive, and HIF is stabilized and able to induce the

OH OH pVHL

OH

O2

O2

O2

genes, including Epo.

O2

**α**

OH OH

O2

**α**

PHD FIH

O2

expression of target genes including Epo.

O2

### Epo promotes cell survival through inhibiting apoptosis (FIGURE 3).

In erythroid cells, after binding of Epo to its receptor (EpoR), Janus tyrosine kinase 2 (JAK2) is phosphorylated and thus activated. This leads to engaging secondary signaling molecules such as signal transducer and activator of transcription 5 (STAT5), followed by the activation of Ras mitogen-activated protein kinase (MAPK), ERK-1/-2, and PI3K/Akt (22). Moreover, Epo induced the upregulation of the anti-apoptotic protein BCL-XL (Kilic et al, 2005). The functional significance of these signaling molecules in erythropoiesis is not absolutely clear though. For instance, whereas in one study STAT5 knockout adult mice were largely unaffected in their erythroid lineage (Teglund et al, 1998), in another study STAT5 knockout embryos suffered from severe anemia, showed a reduced number of erythroid progenitors cells, and had higher numbers of apoptotic cells (Socolovsky et al, 1999). Most of these pathways seem also to be functional in the brain (Brines and Cerami, 2005; Kilic et al, 2005). In vitro, inhibition of MAPK and PI3K blocked Epo-mediated protection of rat hippocampal neurons against hypoxia (Siren et al, 2001b). Moreover, using ERK-1/-2 and Akt inhibitors, Kilic et al. showed that activation of these proteins is essential for Epo-mediated neuroprotection in an animal model of focal cerebral ischemia. The role of STAT5 in Epo-induced neuroprotection is, however, controversial. STAT5 phosphorylation has been shown to occur in hippocampal CA1 neurons after transient global cerebral ischemia in rats (Zhang et al, 2007). Therefore, the authors concluded that STAT5 plays a role in Epo-mediated neuroprotection. However, in a very recent study, in an in vitro model of glutamate toxicity using hippocampal neuronal culture from STAT5 knockout mouse

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 427

number of neuronal progenitor cells (Yu et al, 2002). A comprehensive description of the role of the Epo/EpoR system in development is found elsewhere (Arcasoy, 2008; Dame et al,

For a long time, Epo has been used clinically in patients suffering from anemia due to endstage renal failure. In addition to the correction of anemia, these patients showed improved cognitive abilities (Siren and Ehrenreich, 2001). Initially, since it was believed that systemic Epo cannot pass through the blood-brain-barrier due to its large size (Recny et al, 1987) and brain-derived Epo production and expression of EpoR in the CNS were not yet discovered, the positive effect on cognition was attributed to the improved oxygen-carrying capacity of the blood after Epo-induced erythropoiesis. However, since later studies have shown that both Epo and its receptor are expressed in different regions of the brain by different cell types (Genc et al, 2004; Marti, 2004), the hypothesis was established that locally produced as well as exogenously added Epo could directly influence cognitive function. Interestingly, the expression level of Epo and EpoR is especially high in regions of the brain known to be particularly sensitive to acute hypoxia (Lipton, 1999), the hippocampus and the telencephalon (Digicaylioglu et al, 1995), suggesting that Epo might act as a protective agent against hypoxia. Indeed, infusion of soluble EpoR (capable of binding with endogenous EPO, thus sequestering it) into the brain of gerbils, which were subjected to a mild form of ischemia that normally does not cause neuronal damage, resulted in neuronal death in the hippocampus, clearly showing that endogenous Epo has a neuroprotective effect (Sakanaka

Besides neuroprotection under hypoxic conditions, Epo also has a neurotrophic function in normoxic neurons. This was first demonstrated by Konishi and co-workers showing that Epo augments the activity of choline acetyltransferase in primary cultured mouse septal neurons (Konishi et al, 1993). Epo promoted the regeneration of septal cholinergic neurons in adult rats that had undergone fimbria-fornix transections. In addition and similar to its anti-apoptotic role in erythropoiesis, Epo promoted the survival and differentiation of dopaminergic precursor neurons in vitro (Studer et al, 2000). Moreover, hypoxia-induced Epo production appeared to directly act on neuronal stem cells in the forebrain, showing that Epo plays a direct role in neurogenesis after hypoxia (Shingo et al, 2001). In addition, Epo also acts indirectly by inducing brain-derived neurotrophic factor (BDNF) expression (Wang et al, 2004), which in turn augmented the effect of Epo on neurogenesis. These data show that Epo is not only involved in neuroprotection, but also in neuronal survival,

Besides its direct effects on neurons, Epo-induced neuroprotection may be attributed to an improvement in brain perfusion by promoting new vessel growth. Anagnostou et al demonstrated mitogenic and chemotactic effects of Epo on human umbilical vein and bovine adrenal capillary endothelial cells (Anagnostou et al, 1990). Moreover, Epo stimulated vessel outgrowth of rat aortic rings (Carlini et al, 1995), suggesting that Epo has angiogenic effects. This was further supported by the observation that Epo injection into the

2001).

et al, 1998).

**3.5.2 Neurotrophic function and neurogenesis** 

differentiation, and neurogenesis.

**3.5.3 Angiogenesis and vascular permeability** 

**3.5.1 Neuroprotection** 

fetuses, STAT5 was not required for Epo-mediated neuroprotection (Byts et al, 2008). However, STAT5 was indispensable for the neurotrophic function of Epo. A unique pathway for the brain seems to be that activation of EpoR induces nuclear factor- κB (NF-κB) translocation into the nucleus and that this effect is important for Epo-mediated neuroprotection (Digicaylioglu and Lipton, 2001). Interestingly, Epo-induced NF-κB translocation was observed only in neuronal cells and not in astrocytes. Thus it appears likely that NF-κB, in the nucleus, induces the expression of neuroprotective and antiapoptotic proteins. However, some differences exist between the signaling cascade activated by Epo in the CNS and in erythroid cells. For instance, in one study, BCL-XL has been found to be important in Epo-mediated protection of erythroid but not neuronal cells (Rischer et al, 2002). Additionally, Epo has been found to activate phospholipase C-gamma (PLCγ) (Marreo et al, 1998) and thus can directly influence neuronal activity (Koshimura et al, 1999) and neurotransmitter release (Kawakami et al, 2000) by modulating intracellular calcium concentrations in neurons.

Fig. 3. Epo Signaling

### **3.5 Epo function in the CNS**

For almost a century, Epo was thought to be involved in the process of erythropoiesis only. Through its antiapoptotic action, it enables committed erythroid progenitor cells to survive and mature (Jelkmann, 1992). However, during the last decade, it became evident that Epo is implicated in other processes such as neuroprotection, neurogenesis, and angiogenesis, and plays an important role as neurotrophic as well as immunomodulatory factor. The important role of Epo in the CNS is also evident from studies with EpoR knockout mice. As a result of EpoR deficiency, these mice show massive apoptosis and a reduction in the number of neuronal progenitor cells (Yu et al, 2002). A comprehensive description of the role of the Epo/EpoR system in development is found elsewhere (Arcasoy, 2008; Dame et al, 2001).

### **3.5.1 Neuroprotection**

426 Advances in the Preclinical Study of Ischemic Stroke

fetuses, STAT5 was not required for Epo-mediated neuroprotection (Byts et al, 2008). However, STAT5 was indispensable for the neurotrophic function of Epo. A unique pathway for the brain seems to be that activation of EpoR induces nuclear factor- κB (NF-κB) translocation into the nucleus and that this effect is important for Epo-mediated neuroprotection (Digicaylioglu and Lipton, 2001). Interestingly, Epo-induced NF-κB translocation was observed only in neuronal cells and not in astrocytes. Thus it appears likely that NF-κB, in the nucleus, induces the expression of neuroprotective and antiapoptotic proteins. However, some differences exist between the signaling cascade activated by Epo in the CNS and in erythroid cells. For instance, in one study, BCL-XL has been found to be important in Epo-mediated protection of erythroid but not neuronal cells (Rischer et al, 2002). Additionally, Epo has been found to activate phospholipase C-gamma (PLCγ) (Marreo et al, 1998) and thus can directly influence neuronal activity (Koshimura et al, 1999) and neurotransmitter release (Kawakami et al, 2000) by modulating intracellular calcium

For almost a century, Epo was thought to be involved in the process of erythropoiesis only. Through its antiapoptotic action, it enables committed erythroid progenitor cells to survive and mature (Jelkmann, 1992). However, during the last decade, it became evident that Epo is implicated in other processes such as neuroprotection, neurogenesis, and angiogenesis, and plays an important role as neurotrophic as well as immunomodulatory factor. The important role of Epo in the CNS is also evident from studies with EpoR knockout mice. As a result of EpoR deficiency, these mice show massive apoptosis and a reduction in the

concentrations in neurons.

Fig. 3. Epo Signaling

**3.5 Epo function in the CNS** 

For a long time, Epo has been used clinically in patients suffering from anemia due to endstage renal failure. In addition to the correction of anemia, these patients showed improved cognitive abilities (Siren and Ehrenreich, 2001). Initially, since it was believed that systemic Epo cannot pass through the blood-brain-barrier due to its large size (Recny et al, 1987) and brain-derived Epo production and expression of EpoR in the CNS were not yet discovered, the positive effect on cognition was attributed to the improved oxygen-carrying capacity of the blood after Epo-induced erythropoiesis. However, since later studies have shown that both Epo and its receptor are expressed in different regions of the brain by different cell types (Genc et al, 2004; Marti, 2004), the hypothesis was established that locally produced as well as exogenously added Epo could directly influence cognitive function. Interestingly, the expression level of Epo and EpoR is especially high in regions of the brain known to be particularly sensitive to acute hypoxia (Lipton, 1999), the hippocampus and the telencephalon (Digicaylioglu et al, 1995), suggesting that Epo might act as a protective agent against hypoxia. Indeed, infusion of soluble EpoR (capable of binding with endogenous EPO, thus sequestering it) into the brain of gerbils, which were subjected to a mild form of ischemia that normally does not cause neuronal damage, resulted in neuronal death in the hippocampus, clearly showing that endogenous Epo has a neuroprotective effect (Sakanaka et al, 1998).

### **3.5.2 Neurotrophic function and neurogenesis**

Besides neuroprotection under hypoxic conditions, Epo also has a neurotrophic function in normoxic neurons. This was first demonstrated by Konishi and co-workers showing that Epo augments the activity of choline acetyltransferase in primary cultured mouse septal neurons (Konishi et al, 1993). Epo promoted the regeneration of septal cholinergic neurons in adult rats that had undergone fimbria-fornix transections. In addition and similar to its anti-apoptotic role in erythropoiesis, Epo promoted the survival and differentiation of dopaminergic precursor neurons in vitro (Studer et al, 2000). Moreover, hypoxia-induced Epo production appeared to directly act on neuronal stem cells in the forebrain, showing that Epo plays a direct role in neurogenesis after hypoxia (Shingo et al, 2001). In addition, Epo also acts indirectly by inducing brain-derived neurotrophic factor (BDNF) expression (Wang et al, 2004), which in turn augmented the effect of Epo on neurogenesis. These data show that Epo is not only involved in neuroprotection, but also in neuronal survival, differentiation, and neurogenesis.

### **3.5.3 Angiogenesis and vascular permeability**

Besides its direct effects on neurons, Epo-induced neuroprotection may be attributed to an improvement in brain perfusion by promoting new vessel growth. Anagnostou et al demonstrated mitogenic and chemotactic effects of Epo on human umbilical vein and bovine adrenal capillary endothelial cells (Anagnostou et al, 1990). Moreover, Epo stimulated vessel outgrowth of rat aortic rings (Carlini et al, 1995), suggesting that Epo has angiogenic effects. This was further supported by the observation that Epo injection into the

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 429

mediated by the extracellular pathways (Banks et al, 2004). However, variations in physiological serum Epo level may not result in significant changes of Epo levels within the brain, since no correlation between serum and liquor Epo concentrations was found when the BBB is intact (Marti et al, 1997). In summary, one can conclude that endogenously produced Epo (by kidney or liver) has only a marginal influence on brain Epo availability, whereas high dosages of therapeutically administered r-hu Epo can penetrate even the intact BBB (Marti et al, 1997). Accordingly, many studies are currently ongoing to test the

The first hint came from the observation that the expression of Epo and its receptor in the brain is upregulated upon cerebral ischemia (Bernaudin et al, 1999; Siren et al, 2001). Several in vivo experiments confirmed this hypothesis. Intracerebroventricular injection of Epo 24 h before permanent occlusion of the MCA in mice reduced infarct volume significantly. Similarly, infusion of Epo in the lateral ventricles of gerbils in a global ischemia model rescued hippocampal CA1 neurons and increased the number of synapses in the same region (Sakanaka et al, 1998). Moreover, in another experimental rodent model of cerebral ischemia where the MCA is transiently occluded, systemic administration of Epo also reduced the infarct size (Brines et al, 2000). Significantly, this protective effect of Epo was retained even when Epo was applied 6 h after the onset of the cerebral ischemia. In addition, brain-specific overexpression of Epo reduced infarct size in mice subjected to transient cerebral ischemia (Kilic et al, 2005). Other studies, where the functional outcome of Epo treatment was investigated, have shown that Epo not only reduces infarct volume but also improves the learning ability in gerbils and reduces the navigation disability in rats (Sadamoto et al, 1998; Sakanaka et al, 1998). Epo has also been shown to be protective in models of hemorrhagic stroke where the interruption of the cerebral blood flow is due to subarachnoid or cerebral hemorrhage (Alafaci et al, 2000; Grasso et al, 2002). The abovementioned studies prompted the initiation of clinical trials in stroke patients. The safety and proof-of-concept phases of the Göttingen-Epo-Stroke Study have shown Epo to be safe and to improve the patient functional outcome after stroke (Ehrenreich et al, 2002). Although good evidence for direct neuroprotection exists, the observed brain-protective effect of Epo could also be attributed to its effect on astrocytes. Astrocytes protect neurons from oxidative stress by neutralizing reactive oxygen species (Dringen and Hirrlinger, 2003). It has been reported that activated astrocytes in ischemic human brain express increased levels of EpoR (Siren et al, 2001). Since Epo enhances brain glutathione peroxidase activity (Kumral et al, 2005), Epo, by binding to EpoR on the surface of activated astrocytes, might contribute to the astrocyte-mediated neuroprotective effect against ischemia-induced free-radical formation.

Clinical studies are ongoing to test the safety and efficacy of EPO for the treatment of different neurological diseases. In the recent multicenter Epo stroke trial (Ehreich et al, 2009), adult stroke patients receiving Epo after tissue-plasminogen activator (t-PA)-induced thrombolysis reported increased mortality, intra-cerebral hemorrhage, brain edema, and thromboembolic events. The increased death rate in the rtPA population is still unexplained and may result from a combinant of factors and/or potential rtPA-EPO interactions. In contrast, in non rtPA population, the tendency toward a higher death rate in the EPO group

therapeutic potential of Epo in many CNS diseases.

**3.7 Safety concerns with the clinical use of EPO** 

**3.6 Epo in stroke** 

mouse uterine cavity stimulated neovascularization in the endometrium (Yasuda et al, 1998). Similarly, neovascularization was stimulated in the chick embryo chorioallantonic membrane upon Epo administration (Ribatti et al, 1999a). The angiogenic effect of Epo was also found in the brain, since capillary endothelial cells express two forms of EpoR mRNA and Epo showed a dose-dependent mitogenic activity on brain capillary endothelial cells (Yamaji et al, 1996). This angiogenic effect was finally confirmed in mice genetically engineered to lack either Epo or its receptor (EpoR) where mutant embryos suffer from severe defects in angiogenesis (Kertesz et al, 2004). In addition to its angiogenic effect, Epo is involved in the regulation of vascular permeability. In an in vitro model of the blood-brain barrier (BBB), Epo treatment protected bovine brain endothelial cells against VEGF-induced increase in vascular permeability (Martinez-Estrada et al, 2003). This suggests that the protective effect of Epo on the brain could be mediated by stimulating angiogenesis as well as by protecting the BBB.

### **3.5.4 Anti-inflammation**

Inflammatory processes play a major role in the pathogenesis of cerebral ischemia, where Epo is protective. Inflammation results in influx of leukocytes from the blood into the brain and in activation of resident microglial cells (Dirnagl et al, 1999). These cells produce inflammatory mediators and cytokines leading to barrier damage, microvascular occlusion, and thus the aggravation of the injury (Witko-Sarsat et al, 2000). In an animal model of cerebral ischemia, administration of Epo resulted in the reduction of the local production of TNF, IL-6, and the chemokine MCP-1, all markers of inflammation, subsequently leading to a marked reduction of infarct size. These results indicate that Epo has an anti-inflammatory effect that contributes to its direct neuroprotective effect during cerebral ischemia (Villa et al, 2003). Since Epo did not reduce cytokine production in response to LPS applied directly in vivo and in vitro, the authors concluded that the observed antiinflammatory effect is due to inhibiting neuronal apoptosis and not to a direct effect on inflammatory cells. Epo might reduce leukocyte transmigration through endothelial cells, since Epo enhances the resistance of endothelial cells toward ischemia (Chong et al, 2002). The protective effect of Epo on oligodendrocytes against cytotoxicity induced by inflammatory stimuli (Genc et al, 2006) could explain the beneficial effect of Epo in case of MS where oligodendrocytes play a crucial role in the pathogenesis of the disease.

### **3.5.5 Transport through BBB**

An important prerequisite for considering Epo as a therapeutic agent in CNS diseases is to answer the question as of whether Epo, administered systemically, is able to cross the BBB. Brines et al. (Brines et al, 2000) injected mice with biotinylated Epo and subsequently visualized brain section with peroxidase-labeled streptavidin. Indeed, a signal for biotin was detected in a region surrounding the capillaries extending into brain parenchyma. The authors concluded that Epo crosses the BBB. However, biotin might not be an ideal tool to study BBB permeability since it is rapidly transported across the BBB (Shi et al, 1993; Spector and Mock, 1987), and, therefore, even a small amount of free biotin in the blood will cross the BBB leading to false results. Since the authors detected EpoR in the brain capillaries, they attributed Epo transport through BBB to transcytosis. This hypothesis was later challenged by the observation that radiolabeled Epo and albumin crossed the BBB and entered the brain parenchyma in similar kinetics, showing that the transport of Epo across BBB is rather mediated by the extracellular pathways (Banks et al, 2004). However, variations in physiological serum Epo level may not result in significant changes of Epo levels within the brain, since no correlation between serum and liquor Epo concentrations was found when the BBB is intact (Marti et al, 1997). In summary, one can conclude that endogenously produced Epo (by kidney or liver) has only a marginal influence on brain Epo availability, whereas high dosages of therapeutically administered r-hu Epo can penetrate even the intact BBB (Marti et al, 1997). Accordingly, many studies are currently ongoing to test the therapeutic potential of Epo in many CNS diseases.

### **3.6 Epo in stroke**

428 Advances in the Preclinical Study of Ischemic Stroke

mouse uterine cavity stimulated neovascularization in the endometrium (Yasuda et al, 1998). Similarly, neovascularization was stimulated in the chick embryo chorioallantonic membrane upon Epo administration (Ribatti et al, 1999a). The angiogenic effect of Epo was also found in the brain, since capillary endothelial cells express two forms of EpoR mRNA and Epo showed a dose-dependent mitogenic activity on brain capillary endothelial cells (Yamaji et al, 1996). This angiogenic effect was finally confirmed in mice genetically engineered to lack either Epo or its receptor (EpoR) where mutant embryos suffer from severe defects in angiogenesis (Kertesz et al, 2004). In addition to its angiogenic effect, Epo is involved in the regulation of vascular permeability. In an in vitro model of the blood-brain barrier (BBB), Epo treatment protected bovine brain endothelial cells against VEGF-induced increase in vascular permeability (Martinez-Estrada et al, 2003). This suggests that the protective effect of Epo on the brain could be mediated by stimulating angiogenesis as well

Inflammatory processes play a major role in the pathogenesis of cerebral ischemia, where Epo is protective. Inflammation results in influx of leukocytes from the blood into the brain and in activation of resident microglial cells (Dirnagl et al, 1999). These cells produce inflammatory mediators and cytokines leading to barrier damage, microvascular occlusion, and thus the aggravation of the injury (Witko-Sarsat et al, 2000). In an animal model of cerebral ischemia, administration of Epo resulted in the reduction of the local production of TNF, IL-6, and the chemokine MCP-1, all markers of inflammation, subsequently leading to a marked reduction of infarct size. These results indicate that Epo has an anti-inflammatory effect that contributes to its direct neuroprotective effect during cerebral ischemia (Villa et al, 2003). Since Epo did not reduce cytokine production in response to LPS applied directly in vivo and in vitro, the authors concluded that the observed antiinflammatory effect is due to inhibiting neuronal apoptosis and not to a direct effect on inflammatory cells. Epo might reduce leukocyte transmigration through endothelial cells, since Epo enhances the resistance of endothelial cells toward ischemia (Chong et al, 2002). The protective effect of Epo on oligodendrocytes against cytotoxicity induced by inflammatory stimuli (Genc et al, 2006) could explain the beneficial effect of Epo in case of MS where oligodendrocytes play a

An important prerequisite for considering Epo as a therapeutic agent in CNS diseases is to answer the question as of whether Epo, administered systemically, is able to cross the BBB. Brines et al. (Brines et al, 2000) injected mice with biotinylated Epo and subsequently visualized brain section with peroxidase-labeled streptavidin. Indeed, a signal for biotin was detected in a region surrounding the capillaries extending into brain parenchyma. The authors concluded that Epo crosses the BBB. However, biotin might not be an ideal tool to study BBB permeability since it is rapidly transported across the BBB (Shi et al, 1993; Spector and Mock, 1987), and, therefore, even a small amount of free biotin in the blood will cross the BBB leading to false results. Since the authors detected EpoR in the brain capillaries, they attributed Epo transport through BBB to transcytosis. This hypothesis was later challenged by the observation that radiolabeled Epo and albumin crossed the BBB and entered the brain parenchyma in similar kinetics, showing that the transport of Epo across BBB is rather

as by protecting the BBB.

**3.5.4 Anti-inflammation** 

crucial role in the pathogenesis of the disease.

**3.5.5 Transport through BBB** 

The first hint came from the observation that the expression of Epo and its receptor in the brain is upregulated upon cerebral ischemia (Bernaudin et al, 1999; Siren et al, 2001). Several in vivo experiments confirmed this hypothesis. Intracerebroventricular injection of Epo 24 h before permanent occlusion of the MCA in mice reduced infarct volume significantly. Similarly, infusion of Epo in the lateral ventricles of gerbils in a global ischemia model rescued hippocampal CA1 neurons and increased the number of synapses in the same region (Sakanaka et al, 1998). Moreover, in another experimental rodent model of cerebral ischemia where the MCA is transiently occluded, systemic administration of Epo also reduced the infarct size (Brines et al, 2000). Significantly, this protective effect of Epo was retained even when Epo was applied 6 h after the onset of the cerebral ischemia. In addition, brain-specific overexpression of Epo reduced infarct size in mice subjected to transient cerebral ischemia (Kilic et al, 2005). Other studies, where the functional outcome of Epo treatment was investigated, have shown that Epo not only reduces infarct volume but also improves the learning ability in gerbils and reduces the navigation disability in rats (Sadamoto et al, 1998; Sakanaka et al, 1998). Epo has also been shown to be protective in models of hemorrhagic stroke where the interruption of the cerebral blood flow is due to subarachnoid or cerebral hemorrhage (Alafaci et al, 2000; Grasso et al, 2002). The abovementioned studies prompted the initiation of clinical trials in stroke patients. The safety and proof-of-concept phases of the Göttingen-Epo-Stroke Study have shown Epo to be safe and to improve the patient functional outcome after stroke (Ehrenreich et al, 2002). Although good evidence for direct neuroprotection exists, the observed brain-protective effect of Epo could also be attributed to its effect on astrocytes. Astrocytes protect neurons from oxidative stress by neutralizing reactive oxygen species (Dringen and Hirrlinger, 2003). It has been reported that activated astrocytes in ischemic human brain express increased levels of EpoR (Siren et al, 2001). Since Epo enhances brain glutathione peroxidase activity (Kumral et al, 2005), Epo, by binding to EpoR on the surface of activated astrocytes, might contribute to the astrocyte-mediated neuroprotective effect against ischemia-induced free-radical formation.

### **3.7 Safety concerns with the clinical use of EPO**

Clinical studies are ongoing to test the safety and efficacy of EPO for the treatment of different neurological diseases. In the recent multicenter Epo stroke trial (Ehreich et al, 2009), adult stroke patients receiving Epo after tissue-plasminogen activator (t-PA)-induced thrombolysis reported increased mortality, intra-cerebral hemorrhage, brain edema, and thromboembolic events. The increased death rate in the rtPA population is still unexplained and may result from a combinant of factors and/or potential rtPA-EPO interactions. In contrast, in non rtPA population, the tendency toward a higher death rate in the EPO group

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 431

is regulated at both the transcriptional and post-transcriptional levels and this regulation is important to meet the demands of plasma, because albumin is not stored in the liver in large amounts. The rate of albumin transcription is affected by several conditions such as trauma, sepsis, hepatic diseases, diabetes and fasting. The change in interstitial colloid oncotic pressure is thought to be the predominant factor for regulation of albumin synthesis. Albumin homeostasis is maintained by balanced catabolism occurring in all tissues but most of the albumin (40-60 %) is degraded in the muscle and skin. However, the liver (15 %), kidney (10 %) and gastro-intestinal tract (10 %) are also responsible for albumin degradation. Albumin leaks from plasma at a rate of 5 % per hour and is returned to the vascular space at an equivalent rate through the lymphatic system. Albumin is also diffused into CSF and ISF compartments of the central nervous system (CNS) from blood circulation of the brain (Nicholson et al, 2000). Blood derived albumin in CSF and interstitial fluid (ISF

is implicated in normal as well as many pathophysiological conditions of the brain.

CSF originates from choroid plexus in the ventricles. CSF flows through cisternae and subarachnoid space and finally drains through the arachnoid villi into venous blood. CSF has several important functions; it mainly helps to provide mechanical support for the brain. CSF also acts as a drainage pathway for the brain, by providing a ' sink ' into which products of metabolism or synaptic activity are diluted and subsequently removed. Also, it acts as a route of communication within the CNS, i.e., it carries hormones, nutrients and transmitters between different areas of the brain. CSF albumin is predominantly a blood derived protein and it is mainly entered from the leptomeningeal blood CSF barrier (BCSFB) or from choroid plexus BCSFB (Johanson et al, 2008). The albumin quotient (Q alb ) in the CSF is approximately 30-80 % of the total protein. The altered Albumin CSF/serum ratio (QAlb) is the indicator of the dysfunction of the BCSFB. CSF serum Q alb, along with other blood derived proteins in CSF, is widely used in the diagnosis of neurological diseases (Reiber, 1998, 2003; Reiber and Peter, 2001). The exact role of albumin in CSF is not fully known, but it is proposed that albumin could be involved in the maintenance of CSF oncotic pressure, delivery of a wide range of molecules that are important for normal brain function and in the removal of some of the harmful molecules from the brain. The exact roles of albumin in CSF and in the brain function are not fully understood. However, many recent studies indicate that albumin might have a neuroprotective role via multiple mechanisms in

Ischemic stroke is an acute cerebrovascular disease resulting from a transient or permanent reduction in the cerebral blood flow (CBF). It mainly occurs due to blockade of the major cerebral blood vessels by a local thrombus or an embolus. Ischemia causes reduction in the oxygen and nutrient supply to the brain areas which leads to neuronal cell damage or cell death. It can cause long-term disabilities such as muscle paralysis, cognitive deficits, language deficits, emotional deficits and even coma or death. Stroke is the third leading cause of death worldwide after coronary heart disease and cancer (Lloyd-Jones et al, 2009) and ischemic stroke comprises approximately 87 % of all types of brain strokes. The only approved treatment with intravenous fibrinolytic such as tissue plasminogen activator (tPA) within 3 h of stroke onset yields reperfusion and clinical benefits (rt -PA Stroke Study

**4.2 Albumin in the CSF** 

different pathophysiological conditions.

**4.3 Albumin induced neuroprotection in experimental stroke** 

might be explained by higher stroke severity of dead patients on inclusion (before any study medication was applied). Moreover, the mechanism of action of EPO is different from the clot-dissolving strategy pursued by thrombolysis. It would, therefore, have been most attractive to see that the neuroprotective approach using EPO, aimed at salvaging potentially viable brain tissue from spreading of death signals, and thrombolysis, targeting reopening of the feeding artery, had provided additive beneficial outcome. However, the unexpected observation that a combination of EPO and rtPA is not advantageous, and can even be detrimental, poses at present a contraindication for acute EPO treatment in patients receiving rtPA.

### **4. Albumin**

Albumin is the most abundant plasma protein synthesized mainly in the liver. Albumin is also a major component of most extracellular fluids including cerebrospinal fluid (CSF), interstitial fluid (ISF) and lymph. It is a non-glycosylated and negatively charged protein with high ligand binding and transport capacity. It has multifunctional properties which include the maintenance of colloid osmotic pressure of plasma, transportation of hormones, fatty acids, drugs and metabolites, regulation of microvascular permeability, antioxidant activity, anti-thrombotic activity and anti-inflammatory activity (Evans, 2002; Garcovich et al, 2009). Owing to its multifunctional properties it has been widely used in therapeutics related to hepatology. The volumeexpanding property of albumin, in combination with other therapeutic approaches, has been used for the clinical benefit of patients with liver cirrhosis. Also, human serum albumin (HSA) as an iso-oncotic (4-5%) solution has been used to combat blood volume deficits and as a hyperoncotic (20-25%) solution has been used for restoration of oncotic deficits (Arroyo, 2002; Garcovich et al, 2009). Albumin has been shown to play a crucial role in the microcirculation of many organs including brain. Owing to its strong hemodynamic and binding capacity, it has been implicated in physiological and many disease conditions of the brain. Albumin has been implicated in neurological diseases such as ischemic stroke, Alzheimer's disease and epilepsy. High-dose human albumin is robustly neuroprotective in preclinical ischemia models and it is currently in Phase III clinical trials for acute ischemic stroke (Ginsberg, 2008). Albumin also has the potential to produce direct neuroprotective action on neuronal and glial cells.

### **4.1 Albumin synthesis and distribution**

Albumin protein contains a single polypeptide chain of 585 amino acids with a molecular mass of approximately 67 kDa.

In the mouse liver, the albumin gene becomes active during early foetal stages and the transcript levels gradually increase after birth until high levels are reached in the adult animal (Tilghman and Belayew, 1982). Albumin is synthesized as preproalbumin in the liver, which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce albumin. Albumin synthesis predominantly occurs in the liver at the rate of 10-15 g/day. In healthy human adults, total albumin content is approximately 250-300 g/70 kg of the body weight and the majority of synthesized albumin (40-45 %) is maintained in the plasma. A very small amount of albumin is stored in liver ( < 2 g) and the remaining amount is located in the muscle and skin (Quinlan et al, 2005). Albumin synthesis is regulated at both the transcriptional and post-transcriptional levels and this regulation is important to meet the demands of plasma, because albumin is not stored in the liver in large amounts. The rate of albumin transcription is affected by several conditions such as trauma, sepsis, hepatic diseases, diabetes and fasting. The change in interstitial colloid oncotic pressure is thought to be the predominant factor for regulation of albumin synthesis. Albumin homeostasis is maintained by balanced catabolism occurring in all tissues but most of the albumin (40-60 %) is degraded in the muscle and skin. However, the liver (15 %), kidney (10 %) and gastro-intestinal tract (10 %) are also responsible for albumin degradation. Albumin leaks from plasma at a rate of 5 % per hour and is returned to the vascular space at an equivalent rate through the lymphatic system. Albumin is also diffused into CSF and ISF compartments of the central nervous system (CNS) from blood circulation of the brain (Nicholson et al, 2000). Blood derived albumin in CSF and interstitial fluid (ISF is implicated in normal as well as many pathophysiological conditions of the brain.

### **4.2 Albumin in the CSF**

430 Advances in the Preclinical Study of Ischemic Stroke

might be explained by higher stroke severity of dead patients on inclusion (before any study medication was applied). Moreover, the mechanism of action of EPO is different from the clot-dissolving strategy pursued by thrombolysis. It would, therefore, have been most attractive to see that the neuroprotective approach using EPO, aimed at salvaging potentially viable brain tissue from spreading of death signals, and thrombolysis, targeting reopening of the feeding artery, had provided additive beneficial outcome. However, the unexpected observation that a combination of EPO and rtPA is not advantageous, and can even be detrimental, poses at present a contraindication for acute EPO treatment in patients

Albumin is the most abundant plasma protein synthesized mainly in the liver. Albumin is also a major component of most extracellular fluids including cerebrospinal fluid (CSF), interstitial fluid (ISF) and lymph. It is a non-glycosylated and negatively charged protein with high ligand binding and transport capacity. It has multifunctional properties which include the maintenance of colloid osmotic pressure of plasma, transportation of hormones, fatty acids, drugs and metabolites, regulation of microvascular permeability, antioxidant activity, anti-thrombotic activity and anti-inflammatory activity (Evans, 2002; Garcovich et al, 2009). Owing to its multifunctional properties it has been widely used in therapeutics related to hepatology. The volumeexpanding property of albumin, in combination with other therapeutic approaches, has been used for the clinical benefit of patients with liver cirrhosis. Also, human serum albumin (HSA) as an iso-oncotic (4-5%) solution has been used to combat blood volume deficits and as a hyperoncotic (20-25%) solution has been used for restoration of oncotic deficits (Arroyo, 2002; Garcovich et al, 2009). Albumin has been shown to play a crucial role in the microcirculation of many organs including brain. Owing to its strong hemodynamic and binding capacity, it has been implicated in physiological and many disease conditions of the brain. Albumin has been implicated in neurological diseases such as ischemic stroke, Alzheimer's disease and epilepsy. High-dose human albumin is robustly neuroprotective in preclinical ischemia models and it is currently in Phase III clinical trials for acute ischemic stroke (Ginsberg, 2008). Albumin also has the potential to

Albumin protein contains a single polypeptide chain of 585 amino acids with a molecular

In the mouse liver, the albumin gene becomes active during early foetal stages and the transcript levels gradually increase after birth until high levels are reached in the adult animal (Tilghman and Belayew, 1982). Albumin is synthesized as preproalbumin in the liver, which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce albumin. Albumin synthesis predominantly occurs in the liver at the rate of 10-15 g/day. In healthy human adults, total albumin content is approximately 250-300 g/70 kg of the body weight and the majority of synthesized albumin (40-45 %) is maintained in the plasma. A very small amount of albumin is stored in liver ( < 2 g) and the remaining amount is located in the muscle and skin (Quinlan et al, 2005). Albumin synthesis

produce direct neuroprotective action on neuronal and glial cells.

**4.1 Albumin synthesis and distribution** 

mass of approximately 67 kDa.

receiving rtPA.

**4. Albumin** 

CSF originates from choroid plexus in the ventricles. CSF flows through cisternae and subarachnoid space and finally drains through the arachnoid villi into venous blood. CSF has several important functions; it mainly helps to provide mechanical support for the brain. CSF also acts as a drainage pathway for the brain, by providing a ' sink ' into which products of metabolism or synaptic activity are diluted and subsequently removed. Also, it acts as a route of communication within the CNS, i.e., it carries hormones, nutrients and transmitters between different areas of the brain. CSF albumin is predominantly a blood derived protein and it is mainly entered from the leptomeningeal blood CSF barrier (BCSFB) or from choroid plexus BCSFB (Johanson et al, 2008). The albumin quotient (Q alb ) in the CSF is approximately 30-80 % of the total protein. The altered Albumin CSF/serum ratio (QAlb) is the indicator of the dysfunction of the BCSFB. CSF serum Q alb, along with other blood derived proteins in CSF, is widely used in the diagnosis of neurological diseases (Reiber, 1998, 2003; Reiber and Peter, 2001). The exact role of albumin in CSF is not fully known, but it is proposed that albumin could be involved in the maintenance of CSF oncotic pressure, delivery of a wide range of molecules that are important for normal brain function and in the removal of some of the harmful molecules from the brain. The exact roles of albumin in CSF and in the brain function are not fully understood. However, many recent studies indicate that albumin might have a neuroprotective role via multiple mechanisms in different pathophysiological conditions.

### **4.3 Albumin induced neuroprotection in experimental stroke**

Ischemic stroke is an acute cerebrovascular disease resulting from a transient or permanent reduction in the cerebral blood flow (CBF). It mainly occurs due to blockade of the major cerebral blood vessels by a local thrombus or an embolus. Ischemia causes reduction in the oxygen and nutrient supply to the brain areas which leads to neuronal cell damage or cell death. It can cause long-term disabilities such as muscle paralysis, cognitive deficits, language deficits, emotional deficits and even coma or death. Stroke is the third leading cause of death worldwide after coronary heart disease and cancer (Lloyd-Jones et al, 2009) and ischemic stroke comprises approximately 87 % of all types of brain strokes. The only approved treatment with intravenous fibrinolytic such as tissue plasminogen activator (tPA) within 3 h of stroke onset yields reperfusion and clinical benefits (rt -PA Stroke Study

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 433

and subsequently cellular uptake. It has been observed that cortical neurons with preserved structural features had taken up human albumin. Thus, it is reasonable to speculate that treatment with human albumin could also provide direct neuronal protection (Remmers et al, 1999). For the effective treatment of ischemic stroke, treatment should be started within a narrow therapeutic window of 3 h. Moderate-dose albumin therapy (1.25 g/kg intravenously) markedly provides neuroprotection even when treatment is delayed up to 4 h after onset of ischemia (Belayev et al, 2001). Albumin treatment has also been found to be neuroprotective in other models of focal ischemia. Prompt albumin therapy improved neurological function and blood-brain barrier integrity after acute intracortical hematoma (ICH) (Belayev et al, 2005). In a model of laser-induced cortical arteriolar thrombosis, highdose albumin therapy induced a prompt, sustained improvement in microvascular hemodynamics distal to a cortical arteriolar thrombosis (Nimmagadda et al, 2008). In acute ischemic stroke, albumin combination therapy can attenuate the deleterious effects of tPA (Tang et al, 2009). Furthermore, albumin (1.25 g/kg) treatment maintains serum albumin at a higher level and attenuates cortex and hippocampus vascular endothelial growth factor (VEGF) expression at 6 h and 1 day after MCAO. This could partially contribute to the protective effects of albumin on reduction of brain edema and infarct size in the early stage of ischemia (Yao et al, 2010). The above mentioned studies prove that in experimental transient ischemia albumin provides neuroprotection via different indirect and direct mechanisms. Albumin has been found to be effective in other models of stroke such as permanent MCAO, global ischemia induced by bilateral common carotid occlusion (BCCO) and traumatic brain injury (TBI). In permanent MCAO, rats treated with 2 g/kg/day concentrated (25%) albumin begun after 30 min of ischemia showed diminished brain edema and infarct volume up to 6 days (Matsui et al, 1993). Furthermore, albumin (1.25 and 2.5 g/kg) significantly reduced cortical and striatal infarct areas and increased cortical perfusion in the permanent ischemia model (Liu et al, 2001). In transient global ischemia, HSA-treated rats showed signifi cantly improved neurological deficits throughout a 7-day survival period along with increases in numbers of surviving CA1 hippocampal pyramidal neurons compared to saline-treated animals (Belayev et al, 1999b). In TBI, 15 min after trauma, HSA administration signifi cantly improved neurological defi cits and also significantly reduced total contusion area (Belayev et al, 1999a). These experimental trials altogether indicated significant neuroprotective roles of albumin in different models of ischemic stroke and encouraged the further development of this important molecule for

possible treatment of ischemic stroke in humans.

**4.4 Albumin in clinical trials for ischemic stroke** 

The Albumin In Acute Stroke (ALIAS) Pilot Clinical Trial was conducted during 2001 – 2005 at two clinical sites (Universities of Calgary and Miami). This study was designed to investigate the safety and tolerability of albumin therapy in acute ischemic stroke. The ALIAS Pilot Clinical Trial used a multiple-tier, open-label, dose-escalation design. Human albumin (25%) in doses ranging up to 2.05 g/kg was well tolerated by patients with acute ischemic stroke without major dose-limiting complications. Concurrent tPA therapy did not affect the safety profile of albumin (Ginsberg et al, 2006a). Also, in this pilot trial the neuroprotective efficacy of albumin was evaluated and was found to be neuroprotective after ischemic stroke (Palesch et al, 2006). Based on the encouraging results of pilot trials of

Group, 1995; Hacke et al, 2004; Juttler et al, 2006). However, the goal is to discover a neuroprotective drug which can inhibit reperfusion injury and provide neuroprotection within a wide therapeutic window. Hemodilution is an old approach which has been investigated for many decades as a potential therapy for ischemic stroke. Infusion of dextran has been shown to increase CBF of both the normal and ischemic brain, either by decreasing blood viscosity or by vasodilation in response to diminished oxygen delivery (Wood and Kee, 1985; Korosue and Heros, 1992). Despite neuroprotective benefi ts in experimental setups, several clinical trials of hemodilution in ischemic stroke have nonetheless proven negative or inconclusive (Scandinavian Stroke Study Group, 1987; Italian Acute Stroke Study Group, 1988; The Hemodilution in Stroke Study Group,1989). Subsequently, albumin has emerged as an alternative hemodiluting agent to dextran owing to its volume expanding properties (Sundt et al, 1967; Little et al, 1981; Emerson, 1989). However, only recently it has been rigorously evaluated for its anti-ischemic neuroprotective efficacy. Transient focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) is the most widely used model to study molecular mechanisms of cerebral ischemiareperfusion injury and to screen neuroprotective drugs. It is less invasive with a low rate of mortality and a low coefficient of variation in lesion size (Longa et al, 1989; Belayev et al, 1997b). In the rat MCAO model, Cole et al. reported that 5 % albumin administration at the onset of ischemia reduced ischemic brain injury as evidenced by reduced hematocrit, infarct volume and cerebral edema (Cole et al, 1990). In another study, administration of concentrated (20%) HSA (1% body weight, intravenously) to rats at the onset of recirculation induced substantial diminution of infarct volume together with a marked reduction of brain edema. Thus, it is proposed that albumin might modify water homeostasis and ultimately reduce edema of the ischemic brain (Belayev et al, 1997a). These two initial studies suggested that albumin therapy at the onset of ischemia or reperfusion induces neuroprotection.In a detailed study using magnetic resonance imaging, by means of diffusion-weighted magnetic resonance imaging (DWI), 25% Human Serum Albumin (HSA) solution (1% by body weight) administered immediately after reperfusion was associated with DWI normalization and a mitigation of pannecrotic changes within zones of residual injury at 24 h of injury. Albumin therapy lowered the hematocrit on average by 37% and raised plasma colloid oncotic pressure by 56%, improved the neurological score and reduced brain swelling throughout the 3-day survival period (Belayev et al, 1998). Similar treatment also improved local CBF as measured autoradiographically with 14Ciodoantipyrine after 1 h of recirculation (Huh et al, 1998). Using laser scanning confocal microscopy and laser Doppler perfusion imaging, it was found that a beneficial effect of albumin therapy was attributed to reversal of stagnation, thrombosis and corpuscular adherence within cortical venules in the reperfusion phase after focal ischemia (Belayev et al, 2002). It was also reported that after 1 h of reperfusion, 1.25 g/kg intravenous HSA administration increased replenishment of polyunsaturated fatty acid (PUFA) lost from cellular membranes during ischemia (Rodriguez de Turco et al, 2002). These studies collectively indicate that albumin induced neuroprotection is attributed to properties such as reversal of thrombosis, improvement in microvascular blood perfusion, reduction in brain swelling and replenishment of PUFA in brain. All these actions could indicate that actions of albumin are confined in vascular space. However, it has been shown that treatment with human albumin following 2 h of MCAO also leads to albumin extravasations

Group, 1995; Hacke et al, 2004; Juttler et al, 2006). However, the goal is to discover a neuroprotective drug which can inhibit reperfusion injury and provide neuroprotection within a wide therapeutic window. Hemodilution is an old approach which has been investigated for many decades as a potential therapy for ischemic stroke. Infusion of dextran has been shown to increase CBF of both the normal and ischemic brain, either by decreasing blood viscosity or by vasodilation in response to diminished oxygen delivery (Wood and Kee, 1985; Korosue and Heros, 1992). Despite neuroprotective benefi ts in experimental setups, several clinical trials of hemodilution in ischemic stroke have nonetheless proven negative or inconclusive (Scandinavian Stroke Study Group, 1987; Italian Acute Stroke Study Group, 1988; The Hemodilution in Stroke Study Group,1989). Subsequently, albumin has emerged as an alternative hemodiluting agent to dextran owing to its volume expanding properties (Sundt et al, 1967; Little et al, 1981; Emerson, 1989). However, only recently it has been rigorously evaluated for its anti-ischemic neuroprotective efficacy. Transient focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) is the most widely used model to study molecular mechanisms of cerebral ischemiareperfusion injury and to screen neuroprotective drugs. It is less invasive with a low rate of mortality and a low coefficient of variation in lesion size (Longa et al, 1989; Belayev et al, 1997b). In the rat MCAO model, Cole et al. reported that 5 % albumin administration at the onset of ischemia reduced ischemic brain injury as evidenced by reduced hematocrit, infarct volume and cerebral edema (Cole et al, 1990). In another study, administration of concentrated (20%) HSA (1% body weight, intravenously) to rats at the onset of recirculation induced substantial diminution of infarct volume together with a marked reduction of brain edema. Thus, it is proposed that albumin might modify water homeostasis and ultimately reduce edema of the ischemic brain (Belayev et al, 1997a). These two initial studies suggested that albumin therapy at the onset of ischemia or reperfusion induces neuroprotection.In a detailed study using magnetic resonance imaging, by means of diffusion-weighted magnetic resonance imaging (DWI), 25% Human Serum Albumin (HSA) solution (1% by body weight) administered immediately after reperfusion was associated with DWI normalization and a mitigation of pannecrotic changes within zones of residual injury at 24 h of injury. Albumin therapy lowered the hematocrit on average by 37% and raised plasma colloid oncotic pressure by 56%, improved the neurological score and reduced brain swelling throughout the 3-day survival period (Belayev et al, 1998). Similar treatment also improved local CBF as measured autoradiographically with 14Ciodoantipyrine after 1 h of recirculation (Huh et al, 1998). Using laser scanning confocal microscopy and laser Doppler perfusion imaging, it was found that a beneficial effect of albumin therapy was attributed to reversal of stagnation, thrombosis and corpuscular adherence within cortical venules in the reperfusion phase after focal ischemia (Belayev et al, 2002). It was also reported that after 1 h of reperfusion, 1.25 g/kg intravenous HSA administration increased replenishment of polyunsaturated fatty acid (PUFA) lost from cellular membranes during ischemia (Rodriguez de Turco et al, 2002). These studies collectively indicate that albumin induced neuroprotection is attributed to properties such as reversal of thrombosis, improvement in microvascular blood perfusion, reduction in brain swelling and replenishment of PUFA in brain. All these actions could indicate that actions of albumin are confined in vascular space. However, it has been shown that treatment with human albumin following 2 h of MCAO also leads to albumin extravasations and subsequently cellular uptake. It has been observed that cortical neurons with preserved structural features had taken up human albumin. Thus, it is reasonable to speculate that treatment with human albumin could also provide direct neuronal protection (Remmers et al, 1999). For the effective treatment of ischemic stroke, treatment should be started within a narrow therapeutic window of 3 h. Moderate-dose albumin therapy (1.25 g/kg intravenously) markedly provides neuroprotection even when treatment is delayed up to 4 h after onset of ischemia (Belayev et al, 2001). Albumin treatment has also been found to be neuroprotective in other models of focal ischemia. Prompt albumin therapy improved neurological function and blood-brain barrier integrity after acute intracortical hematoma (ICH) (Belayev et al, 2005). In a model of laser-induced cortical arteriolar thrombosis, highdose albumin therapy induced a prompt, sustained improvement in microvascular hemodynamics distal to a cortical arteriolar thrombosis (Nimmagadda et al, 2008). In acute ischemic stroke, albumin combination therapy can attenuate the deleterious effects of tPA (Tang et al, 2009). Furthermore, albumin (1.25 g/kg) treatment maintains serum albumin at a higher level and attenuates cortex and hippocampus vascular endothelial growth factor (VEGF) expression at 6 h and 1 day after MCAO. This could partially contribute to the protective effects of albumin on reduction of brain edema and infarct size in the early stage of ischemia (Yao et al, 2010). The above mentioned studies prove that in experimental transient ischemia albumin provides neuroprotection via different indirect and direct mechanisms. Albumin has been found to be effective in other models of stroke such as permanent MCAO, global ischemia induced by bilateral common carotid occlusion (BCCO) and traumatic brain injury (TBI). In permanent MCAO, rats treated with 2 g/kg/day concentrated (25%) albumin begun after 30 min of ischemia showed diminished brain edema and infarct volume up to 6 days (Matsui et al, 1993). Furthermore, albumin (1.25 and 2.5 g/kg) significantly reduced cortical and striatal infarct areas and increased cortical perfusion in the permanent ischemia model (Liu et al, 2001). In transient global ischemia, HSA-treated rats showed signifi cantly improved neurological deficits throughout a 7-day survival period along with increases in numbers of surviving CA1 hippocampal pyramidal neurons compared to saline-treated animals (Belayev et al, 1999b). In TBI, 15 min after trauma, HSA administration signifi cantly improved neurological defi cits and also significantly reduced total contusion area (Belayev et al, 1999a). These experimental trials altogether indicated significant neuroprotective roles of albumin in different models of ischemic stroke and encouraged the further development of this important molecule for possible treatment of ischemic stroke in humans.

### **4.4 Albumin in clinical trials for ischemic stroke**

The Albumin In Acute Stroke (ALIAS) Pilot Clinical Trial was conducted during 2001 – 2005 at two clinical sites (Universities of Calgary and Miami). This study was designed to investigate the safety and tolerability of albumin therapy in acute ischemic stroke. The ALIAS Pilot Clinical Trial used a multiple-tier, open-label, dose-escalation design. Human albumin (25%) in doses ranging up to 2.05 g/kg was well tolerated by patients with acute ischemic stroke without major dose-limiting complications. Concurrent tPA therapy did not affect the safety profile of albumin (Ginsberg et al, 2006a). Also, in this pilot trial the neuroprotective efficacy of albumin was evaluated and was found to be neuroprotective after ischemic stroke (Palesch et al, 2006). Based on the encouraging results of pilot trials of

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 435

neurotrophic factor is dependent on caveola and the adaptor protein cytosolic adaptor protein disabled (Dab-1) in cultured astrocytes (Bento -Abreu et al, 2009). These studies indicate that albumin could play a role in neuronal differentiation and development. Albumin also induces calcium mobilisation in cultured as well as brain astrocytes (Manning and Sontheimer, 1997; Nadal et al, 1998; Hooper et al, 2005). Albumin elicits calcium entry in the microglia which promotes proliferation of the microglia (Hooper et al, 2005). Astrocyte calcium signalling caused by albumin could have important physiological and pathophysiological consequences when the blood-brain barrier breaks down and allows albumin to enter the CNS. It is reported that albumin leakage induced by blood-brain barrier breaks is followed by albumin uptake into astrocytes which is responsible for epileptogenesis in rats (Ivens et al, 2007; van Vliet et al, 2007). Albumin causes downregulation of Kir current which results in the abnormal accumulation of [K+] o and consequent NMDA-receptor dependent pathological plasticity which isresponsible for epileptogenesis (Ivens et al, 2007). Recently, it was shown that albumin activates astrocytes and microglia producing infl ammatory responses via the mitogen-activated protein kinase pathway and these effects could be involved both in the mechanism of cellular injury and repair (Ralay Ranaivo and Wainwright, 2010). Altogether these fi ndings suggest that the majority of the effects of albumin on astrocytes, microglia and neuronal cells seem to be benefi cial; however, at augmented levels it could contribute towards astrocyte dysfunction. The direct effects of albumin on neuronal and glial cells necessitate further detailed

investigation in individual pathological conditions.

**5. Antithrombin III** 

**4.6 Endogenous albumin and neuroprotection: possible new paradigm** 

synthesized albumin is an unexplored area and warrants further investigations.

Antithrombin III (ATIII) is a single-chain glycoprotein in plasma and belongs to the family of the serpins. It is synthesized in liver parenchymal cells, and it plays a central role in regulating haemostasis. When bound to glycosaminoglycans, it is an important inhibitor of several serine protease, including factors Xa, IXa, XIa, and thrombin (Bauer and Rosenberg, 1991), wich are involved in blood coagulation. Equimolar, irreversible complexes are formed between ATIII and the enzymes. Heparin and heparan sulfate glycoproteins (HSPGs) bind to multiple sites of the ATIII molecule resulting in a steric reconfiguration, thereby

Although albumin is mainly synthesized in the liver, mRNA expression level of albumin has been found in many non-hepatic rat tissues such as lungs, heart, kidney and pancreas, but not in the brain (Nahon et al, 1988). Also, non-hepatic albumin expression at the protein level is rarely confirmed. A recent study suggests that human brain microglia cells can express albumin both at mRNA and protein levels; furthermore, this expression is increased by amyloid beta (Aβ) and lipopolysaccharide treatment (Ahn et al, 2008). It is suggested that enhanced levels of albumin and subsequent secretion by microglia could be implicated in A β removal from the brain (Ahn et al, 2008). We have also found upregulation of albumin at both mRNA and protein levels in ischemic rat brain. Upregulation of albumin in ischemic brain could play a neuroprotective role against altered brain functions (Prajapati et al, 2010). These results indicate that de novo synthesis of albumin also occurs in the brain tissue. However, possible intracellular and extracellular neuroprotective actions of endogenously

albumin, the National Institutes of Health has funded a randomized multicenter placebocontrolled effi cacy trial – the ALIAS Phase III Trial. A randomised, multicenter, doubleblind, placebo controlled trial (ALIAS Phase III Trial, www.clinicaltrials. gov; NCT00235495) is currently being conducted at approximately 70 clinical sites in North America (Ginsberg et al, 2006b; Hill et al, 2011).

### **4.5 Direct neuroprotection by albumin: Mechanisms**

The neuroprotective mechanisms of albumin in ischemic stroke and AD are largely attributed to its hemodynamic properties and binding properties. However, in different in vitro systems, albumin has been reported to possess several direct neuroprotective actions. (Figure 4)

Albumin could produce various neuroprotective actions in the intravascular compartment, cerebrospinal fluid-interstitial fluid compartment and intracellular compartment.

HSA and its N-terminal tetrapeptide DAHK can block oxidant-driven cultured neuronal injury produced by hydrogen peroxide and copper/ascorbic acid (Gum et al, 2004). Furthermore, bovine serum albumin has been found to be neuroprotective by reducing both the DNA damage and apoptosis rates in cultured cortical neurons and these effects are probably due to its antioxidant activity (Baltanas et al, 2009). Albumin has been reported to play an important role in astrocyte functions. It is shown that albumin affects metabolism of cultured astrocytes (Tabernero et al, 1999). Albumin up on transcytosis into cultured astrocytes stimulates the synthesis of neurotrophic factor oleic acid which promotes neuronal differentiation (Tabernero et al, 2002). Megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid (Bento - Abreu et al, 2008). Also, this megalin induced albumin transcytosis and synthesis of

Fig. 4. Overview of possible neuroprotective mechanisms of albumin.

neurotrophic factor is dependent on caveola and the adaptor protein cytosolic adaptor protein disabled (Dab-1) in cultured astrocytes (Bento -Abreu et al, 2009). These studies indicate that albumin could play a role in neuronal differentiation and development. Albumin also induces calcium mobilisation in cultured as well as brain astrocytes (Manning and Sontheimer, 1997; Nadal et al, 1998; Hooper et al, 2005). Albumin elicits calcium entry in the microglia which promotes proliferation of the microglia (Hooper et al, 2005). Astrocyte calcium signalling caused by albumin could have important physiological and pathophysiological consequences when the blood-brain barrier breaks down and allows albumin to enter the CNS. It is reported that albumin leakage induced by blood-brain barrier breaks is followed by albumin uptake into astrocytes which is responsible for epileptogenesis in rats (Ivens et al, 2007; van Vliet et al, 2007). Albumin causes downregulation of Kir current which results in the abnormal accumulation of [K+] o and consequent NMDA-receptor dependent pathological plasticity which isresponsible for epileptogenesis (Ivens et al, 2007). Recently, it was shown that albumin activates astrocytes and microglia producing infl ammatory responses via the mitogen-activated protein kinase pathway and these effects could be involved both in the mechanism of cellular injury and repair (Ralay Ranaivo and Wainwright, 2010). Altogether these fi ndings suggest that the majority of the effects of albumin on astrocytes, microglia and neuronal cells seem to be benefi cial; however, at augmented levels it could contribute towards astrocyte dysfunction. The direct effects of albumin on neuronal and glial cells necessitate further detailed investigation in individual pathological conditions.

### **4.6 Endogenous albumin and neuroprotection: possible new paradigm**

Although albumin is mainly synthesized in the liver, mRNA expression level of albumin has been found in many non-hepatic rat tissues such as lungs, heart, kidney and pancreas, but not in the brain (Nahon et al, 1988). Also, non-hepatic albumin expression at the protein level is rarely confirmed. A recent study suggests that human brain microglia cells can express albumin both at mRNA and protein levels; furthermore, this expression is increased by amyloid beta (Aβ) and lipopolysaccharide treatment (Ahn et al, 2008). It is suggested that enhanced levels of albumin and subsequent secretion by microglia could be implicated in A β removal from the brain (Ahn et al, 2008). We have also found upregulation of albumin at both mRNA and protein levels in ischemic rat brain. Upregulation of albumin in ischemic brain could play a neuroprotective role against altered brain functions (Prajapati et al, 2010). These results indicate that de novo synthesis of albumin also occurs in the brain tissue. However, possible intracellular and extracellular neuroprotective actions of endogenously synthesized albumin is an unexplored area and warrants further investigations.

### **5. Antithrombin III**

434 Advances in the Preclinical Study of Ischemic Stroke

albumin, the National Institutes of Health has funded a randomized multicenter placebocontrolled effi cacy trial – the ALIAS Phase III Trial. A randomised, multicenter, doubleblind, placebo controlled trial (ALIAS Phase III Trial, www.clinicaltrials. gov; NCT00235495) is currently being conducted at approximately 70 clinical sites in North America (Ginsberg

The neuroprotective mechanisms of albumin in ischemic stroke and AD are largely attributed to its hemodynamic properties and binding properties. However, in different in vitro systems, albumin has been reported to possess several direct neuroprotective actions.

Albumin could produce various neuroprotective actions in the intravascular compartment,

HSA and its N-terminal tetrapeptide DAHK can block oxidant-driven cultured neuronal injury produced by hydrogen peroxide and copper/ascorbic acid (Gum et al, 2004). Furthermore, bovine serum albumin has been found to be neuroprotective by reducing both the DNA damage and apoptosis rates in cultured cortical neurons and these effects are probably due to its antioxidant activity (Baltanas et al, 2009). Albumin has been reported to play an important role in astrocyte functions. It is shown that albumin affects metabolism of cultured astrocytes (Tabernero et al, 1999). Albumin up on transcytosis into cultured astrocytes stimulates the synthesis of neurotrophic factor oleic acid which promotes neuronal differentiation (Tabernero et al, 2002). Megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid (Bento - Abreu et al, 2008). Also, this megalin induced albumin transcytosis and synthesis of

Brain cells

Neuroprotection

Prevents DNA damage

Protein

Anti-oxidant activity

cerebrospinal fluid-interstitial fluid compartment and intracellular compartment.

CSF/ISF

Neuroprotection mRNA

Removal of toxic substance

Fig. 4. Overview of possible neuroprotective mechanisms of albumin.

Neuroprotection

Neuroprotection

et al, 2006b; Hill et al, 2011).

Blood Vessels

Brain swellig Blood-element adhesion Thrombosis Blood perfusion

Blood derived albumin Endogenous brain cell albumin

Free fatty acid

(Figure 4)

**4.5 Direct neuroprotection by albumin: Mechanisms** 

Antithrombin III (ATIII) is a single-chain glycoprotein in plasma and belongs to the family of the serpins. It is synthesized in liver parenchymal cells, and it plays a central role in regulating haemostasis. When bound to glycosaminoglycans, it is an important inhibitor of several serine protease, including factors Xa, IXa, XIa, and thrombin (Bauer and Rosenberg, 1991), wich are involved in blood coagulation. Equimolar, irreversible complexes are formed between ATIII and the enzymes. Heparin and heparan sulfate glycoproteins (HSPGs) bind to multiple sites of the ATIII molecule resulting in a steric reconfiguration, thereby

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 437

thrombin include stimulation of neutrophil/monocyte adhesion, action as a chemotactic factor for polymorphonuclear leukocytes (Esmon, 2000), and increased expression of the recently discovered inhibitor of fibrinolysis, thrombin-activable fibrinolysis inhibitor (TAFI) (Opal, 2000). Thrombin also stimulates the increased expression of IL-8 and plays an important role in ischemia-induced leukocyte rolling and adhesion (Kaur et al, 2001; Ludwicka-Bradley et al, 2000; Rabiet et al, 1994). Thus, the ability of ATIII to inhibit the actions of both factor Xa and thrombin gives it the potential to block, in part or fully, a wide

In the late 1980s, initial publications reported on the property of ATIII to stimulate prostacyclin release from endothelial cells independent of thrombin interaction. Although some recent studies question such a mechanism, at least in vitro, a number of papers make an in vivo contribution of prostacyclin release as part of the ATIII anti-inflammatory properties likely (Uchiba and Okajima, 2001). Independent of ATIII's anticoagulatory activities at multiple points of action, a variety of studies provide evidence for a potent anti-inflammatory ATIII effect, wich can only be induced by high ATIII plasma activities in the range of 150%-200% (Harada et al, 1999; Hoffmann et al, 2000; Okajiama and Uchiba, 1998; Uchiba et al, 1996, 1998). These anti-inflammatory ATIII actions may be mainly mediated by an interaction of ATIII with the endothelium (Hoffmann et al, 2002), thereby producing a profound increase in endothelial prostacyclin production (Figure 5C)

ATIII expresses the ability to inhibit leukocyte rolling and adhesion, wich are hallmarks of inflammatory reactions, and have explored the mechanisms underlying these effects. Ostrovsky et al (Ostrovsky et al, 1997) showed that ATIII administration significantly reduced neutrophil rolling and adhesion to pretreatment levels in a feline mesentery ischaemia-reperfusion model. Nevière et al (Nevière et al, 2001) and Hoffmann et al (Hoffmann et al, 2000) showed that the beneficial effects resulting from ATIII's blocking of leukocyte-endothelium interactions were eliminated when indomethacin, a cyclo-oxygenase inhibitor that blocks prostacyclin production, was added to the treatment. The administration of recombinant hirudin did not result in comparable beneficial effects, supporting the thrombin-indipendent mode of action of ATIII. As a consequence of the limited activated leukocyte-endothelium interaction, the severity of subsequent capillary leakage, disturbance of microcirculation, and organ damage were significantly reduced. A report by Yamashiro et al (Yamashiro et al, 2001) suggests direct affects of ATIII on leukocytes and endothelium by demonstrating the downmodulation of P-selectin by ATIII in the LPS-stimulated endothelium, thereby diminishing leukocyte rolling and subsequent transmigration. Support of this hypothesis has also been provided by the work of Souter et al (Souter et al, 2001) showing that the addition of ATIII to LPS-treated whole blood, HUVEC, and mononuclear cells inhibited production of both IL-6 and tissue factor; recombinant hirudin, a specific thrombin inhibitor, did not reduce the production of IL-6 or tissue factor, again suggesting that the observed inhibition by ATIII was not due solely to its

range of proinflammatory events (Seegers, 1978).

(Yamauchi et al, 1989).

**5.2 Coagulation-indipendent anti-inflammatory effects of ATIII** 

**5.3 Effects of ATIII on leukocyte-endothelium interactions** 

ability ti inhibit thrombin (figure 5B and C).

increasing the interaction between ATIII and the activated enzymes. It is believed that much of the physiological inactivation of enzymes by ATIII occurs in the endothelium, mediated by heparan sulfate (Figure 5) (Mammen, 1998).

Fig. 5. Role of heparan sulfate proteoglycan (HSPG) in inhibition of thrombin, in induction of prostacyclin, and inhibition of cytokine and tissue factor release from endothelial cells by antithrombin III.

A large number of recent studies have shown that ATIII has anti-inflammatory actions, (Cuomo et al, 2007) which are independent of its effects on coagulation. These effects include the heterologous deactivation of activated leukocytes and the interaction with the endothelium, thereby reducing vessel wall transmigration and subsequent tissue and organ damage. Thus, ATIII may have two distinct and indipendent actions in patients with cerebral ischemia: (1) interference with pathologic coagulation, and (2) inhibition of inflammation.

### **5.1 Effects of ATIII on abnormal coagulation**

The prothrombotic, proinflammatory state of stroke results in a promotion of thrombin formation and fibrin deposition at the vascular wall, as well as in the formetion of plateletleukocyte coaggregates, leading to severe disturbance of the microcirculation, capillary leakage and tissue damage (Piazza et al, 2010). Many of the events involved in this proinflammatory state have been shown to be inhibited by ATIII. The inhibition of factor Xa by ATIII may be particularly important for protection against, and treatment of, inflammation. This activated clotting factor has a number of proinflammatory effects, including stimulation if the production of IL-6, IL-8, MCP-1, E-selectin, and the soluble adhesion molecules ICAM-1 and vascular cell adhesion molecule (VCAM)-1, wich can be experimentally blocked by ATIII (Senden et al, 1998). The proinflammatory functions of

increasing the interaction between ATIII and the activated enzymes. It is believed that much of the physiological inactivation of enzymes by ATIII occurs in the endothelium, mediated

> Tissue Factor IL-6

> > ATIII

PGI

☈

ATIII

LPS

HSPG

by heparan sulfate (Figure 5) (Mammen, 1998).

*A B C*

Endothelium Endothelium

☈ LPS

NFkB

Fig. 5. Role of heparan sulfate proteoglycan (HSPG) in inhibition of thrombin, in induction of prostacyclin, and inhibition of cytokine and tissue factor release from endothelial cells by

A large number of recent studies have shown that ATIII has anti-inflammatory actions, (Cuomo et al, 2007) which are independent of its effects on coagulation. These effects include the heterologous deactivation of activated leukocytes and the interaction with the endothelium, thereby reducing vessel wall transmigration and subsequent tissue and organ damage. Thus, ATIII may have two distinct and indipendent actions in patients with cerebral ischemia: (1) interference with pathologic coagulation, and (2) inhibition of

The prothrombotic, proinflammatory state of stroke results in a promotion of thrombin formation and fibrin deposition at the vascular wall, as well as in the formetion of plateletleukocyte coaggregates, leading to severe disturbance of the microcirculation, capillary leakage and tissue damage (Piazza et al, 2010). Many of the events involved in this proinflammatory state have been shown to be inhibited by ATIII. The inhibition of factor Xa by ATIII may be particularly important for protection against, and treatment of, inflammation. This activated clotting factor has a number of proinflammatory effects, including stimulation if the production of IL-6, IL-8, MCP-1, E-selectin, and the soluble adhesion molecules ICAM-1 and vascular cell adhesion molecule (VCAM)-1, wich can be experimentally blocked by ATIII (Senden et al, 1998). The proinflammatory functions of

Thrombin

ATIII ATIII

HSPG

antithrombin III.

inflammation.

**5.1 Effects of ATIII on abnormal coagulation** 

ATIII

thrombin include stimulation of neutrophil/monocyte adhesion, action as a chemotactic factor for polymorphonuclear leukocytes (Esmon, 2000), and increased expression of the recently discovered inhibitor of fibrinolysis, thrombin-activable fibrinolysis inhibitor (TAFI) (Opal, 2000). Thrombin also stimulates the increased expression of IL-8 and plays an important role in ischemia-induced leukocyte rolling and adhesion (Kaur et al, 2001; Ludwicka-Bradley et al, 2000; Rabiet et al, 1994). Thus, the ability of ATIII to inhibit the actions of both factor Xa and thrombin gives it the potential to block, in part or fully, a wide range of proinflammatory events (Seegers, 1978).

### **5.2 Coagulation-indipendent anti-inflammatory effects of ATIII**

In the late 1980s, initial publications reported on the property of ATIII to stimulate prostacyclin release from endothelial cells independent of thrombin interaction. Although some recent studies question such a mechanism, at least in vitro, a number of papers make an in vivo contribution of prostacyclin release as part of the ATIII anti-inflammatory properties likely (Uchiba and Okajima, 2001). Independent of ATIII's anticoagulatory activities at multiple points of action, a variety of studies provide evidence for a potent anti-inflammatory ATIII effect, wich can only be induced by high ATIII plasma activities in the range of 150%-200% (Harada et al, 1999; Hoffmann et al, 2000; Okajiama and Uchiba, 1998; Uchiba et al, 1996, 1998). These anti-inflammatory ATIII actions may be mainly mediated by an interaction of ATIII with the endothelium (Hoffmann et al, 2002), thereby producing a profound increase in endothelial prostacyclin production (Figure 5C) (Yamauchi et al, 1989).

### **5.3 Effects of ATIII on leukocyte-endothelium interactions**

ATIII expresses the ability to inhibit leukocyte rolling and adhesion, wich are hallmarks of inflammatory reactions, and have explored the mechanisms underlying these effects. Ostrovsky et al (Ostrovsky et al, 1997) showed that ATIII administration significantly reduced neutrophil rolling and adhesion to pretreatment levels in a feline mesentery ischaemia-reperfusion model. Nevière et al (Nevière et al, 2001) and Hoffmann et al (Hoffmann et al, 2000) showed that the beneficial effects resulting from ATIII's blocking of leukocyte-endothelium interactions were eliminated when indomethacin, a cyclo-oxygenase inhibitor that blocks prostacyclin production, was added to the treatment. The administration of recombinant hirudin did not result in comparable beneficial effects, supporting the thrombin-indipendent mode of action of ATIII. As a consequence of the limited activated leukocyte-endothelium interaction, the severity of subsequent capillary leakage, disturbance of microcirculation, and organ damage were significantly reduced. A report by Yamashiro et al (Yamashiro et al, 2001) suggests direct affects of ATIII on leukocytes and endothelium by demonstrating the downmodulation of P-selectin by ATIII in the LPS-stimulated endothelium, thereby diminishing leukocyte rolling and subsequent transmigration. Support of this hypothesis has also been provided by the work of Souter et al (Souter et al, 2001) showing that the addition of ATIII to LPS-treated whole blood, HUVEC, and mononuclear cells inhibited production of both IL-6 and tissue factor; recombinant hirudin, a specific thrombin inhibitor, did not reduce the production of IL-6 or tissue factor, again suggesting that the observed inhibition by ATIII was not due solely to its ability ti inhibit thrombin (figure 5B and C).

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 439

intracellular pathways leading to transcription factor activation and the generation of cytokines and chemokines (Figure 6) (Vogel et al., 2003; Takeda and Akira, 2005). Each TLR family member, with the exception of TLR3, initiates intracellular signaling via recruitment of the intracellular Toll-interleukin 1 receptor (TIR)–domain-containing adaptor MyD88. When recruited to plasma membrane-associated TLRs, either directly (TLRs 5 and 11) or via the TIRAP adaptor (TLRs 1, 2, 4, 6), MyD88 enlists members of the IRAK family, including IRAK1, IRAK2, and IRAK4, to begin a process of auto- and cross-phosphorylation among the IRAK molecules. Once phosphorylated, IRAKs dissociate from MyD88 and bind TRAF6, an E3 ligase. TRAF6 in turn activates TAK1 which itself activates the IKK complex and MAPKKs. The IKK complex, composed of IKKα, IKKβ and the regulatory subunit IKKγ/NEMO, phosphorylates IκB proteins. This phosphorylation is necessary for the ubiquitination and proteosomal degradation of IκBs and the subsequent nuclear translocation of the transcription factor NFκB. Members of the MAPK family phosphorylate and activate components of the transcription factor AP-1. Together, these transcription factors induce inflammatory cytokine production (e.g. TNFα, IL1). MyD88 is also recruited to the endosomal receptors TLR7 and TLR9 again enlisting members of the IRAK family. Due to the endosomal location of the complex, the phosphorylated IRAKs are able to bind TRAF3 in addition to TRAF6. Activation of TRAF3 leads to phosphorylation, dimerization, and nuclear localization of the transcription factors IRF3, IRF5, and IRF7 with resultant type I interferon (IFN) production. Hence these endosomal TLRs are capable of signaling to NFκB, AP-1 and IRFs, resulting in a diverse genomic response. Endosomal TLR3 is unique among the TLRs because it does not signal through MyD88 but signals instead via recruitment of the Toll-interleukin 1 receptor domain-containing adaptor inducing interferon β (TRIF). TRIF enlists the non-canonical IKKs, TBK1 and IKKε, which activate IRF3. Further, TRIF recruits TRAF6 and RIP-1, which results in activation of MAPK and IKKα/β. Hence TLR3, like the other endosomal receptors, is capable of activating NFκB, AP-1 and IRFs. Of all the TLRs, only TLR4 can recruit either MyD88 (via TIRAP) or TRIF (via TRAM) and can thus induce either the pro-inflammatory cytokines TNFα and IL1 via NFκB or the anti-viral IFNβ via IRF3. The complement of TLR family members expressed by a cell depends on its identity and its activation status. Constitutive expression of TLRs within the brain occurs in microglia and astrocytes and is largely restricted to the circumventricular organs and meninges—areas with direct access to the circulation (Laflamme and Rivest, 2001; Laflamme et al., 2001; Chakravarty and Herkenham, 2005). Human and murine microglia express TLRs 1–9 and generate cytokine profiles specifically tailored by the TLR stimulated (Bsibsi et al., 2002; Olson and Miller, 2004; Jack et al., 2005). Similarly, human and murine astrocytes express multiple TLRs, with particularly prominent TLR3 expression (Bsibsi et al., 2002, 2006; Carpentier et al., 2005; Jack et al., 2005; McKimmie and Fazakerley, 2005). Microglia and astrocytes respond differently to specific TLR engagement reflective of their distinct roles in the brain. Microglia initiate robust cytokine and chemokine responses to stimulation of TLR2 (TNFα, IL-6, IL-10), TLR3 (TNFα, IL-6, IL-10, IL-12, CXCL-10, IFNβ), and TLR4 (TNFα, IL-6, IL-10, CXCL-10, IFNβ), yet astrocytes initiate only minor IL-6 responses to all but TLR3 stimulation (Jack et al., 2005). Microglia express TLR3 and TLR4 at the cell surface while astrocytes express these receptors intracellularly (Bsibsi et al., 2002). The cellular location of TLRs affects their downstream signaling cascades (Kagan et al., 2008), which may explain the different responses of these cells to TLR stimulation. The

### **5.4 Direct effect of ATIII on leukocytes**

Dunzendorfer et al (Dunzendorfer et al, 2000, 2001) have uncovered a second mechanism by which ATIII may inhibit neutrophil migration and adhesion to the endothelium; namely, heterologous deactivation of activated leukocytes by ATIII. These investigators noted that signaling in ATIII-induced neutrophil chemotaxis mimics an IL-8-induced response, ATIII inhibited migration of neutrophils towards IL-8, GRO-alpha, and fMLP; staurosporine, bisindolylmaleimide I, pertussis toxin, and an anti-CXCR1 monoclonal antibody all blocked ATIII-induced neutrophil chemotaxis. However, additional assays did not reveal binding of ATIII to CXCR1. Thus, the results of these studies are generally consistent with the conclusion that the effect of ATIII in neutrophil migration appear to involve a CXCR1 related signaling pathway, its G-proteins, and protein kinase C. Recent findings have shown that the signalling pathway activated by ATIII in leukocytes are different in neutrophils, monocytes, and lymphocytes (Dunzendorfer et al, 2001; Kaneider et al, 2001, 2002). All togheter, these experiments led to the conclusion that ATIII in circulation protects leukocytes from premature activation.

### **5.5 ATIII and nuclear factor-kappaB**

Oelschager et al (Oelshager et al, 2001) showed that ATIII produces a dose-dependent reduction in both LPS and tissue necrosis factor (TNF)-α activation of nuclear factor-kappaB (NF-κB) in cultured monocytes and endothelial cells. Results reported by this working group and by Iampietro et al (Iampietro et al, 2000) indicate that these actions of ATIII block the increase in IL-6, IL-8, TNF, and tissue factor mRNA expression (figure 5B and C). Beyond control of coagulation, ATIII displays anti-inflammatory properties through an interaction with cells, reducing the synthesis and release of proinflammatory mediators, thereby modulating leukocyte activation and their interaction with the vessel wall. As a consequence, tissue damage and organ failure are reduced.

### **6. Toll-like receptors**

The TLRs, so-called because of their homology to the Drosophila Toll receptor, were first characterized in mammals by their ability to recognize pathogen-associated molecular patterns such as those found in the bacterial cell wall components peptidoglycan (TLR2) and lipopolysaccharide (LPS) (TLR4), as well as viral dsRNA (TLR3), ssRNA (TLR7), and nonmethylated cytosine-guanine (CpG) DNA (TLR9). Recently it has been found that in addition to their role in pathogen detection and defense, TLRs act as sentinels of tissue damage and mediate inflammatory responses to aseptic tissue injury. Surfactant, HSP60, components of the extracellular matrix, and fibrinogen have all been shown to activate TLR4, while host HMGB1 and host mRNA and DNA are endogenous ligands of TLR2 (and TLR4), TLR3 and TLR9, respectively. TLRs, upon activation by either pathogen- or host-derived ligands, induce downstream signals that lead to cytokine and chemokine production and thereby initiate inflammatory responses. TLRs are located on antigen presenting cells such as B cells, dendritic cells, monocytes/macrophages and microglia. In addition, these receptors can be expressed by the cerebral endothelium and by cells within the brain parenchyma such as astrocytes, oligodendrocytes, and neurons (Bsibsi et al., 2002; Singh and Jiang, 2004; Jack et al., 2005; Bsibsi et al., 2006). The TLRs signal through common

Dunzendorfer et al (Dunzendorfer et al, 2000, 2001) have uncovered a second mechanism by which ATIII may inhibit neutrophil migration and adhesion to the endothelium; namely, heterologous deactivation of activated leukocytes by ATIII. These investigators noted that signaling in ATIII-induced neutrophil chemotaxis mimics an IL-8-induced response, ATIII inhibited migration of neutrophils towards IL-8, GRO-alpha, and fMLP; staurosporine, bisindolylmaleimide I, pertussis toxin, and an anti-CXCR1 monoclonal antibody all blocked ATIII-induced neutrophil chemotaxis. However, additional assays did not reveal binding of ATIII to CXCR1. Thus, the results of these studies are generally consistent with the conclusion that the effect of ATIII in neutrophil migration appear to involve a CXCR1 related signaling pathway, its G-proteins, and protein kinase C. Recent findings have shown that the signalling pathway activated by ATIII in leukocytes are different in neutrophils, monocytes, and lymphocytes (Dunzendorfer et al, 2001; Kaneider et al, 2001, 2002). All togheter, these experiments led to the conclusion that ATIII in circulation protects

Oelschager et al (Oelshager et al, 2001) showed that ATIII produces a dose-dependent reduction in both LPS and tissue necrosis factor (TNF)-α activation of nuclear factor-kappaB (NF-κB) in cultured monocytes and endothelial cells. Results reported by this working group and by Iampietro et al (Iampietro et al, 2000) indicate that these actions of ATIII block the increase in IL-6, IL-8, TNF, and tissue factor mRNA expression (figure 5B and C). Beyond control of coagulation, ATIII displays anti-inflammatory properties through an interaction with cells, reducing the synthesis and release of proinflammatory mediators, thereby modulating leukocyte activation and their interaction with the vessel wall. As a

The TLRs, so-called because of their homology to the Drosophila Toll receptor, were first characterized in mammals by their ability to recognize pathogen-associated molecular patterns such as those found in the bacterial cell wall components peptidoglycan (TLR2) and lipopolysaccharide (LPS) (TLR4), as well as viral dsRNA (TLR3), ssRNA (TLR7), and nonmethylated cytosine-guanine (CpG) DNA (TLR9). Recently it has been found that in addition to their role in pathogen detection and defense, TLRs act as sentinels of tissue damage and mediate inflammatory responses to aseptic tissue injury. Surfactant, HSP60, components of the extracellular matrix, and fibrinogen have all been shown to activate TLR4, while host HMGB1 and host mRNA and DNA are endogenous ligands of TLR2 (and TLR4), TLR3 and TLR9, respectively. TLRs, upon activation by either pathogen- or host-derived ligands, induce downstream signals that lead to cytokine and chemokine production and thereby initiate inflammatory responses. TLRs are located on antigen presenting cells such as B cells, dendritic cells, monocytes/macrophages and microglia. In addition, these receptors can be expressed by the cerebral endothelium and by cells within the brain parenchyma such as astrocytes, oligodendrocytes, and neurons (Bsibsi et al., 2002; Singh and Jiang, 2004; Jack et al., 2005; Bsibsi et al., 2006). The TLRs signal through common

**5.4 Direct effect of ATIII on leukocytes** 

leukocytes from premature activation.

**5.5 ATIII and nuclear factor-kappaB** 

**6. Toll-like receptors** 

consequence, tissue damage and organ failure are reduced.

intracellular pathways leading to transcription factor activation and the generation of cytokines and chemokines (Figure 6) (Vogel et al., 2003; Takeda and Akira, 2005). Each TLR family member, with the exception of TLR3, initiates intracellular signaling via recruitment of the intracellular Toll-interleukin 1 receptor (TIR)–domain-containing adaptor MyD88. When recruited to plasma membrane-associated TLRs, either directly (TLRs 5 and 11) or via the TIRAP adaptor (TLRs 1, 2, 4, 6), MyD88 enlists members of the IRAK family, including IRAK1, IRAK2, and IRAK4, to begin a process of auto- and cross-phosphorylation among the IRAK molecules. Once phosphorylated, IRAKs dissociate from MyD88 and bind TRAF6, an E3 ligase. TRAF6 in turn activates TAK1 which itself activates the IKK complex and MAPKKs. The IKK complex, composed of IKKα, IKKβ and the regulatory subunit IKKγ/NEMO, phosphorylates IκB proteins. This phosphorylation is necessary for the ubiquitination and proteosomal degradation of IκBs and the subsequent nuclear translocation of the transcription factor NFκB. Members of the MAPK family phosphorylate and activate components of the transcription factor AP-1. Together, these transcription factors induce inflammatory cytokine production (e.g. TNFα, IL1). MyD88 is also recruited to the endosomal receptors TLR7 and TLR9 again enlisting members of the IRAK family. Due to the endosomal location of the complex, the phosphorylated IRAKs are able to bind TRAF3 in addition to TRAF6. Activation of TRAF3 leads to phosphorylation, dimerization, and nuclear localization of the transcription factors IRF3, IRF5, and IRF7 with resultant type I interferon (IFN) production. Hence these endosomal TLRs are capable of signaling to NFκB, AP-1 and IRFs, resulting in a diverse genomic response. Endosomal TLR3 is unique among the TLRs because it does not signal through MyD88 but signals instead via recruitment of the Toll-interleukin 1 receptor domain-containing adaptor inducing interferon β (TRIF). TRIF enlists the non-canonical IKKs, TBK1 and IKKε, which activate IRF3. Further, TRIF recruits TRAF6 and RIP-1, which results in activation of MAPK and IKKα/β. Hence TLR3, like the other endosomal receptors, is capable of activating NFκB, AP-1 and IRFs. Of all the TLRs, only TLR4 can recruit either MyD88 (via TIRAP) or TRIF (via TRAM) and can thus induce either the pro-inflammatory cytokines TNFα and IL1 via NFκB or the anti-viral IFNβ via IRF3. The complement of TLR family members expressed by a cell depends on its identity and its activation status. Constitutive expression of TLRs within the brain occurs in microglia and astrocytes and is largely restricted to the circumventricular organs and meninges—areas with direct access to the circulation (Laflamme and Rivest, 2001; Laflamme et al., 2001; Chakravarty and Herkenham, 2005). Human and murine microglia express TLRs 1–9 and generate cytokine profiles specifically tailored by the TLR stimulated (Bsibsi et al., 2002; Olson and Miller, 2004; Jack et al., 2005). Similarly, human and murine astrocytes express multiple TLRs, with particularly prominent TLR3 expression (Bsibsi et al., 2002, 2006; Carpentier et al., 2005; Jack et al., 2005; McKimmie and Fazakerley, 2005). Microglia and astrocytes respond differently to specific TLR engagement reflective of their distinct roles in the brain. Microglia initiate robust cytokine and chemokine responses to stimulation of TLR2 (TNFα, IL-6, IL-10), TLR3 (TNFα, IL-6, IL-10, IL-12, CXCL-10, IFNβ), and TLR4 (TNFα, IL-6, IL-10, CXCL-10, IFNβ), yet astrocytes initiate only minor IL-6 responses to all but TLR3 stimulation (Jack et al., 2005). Microglia express TLR3 and TLR4 at the cell surface while astrocytes express these receptors intracellularly (Bsibsi et al., 2002). The cellular location of TLRs affects their downstream signaling cascades (Kagan et al., 2008), which may explain the different responses of these cells to TLR stimulation. The

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 441

suggesting that these molecules utilize MyD88 in distinct ways. Hence even minor alterations of these fine-tuned endogenous pathways can have profound effects on cellular responses to TLR engagement. Studies with TLR knockout mice illustrate the endogenous function of TLRs in health and disease. TLR2 and TLR4 have been shown to play detrimental roles in the development of congestive heart failure and cardiac hypertrophy, respectively, by signaling through MyD88 and NFκB (Shishido et al., 2003; Ha et al., 2005). TLR2 has additionally been found to be proatherogenic in hyperlipidemic mice (Tobias and Curtiss, 2007), and TLR4 has been shown to produce inflammatory reactions in adipose tissue and thereby mediates obesity and insulin resistance (Tsukumo et al., 2007; Davis et al., 2008). Conversely, TLR2 and TLR4 activation by hyaluronic acid protects lung tissue from non-infectious injury (Jiang et al., 2006), and TLR4 has been shown to help maintain lung integrity, and prevent the development of emphysema, by modulating oxidant generation (Zhang et al., 2006). The effects of endogenous TLR stimulation are clearly varied, depending on the cell and tissue type in which the receptors are found and on the disease process in which they are involved. The overwhelming and generally damaging inflammatory response of TLRs to aseptic tissue injury may be a consequence of TLR evolution in response to pathogens. In the setting of pathogen invasion, an inflammatory deluge may be the most effective means to clear microorganisms. The activation and influx of leukocytes, with the concomitant release of free radicals and tissue-destroying enzymes, assails not only the invading pathogen but any host cells that harbor the pathogen. However, when this same powerful response is co-opted by the host to clear and resolve tissue damage, it can destroy the very cells it is meant to save. This damage promoting characteristic is prominently observed following brain ischemia, where inflammation plays

A significant portion of the damage associated with stroke injury is due to the resultant inflammatory response. This aspect of the innate immune response is exemplified by the fact that some anti-inflammatory strategies have been shown to ameliorate ischemic damage

response to stroke is initiated by the detection of injury associated molecules by local cells such as microglia and astrocytes. The response is further promoted by infiltrating neutrophils and macrophages, resulting in the production of inflammatory cytokines, proteolytic enzymes, and other cytotoxic mediators. In the mouse, leukocytes and brain cells (microglia, astrocytes and neurons) express TLRs (Zarember and Godowski, 2002; Olson and Miller, 2004). Hence, injury-associated molecules such as HSP60 and HMGB1 may act as endogenous ligands for TLRs, thereby initiating the damaging inflammatory response to stroke. It is increasingly clear that TLRs do in fact play a role in ischemic damage (Fig.). The pathogenic role of TLRs in ischemic processes was first demonstrated in a mouse model myocardial ischemia/reperfusion injury, because mice lacking functional TLR4 incur less damage than wild type mice (Oyama et al., 2004). Since then, TLR2 has also been shown to cause dysfunction following cardiac ischemia and both have been shown to exacerbate renal ischemic damage, in a MyD88-dependent and a MyD88-independent manner (Sakata et al., 2007; Shigeoka et al., 2007). However, the particular pathway responsible for the damaging effects of TLR activation may differ depending on the cell type or organ affected as TLR4

(Relton et al., 1996; Hara et al., 1997; Spera et al., 1998). The inflammatory

a critical role in both injury progression and resolution.

**6.1 TLRS and ischemic damage** 

Fig. 6. TLRs signaling

inflammatory milieu also plays a critical role in regulating TLR expression. Microglia stimulated with CpG specifically up-regulate TLR9, whereas those stimulated with a synthetic TLR3 ligand suppress all TLRs except TLR3 (Olson and Miller, 2004). Similarly, astrocytes stimulated with LPS up-regulate TLRs 2 and 3 but suppress TLR4, while astrocytes exposed to RNA viruses up-regulate TLR3 and TLR9 (McKimmie and Fazakerley, 2005). Thus microglia and astrocytes initiate a layered and multifaceted response to TLR engagement. Oligodendrocytes and endothelial cells express a relatively limited repertoire of TLRs. Oligodendrocytes express TLRs 2 and 3 (Bsibsi et al., 2002), while cerebral endothelial cells constitutively express TLRs 2, 4, and 9 (Constantin et al., 2004) and increase their expression of these TLRs in response to stressful stimuli, including systemic LPS and cerebral ischemia (Singh and Jiang, 2004; Zhou et al., 2007; Ziegler et al., 2007). In response to LPS, endothelial cells up-regulate E-selectin, an NFκB-dependent molecule, and IFNβ, an IRF3-dependent molecule, indicating that these cells utilize the TLR–NFκB and the TLR– IRF3 signaling pathways (Lloyd-Jones et al., 2008). Neurons express TLR3 and generate inflammatory cytokines (TNFα, IL-6), chemokines (CCL5, CXCL10) and antiviral molecules (IFNβ) in response to dsRNA (Lafon et al., 2006; Prehaud et al., 2005). Neurons also employ TLRs in their development and differentiation. TLRs 3 and 8 are expressed on murine neurons early in development and inhibit neurite outgrowth in a MyD88- and NFκBindependent manner (Ma et al., 2006). TLR2 and TLR4 have been found on adult neural progenitor cells where they appear to elicit opposing effects. While TLR2 activation stimulates neuronal differentiation of these cells, TLR4 activation decreases proliferation and neuronal differentiation, driving these cells toward an astrocytic fate (Rolls et al., 2007). Curiously, both TLRs exert these endogenous effects in a MyD88-dependent manner, suggesting that these molecules utilize MyD88 in distinct ways. Hence even minor alterations of these fine-tuned endogenous pathways can have profound effects on cellular responses to TLR engagement. Studies with TLR knockout mice illustrate the endogenous function of TLRs in health and disease. TLR2 and TLR4 have been shown to play detrimental roles in the development of congestive heart failure and cardiac hypertrophy, respectively, by signaling through MyD88 and NFκB (Shishido et al., 2003; Ha et al., 2005). TLR2 has additionally been found to be proatherogenic in hyperlipidemic mice (Tobias and Curtiss, 2007), and TLR4 has been shown to produce inflammatory reactions in adipose tissue and thereby mediates obesity and insulin resistance (Tsukumo et al., 2007; Davis et al., 2008). Conversely, TLR2 and TLR4 activation by hyaluronic acid protects lung tissue from non-infectious injury (Jiang et al., 2006), and TLR4 has been shown to help maintain lung integrity, and prevent the development of emphysema, by modulating oxidant generation (Zhang et al., 2006). The effects of endogenous TLR stimulation are clearly varied, depending on the cell and tissue type in which the receptors are found and on the disease process in which they are involved. The overwhelming and generally damaging inflammatory response of TLRs to aseptic tissue injury may be a consequence of TLR evolution in response to pathogens. In the setting of pathogen invasion, an inflammatory deluge may be the most effective means to clear microorganisms. The activation and influx of leukocytes, with the concomitant release of free radicals and tissue-destroying enzymes, assails not only the invading pathogen but any host cells that harbor the pathogen. However, when this same powerful response is co-opted by the host to clear and resolve tissue damage, it can destroy the very cells it is meant to save. This damage promoting characteristic is prominently observed following brain ischemia, where inflammation plays a critical role in both injury progression and resolution.

### **6.1 TLRS and ischemic damage**

440 Advances in the Preclinical Study of Ischemic Stroke

inflammatory milieu also plays a critical role in regulating TLR expression. Microglia stimulated with CpG specifically up-regulate TLR9, whereas those stimulated with a synthetic TLR3 ligand suppress all TLRs except TLR3 (Olson and Miller, 2004). Similarly, astrocytes stimulated with LPS up-regulate TLRs 2 and 3 but suppress TLR4, while astrocytes exposed to RNA viruses up-regulate TLR3 and TLR9 (McKimmie and Fazakerley, 2005). Thus microglia and astrocytes initiate a layered and multifaceted response to TLR engagement. Oligodendrocytes and endothelial cells express a relatively limited repertoire of TLRs. Oligodendrocytes express TLRs 2 and 3 (Bsibsi et al., 2002), while cerebral endothelial cells constitutively express TLRs 2, 4, and 9 (Constantin et al., 2004) and increase their expression of these TLRs in response to stressful stimuli, including systemic LPS and cerebral ischemia (Singh and Jiang, 2004; Zhou et al., 2007; Ziegler et al., 2007). In response to LPS, endothelial cells up-regulate E-selectin, an NFκB-dependent molecule, and IFNβ, an IRF3-dependent molecule, indicating that these cells utilize the TLR–NFκB and the TLR– IRF3 signaling pathways (Lloyd-Jones et al., 2008). Neurons express TLR3 and generate inflammatory cytokines (TNFα, IL-6), chemokines (CCL5, CXCL10) and antiviral molecules (IFNβ) in response to dsRNA (Lafon et al., 2006; Prehaud et al., 2005). Neurons also employ TLRs in their development and differentiation. TLRs 3 and 8 are expressed on murine neurons early in development and inhibit neurite outgrowth in a MyD88- and NFκBindependent manner (Ma et al., 2006). TLR2 and TLR4 have been found on adult neural progenitor cells where they appear to elicit opposing effects. While TLR2 activation stimulates neuronal differentiation of these cells, TLR4 activation decreases proliferation and neuronal differentiation, driving these cells toward an astrocytic fate (Rolls et al., 2007). Curiously, both TLRs exert these endogenous effects in a MyD88-dependent manner,

Fig. 6. TLRs signaling

A significant portion of the damage associated with stroke injury is due to the resultant inflammatory response. This aspect of the innate immune response is exemplified by the fact that some anti-inflammatory strategies have been shown to ameliorate ischemic damage (Relton et al., 1996; Hara et al., 1997; Spera et al., 1998). The inflammatory

response to stroke is initiated by the detection of injury associated molecules by local cells such as microglia and astrocytes. The response is further promoted by infiltrating neutrophils and macrophages, resulting in the production of inflammatory cytokines, proteolytic enzymes, and other cytotoxic mediators. In the mouse, leukocytes and brain cells (microglia, astrocytes and neurons) express TLRs (Zarember and Godowski, 2002; Olson and Miller, 2004). Hence, injury-associated molecules such as HSP60 and HMGB1 may act as endogenous ligands for TLRs, thereby initiating the damaging inflammatory response to stroke. It is increasingly clear that TLRs do in fact play a role in ischemic damage (Fig.). The pathogenic role of TLRs in ischemic processes was first demonstrated in a mouse model myocardial ischemia/reperfusion injury, because mice lacking functional TLR4 incur less damage than wild type mice (Oyama et al., 2004). Since then, TLR2 has also been shown to cause dysfunction following cardiac ischemia and both have been shown to exacerbate renal ischemic damage, in a MyD88-dependent and a MyD88-independent manner (Sakata et al., 2007; Shigeoka et al., 2007). However, the particular pathway responsible for the damaging effects of TLR activation may differ depending on the cell type or organ affected as TLR4

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 443

shown that LPS preconditioning changes the response of circulating leukocytes to stroke, attenuating stroke-induced neutrophilia, lymphopenia, and monocyte activation. This altered inflammatory response extends into the brain itself. LPS preconditioning attenuates activation of microglia after stroke and reduces neutrophil infiltration into the ischemic hemisphere. Hence, LPS-induced preservation of microvascular function following MCAO may be due to suppressed lymphocyte adhesion to activated endothelium, either by TNFαinduced suppression of endothelial activation and adhesion molecules (Ginis et al., 1999; Ahmed et al., 2000) or by prevention of cellular inflammatory responses to ischemia (Rosenzweig et al., 2004). One hallmark of LPS preconditioning is suppression of cytotoxic TNFα signaling following stroke. Mice that have been preconditioned with LPS prior to ischemia display a pronounced suppression of the TNFα pathway following stroke, as evinced by reduced TNFα in the serum, decreased levels of cellular TNFR1, and enhanced levels of neutralizing soluble-TNFR1. These mice are thus protected from the cytotoxic effects of TNFα after cerebral ischemia (Rosenzweig et al., 2007). Collectively, these mechanisms lead to a muted TNFα response to ischemic injury and increased cell survival. Recently a new TLR ligand has been shown to induce tolerance to brain ischemia. As with TLR4 and L PS, stimul ation of TLR9 by systemic all y ad mi nistered Cp G oligodeoxynucleotides induces robust protection against brain ischemia in a time and dose dependent manner. CpG pretreatment protects neurons in both in vivo and in vitro models of stroke (Stevens et al., 2008). Notably, the protection afforded by CpG depends on TNFα, as systemic CpG administration acutely and significantly increases serum TNFα, and TNFα knockout mice fail to be protected by CpG preconditioning. Similarities among the known TLR signaling pathways and their shared ability to induce TNFα, itself a potent preconditioning stimulus, suggest that stimulation of TLR4 and TLR9 may induce ischemic tolerance by similar means. The neuroprotective potential of other TLRs has yet to be explored, but this family of molecules may be a rich source of therapeutic targets. The finding that TLRs are mediators of ischemic injury provides insight into the potential mechanisms of LPS- and CpG-induced neuroprotection. In fact, TLR-induced tolerance to subsequent ischemia may occur by the same mechanisms that govern a very similar phenomenon—that of LPS-induced tolerance to subsequent LPS exposure. The latter phenomenon is known as "endotoxin tolerance" and occurs when pretreatment with a low dose of LPS renders cells or whole animals tolerant to the normally detrimental effects of a second, higher dose of LPS. Cells that are tolerant to LPS are defined by their inability to generate TNFα in response to TLR4 activation. Upon TLR4 ligation, LPS tolerant cells, unlike naive cells, do not recruit MyD88 to TLR4, and fail to activate IRAK-1 and NFκB (Medvedev et al., 2002). The TLR4–NFκB signaling axis becomes decommissioned following a primary exposure to LPS via an elaborate negative feedback loop that involves known inhibitors of TLR signaling. Among those inhibitors are Ship-1, which prevents TLR4- MyD88 interaction, IRAK-M, a non-functional IRAK decoy, and TRIM30α, which destabilized the TAK1 complex (Kobayashi et al., 2002; Sly et al., 2004; Shi et al., 2008). Thus, subsequent signaling of TLR4 to NFκB is blocked and inflammatory cytokine production is suppressed. Conversely, secondary exposure causes enhanced IFNβ release, suggesting increased signaling via the TLR4-IRF3 axis (Broad et al., 2007). Thus, pretreatment with LPS causes cells to switch their transcriptional response to TLR4 stimulation by enhancing the IRF3- induced cytokine IFNβ and suppressing the NFκB-induced cytokine TNFα. Similar to

worsens ischemic damage following liver transplant in a MyD88-independent, IRF3 dependent fashion (Zhai et al., 2004; Shen et al., 2005). Importantly, TLR2 and TLR4 have been shown to play a role in cerebral ischemic damage. Mice lacking either functional TLR2 or TLR4 are less susceptible to transient focal cerebral ischemia/reperfusion damage, demonstrating smaller infarcts than wild type controls (Cao et al., 2007; Lehnardt et al., 2007; Ziegler et al., 2007). Further, mice lacking TLR4 incur less damage following global cerebral ischemia and permanent focal ischemia (Caso et al., 2007; Hua et al., 2007). The TLR endogenous ligands HSP 60, HSP70 and HMGB1 are found in the brain following injury (Kinouchi et al., 1993; Faraco et al., 2007; Lehnardt et al., 2008). Hence these molecules may activate TLR2 and TLR4 within the brain itself, leading to the generation of inflammatory mediators such as TNFα, IL1, IL6, and iNOS, all known to be associated with stroke damage.

### **6.2 TLRS and neuroprotection**

In contrast to the detrimental role of TLRs in response to ischemia, stimulation of these receptors prior to ischemia provides robust neuroprotection. TLR4-induced tolerance to cerebral ischemia was first demonstrated with low dose systemic administration of LPS (endotoxin), a cell wall component of gram-negative bacteria, which caused spontaneously hypertensive rats to become tolerant to subsequent ischemic brain damage induced by middle cerebral artery occlusion (MCAO) (Tasaki et al., 1997). Since then, LPS-induced tolerance to brain ischemia has been demonstrated in a mouse model of stroke and in a porcine model of deep hypothermic circulatory arrest (Rosenzweig et al., 2004; Hickey et al., 2007) (for additional information on the dual effects of neuro-immune crosstalk, please refer to Kerschensteiner et al., in this issue). Neuroprotection induced by LPS is time and dose dependent. Tolerance appears by 24 h after LPS administration and extends out to 7 days but is gone by 14 days (Rosenzweig et al., 2007). Protective doses of LPS appear to depend on the animal model of stroke and the route of systemic administration, ranging from 0.02–1 mg/kg (Tasaki et al., 1997; Ahmed et al., 2000; Bordet et al., 2000; Furuya et al., 2005; Hickey et al., 2007; Kunz et al., 2007; Rosenzweig et al., 2007). Tolerance induction has been shown to require new protein synthesis and a modest inflammatory response, as it can be blocked by prior administration of cycloheximide or dexamethasone (Bordet et al., 2000). Specifically, TNFα has been implicated as a mediator of LPS-induced ischemic tolerance because inhibition of TNFα systemically (Tasaki et al., 1997) or within the brain (Rosenzweig et al., 2007) blocks neuroprotection, and mice lacking TNFα fail to be protected by LPS preconditioning (Rosenzweig et al., 2007). In addition to its neuroprotective effects, LPS preconditioning has vasculoprotective efficacy. Nitric oxide appears to play a critical role in the protective effects of LPS. Mice lacking iNOS expression fail to be protected by LPS pretreatment (Kunz et al., 2007), and eNOS expression within the brain is directly correlated to the time window of LPS-induced neuroprotection (Furuya et al., 2005). LPS pretreatment has further been shown to prevent the impairment of endothelial and smooth muscle relaxation normally induced by ischemia/reperfusion injury (Bastide et al., 2003), resulting in normalization of cerebral blood flow in peri-infarct regions lasting out to 24 h after MCAO (Dawson et al., 1999; Furuya et al., 2005). LPS-induced ischemic protection requires an inflammatory response prior to the ischemic event, yet protection occurs through modulation of the inflammatory response following ischemia. Rosenzweig et al. (2004) have

worsens ischemic damage following liver transplant in a MyD88-independent, IRF3 dependent fashion (Zhai et al., 2004; Shen et al., 2005). Importantly, TLR2 and TLR4 have been shown to play a role in cerebral ischemic damage. Mice lacking either functional TLR2 or TLR4 are less susceptible to transient focal cerebral ischemia/reperfusion damage, demonstrating smaller infarcts than wild type controls (Cao et al., 2007; Lehnardt et al., 2007; Ziegler et al., 2007). Further, mice lacking TLR4 incur less damage following global cerebral ischemia and permanent focal ischemia (Caso et al., 2007; Hua et al., 2007). The TLR endogenous ligands HSP 60, HSP70 and HMGB1 are found in the brain following injury (Kinouchi et al., 1993; Faraco et al., 2007; Lehnardt et al., 2008). Hence these molecules may activate TLR2 and TLR4 within the brain itself, leading to the generation of inflammatory mediators such as TNFα, IL1, IL6, and iNOS, all known to be associated with stroke

In contrast to the detrimental role of TLRs in response to ischemia, stimulation of these receptors prior to ischemia provides robust neuroprotection. TLR4-induced tolerance to cerebral ischemia was first demonstrated with low dose systemic administration of LPS (endotoxin), a cell wall component of gram-negative bacteria, which caused spontaneously hypertensive rats to become tolerant to subsequent ischemic brain damage induced by middle cerebral artery occlusion (MCAO) (Tasaki et al., 1997). Since then, LPS-induced tolerance to brain ischemia has been demonstrated in a mouse model of stroke and in a porcine model of deep hypothermic circulatory arrest (Rosenzweig et al., 2004; Hickey et al., 2007) (for additional information on the dual effects of neuro-immune crosstalk, please refer to Kerschensteiner et al., in this issue). Neuroprotection induced by LPS is time and dose dependent. Tolerance appears by 24 h after LPS administration and extends out to 7 days but is gone by 14 days (Rosenzweig et al., 2007). Protective doses of LPS appear to depend on the animal model of stroke and the route of systemic administration, ranging from 0.02–1 mg/kg (Tasaki et al., 1997; Ahmed et al., 2000; Bordet et al., 2000; Furuya et al., 2005; Hickey et al., 2007; Kunz et al., 2007; Rosenzweig et al., 2007). Tolerance induction has been shown to require new protein synthesis and a modest inflammatory response, as it can be blocked by prior administration of cycloheximide or dexamethasone (Bordet et al., 2000). Specifically, TNFα has been implicated as a mediator of LPS-induced ischemic tolerance because inhibition of TNFα systemically (Tasaki et al., 1997) or within the brain (Rosenzweig et al., 2007) blocks neuroprotection, and mice lacking TNFα fail to be protected by LPS preconditioning (Rosenzweig et al., 2007). In addition to its neuroprotective effects, LPS preconditioning has vasculoprotective efficacy. Nitric oxide appears to play a critical role in the protective effects of LPS. Mice lacking iNOS expression fail to be protected by LPS pretreatment (Kunz et al., 2007), and eNOS expression within the brain is directly correlated to the time window of LPS-induced neuroprotection (Furuya et al., 2005). LPS pretreatment has further been shown to prevent the impairment of endothelial and smooth muscle relaxation normally induced by ischemia/reperfusion injury (Bastide et al., 2003), resulting in normalization of cerebral blood flow in peri-infarct regions lasting out to 24 h after MCAO (Dawson et al., 1999; Furuya et al., 2005). LPS-induced ischemic protection requires an inflammatory response prior to the ischemic event, yet protection occurs through modulation of the inflammatory response following ischemia. Rosenzweig et al. (2004) have

damage.

**6.2 TLRS and neuroprotection** 

shown that LPS preconditioning changes the response of circulating leukocytes to stroke, attenuating stroke-induced neutrophilia, lymphopenia, and monocyte activation. This altered inflammatory response extends into the brain itself. LPS preconditioning attenuates activation of microglia after stroke and reduces neutrophil infiltration into the ischemic hemisphere. Hence, LPS-induced preservation of microvascular function following MCAO may be due to suppressed lymphocyte adhesion to activated endothelium, either by TNFαinduced suppression of endothelial activation and adhesion molecules (Ginis et al., 1999; Ahmed et al., 2000) or by prevention of cellular inflammatory responses to ischemia (Rosenzweig et al., 2004). One hallmark of LPS preconditioning is suppression of cytotoxic TNFα signaling following stroke. Mice that have been preconditioned with LPS prior to ischemia display a pronounced suppression of the TNFα pathway following stroke, as evinced by reduced TNFα in the serum, decreased levels of cellular TNFR1, and enhanced levels of neutralizing soluble-TNFR1. These mice are thus protected from the cytotoxic effects of TNFα after cerebral ischemia (Rosenzweig et al., 2007). Collectively, these mechanisms lead to a muted TNFα response to ischemic injury and increased cell survival. Recently a new TLR ligand has been shown to induce tolerance to brain ischemia. As with TLR4 and L PS, stimul ation of TLR9 by systemic all y ad mi nistered Cp G oligodeoxynucleotides induces robust protection against brain ischemia in a time and dose dependent manner. CpG pretreatment protects neurons in both in vivo and in vitro models of stroke (Stevens et al., 2008). Notably, the protection afforded by CpG depends on TNFα, as systemic CpG administration acutely and significantly increases serum TNFα, and TNFα knockout mice fail to be protected by CpG preconditioning. Similarities among the known TLR signaling pathways and their shared ability to induce TNFα, itself a potent preconditioning stimulus, suggest that stimulation of TLR4 and TLR9 may induce ischemic tolerance by similar means. The neuroprotective potential of other TLRs has yet to be explored, but this family of molecules may be a rich source of therapeutic targets. The finding that TLRs are mediators of ischemic injury provides insight into the potential mechanisms of LPS- and CpG-induced neuroprotection. In fact, TLR-induced tolerance to subsequent ischemia may occur by the same mechanisms that govern a very similar phenomenon—that of LPS-induced tolerance to subsequent LPS exposure. The latter phenomenon is known as "endotoxin tolerance" and occurs when pretreatment with a low dose of LPS renders cells or whole animals tolerant to the normally detrimental effects of a second, higher dose of LPS. Cells that are tolerant to LPS are defined by their inability to generate TNFα in response to TLR4 activation. Upon TLR4 ligation, LPS tolerant cells, unlike naive cells, do not recruit MyD88 to TLR4, and fail to activate IRAK-1 and NFκB (Medvedev et al., 2002). The TLR4–NFκB signaling axis becomes decommissioned following a primary exposure to LPS via an elaborate negative feedback loop that involves known inhibitors of TLR signaling. Among those inhibitors are Ship-1, which prevents TLR4- MyD88 interaction, IRAK-M, a non-functional IRAK decoy, and TRIM30α, which destabilized the TAK1 complex (Kobayashi et al., 2002; Sly et al., 2004; Shi et al., 2008). Thus, subsequent signaling of TLR4 to NFκB is blocked and inflammatory cytokine production is suppressed. Conversely, secondary exposure causes enhanced IFNβ release, suggesting increased signaling via the TLR4-IRF3 axis (Broad et al., 2007). Thus, pretreatment with LPS causes cells to switch their transcriptional response to TLR4 stimulation by enhancing the IRF3- induced cytokine IFNβ and suppressing the NFκB-induced cytokine TNFα. Similar to

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 445

stroke-induced IRF signaling. We suggest that pretreatment with TLR ligands reprograms the brain's response to ischemia and alters endogenous stroke-induced TLR signaling by suppression of the NFκB-inducing pathway and upregulation of the IRF-inducing pathway. Reprogramming causes a finely controlled shift in the balance of proinflammatory and antiinflammatory cytokines, and represents an endogenously orchestrated mechanism that protects the organism from additional damage. We further suggest that reprogramming of endogenous TLR signaling, with the subsequent generation of neuroprotective type I IFNs, is a unifying property of the neuroprotected phenotype. The brain has evolved numerous mechanisms that allow it to withstand the shortage of energy and the oxidative stress caused by ischemia. This tolerant state can be induced by prior exposure to LPS or CpG, or by prior exposure to other non-damaging (i.e. sub-threshold) noxious stimuli. For example, mild exposure to ischemia, excitotoxic stimuli, or inflammatory mediators can precondition the brain to better tolerate a subsequent injurious ischemic event. These mild preconditioning exposures herald impending danger and, as such, induce endogenous protective strategies in anticipation of injury. Though the final outcome of tolerance induction is the same—protection of brain tissue from ischemic injury—the effector mechanisms employed by the brain are as diverse as the preconditioning stimuli that induced them. In fact, the phenotype of neuroprotection may be specifically tailored by the nature of the preconditioning stimulus (Stenzel-Poore et al., 2007). For example, preconditioning events that deprive the brain of oxygen or glucose for a short time lead to conservation of energy regulation and mitochondrial integrity during the injurious ischemic episode (Stenzel-Poore et al., 2003; McFalls et al., 2006). Further, as we have described above, preconditioning events that invoke a small inflammatory response lead to altered inflammatory responses to damaging ischemia (Rosenzweig et al., 2004, 2007). It should be emphasized that although significant overlap exists in the cellular processes induced by these diverse stimuli, the pathways that dominate each response are distinct. The first demonstration that a short period of oxygen deprivation could protect the brain from a subsequent extended period of hypoxia occurred in 1943 (Noble, 1943). Since then, hundreds of studies have been undertaken to better understand the underlying mechanisms of "ischemic preconditioning." Though several endogenously protective pathways are induced by the initiating ischemic event, one particular theme is emerging—that of mitochondrial maintenance and energy conservation (Dirnagl and Meisel, 2008). The priming ischemic episode appears to induce cellular pathways that protect mitochondria against stroke induced deficits in the electron transport chain (Dave et al., 2001). These pathways protect mitochondrial membrane potential (Wu et al., 2004), preserve mitochondrial cytochrome c (Zhan et al., 2002), increase mitochondrial sequestration of Ca+ and increase Ca+-ATPase activity. In addition, ischemic preconditioning appears to suppress molecules that regulate ion channels, leading to channel arrest—i.e. reduction in ion permeability through the plasma membrane—which has been shown to reduce the amount of ATP required to maintain ionic homeostasis (Buck and Hochachka, 1993; Stenzel-Poore et al., 2003). Finally, a decrease in the overall cellular metabolic rate limits the stressful effects of oxygen deprivation. The pre preconditioning stimulus suppresses the expression of genes involved in protein turnover, proteasomal degradation, and energy metabolism (Stenzel-Poore et al., 2003). Although ischemic preconditioning has also been shown to help maintain protein structure and function and to suppress the damaging inflammatory response to

endotoxin tolerance, priming TLR9 with its ligand, CpG, induces a state of hyporesponsiveness to subsequent challenge with CpGs (Dalpke et al., 2005). Interestingly, crosstolerance between the two receptors has also been reported, as ligands for TLR9 induce tolerance against a subsequent challenge with a TLR4 ligand (Bagchi et al., 2007; Broad et al., 2007). CpG-pretreated cells not only produce less TNFα when secondarily challenged with LPS, they also produce significantly higher levels of IFNβ (Broad et al., 2007). Together, the aforementioned studies suggest the intriguing possibility that TLR stimulation prior to stroke may reprogram ischemia-induced TLR activation (Fig.). Specifically, administration of LPS or CpG may activate TLR4 and TLR9, respectively, causing a small inflammatory response, with an initial rise in TNFα. Cells would then regulate their inflammatory response through expression of negative feedback inhibitors of the TLR4–NFκB signaling axis that remain present when cells are subsequently exposed to endogenous TLR ligands generated from ischemia-injured tissue. Within this new cellular environment, stimulated TLRs such as TLR2 and TLR4 would be unable to activate NFκB-inducing pathways. Because of this, stroke-induced TLR2 signaling may be blocked completely leading to reduced injury, and stroke-induced TLR4 signaling would shift from NFκB induction to IRF3 induction (Fig.). Suppression of NFκB induction would be expected to protect the brain, as mice lacking the p50 subunit of NFκB suffer less cerebral ischemic damage than wild type mice (Schneider et al., 1999). Enhancement of IRF signaling would also be expected to protect the brain, as IFNβ, a downstream product of IRF3 induction, has been shown to act as an acute neuroprotectant (Liu et al., 2002; Veldhuis et al., 2003a). IFNβ, best known for its anti-viral effects, has potent anti-inflammatory activities as well. Several studies have shown that IFNβ can stabilize the blood–brain barrier, potentially by reducing matrix metalloprotease production by activated glia (Veldhuis et al., 2003b; Kraus et al., 2004; Liuzzi et al., 2004). Similarly, it has been shown to inhibit monocyte migration across human brainderived endothelial cells (Seguin et al., 2003) and reduce cellular infiltration into damaged brain regions (Veldhuis et al., 2003b). On a cellular level, IFNβ has been shown to reduce reactive oxygen species (Lopez-Collazo et al., 1998; Stewart et al., 1998; Hua et al., 2002), suppress inflammatory cytokine production and induce IL-1Ra (Bosca et al., 2000; Palmer et al., 2004), promote nerve growth factor production by astrocytes (Boutros et al., 1997) and protect neurons from toxicity induced by activated microglia (Jin et al., 2007). In addition, systemic administration of IFNβ has been shown to reduce infarct damage in rat and rabbit models of ischemic stroke (Liu et al., 2002; Veldhuis et al., 2003a). Therefore, in the setting of LPS preconditioning, upregulation of this cytokine following stroke would be expected to contribute to neuroprotection. IFNβ may not be the only neuroprotective molecule downstream of IRF signaling. TLR3 signals exclusively through the TRIFdependent pathway and stimulation of TLR3 in human astrocyte cultures induces the expression of several neuroprotective molecules such as brainderived neurotrophic factor, neurotrophin 4, pleiotrophin, and TGFβ2 (Bsibsi et al., 2006), all of which have been implicated in endogenous neuroprotection (Yeh et al., 1998; Endres et al., 2000; Zhang et al., 2005). Astrocytic TLR3 stimulation also results in production of the anti-inflammatory cytokine IL-10 (Bsibsi et al., 2006). Conditioned media from these cultures enhance neuronal survival and suppress astrocyte growth in slice cultures. Interestingly, LPS stimulation of macrophages has been shown to upregulate TLR3 expression (Nhu et al., 2006), inviting the possibility that LPS preconditioning may upregulate TLR3 in the brain, further enhancing

endotoxin tolerance, priming TLR9 with its ligand, CpG, induces a state of hyporesponsiveness to subsequent challenge with CpGs (Dalpke et al., 2005). Interestingly, crosstolerance between the two receptors has also been reported, as ligands for TLR9 induce tolerance against a subsequent challenge with a TLR4 ligand (Bagchi et al., 2007; Broad et al., 2007). CpG-pretreated cells not only produce less TNFα when secondarily challenged with LPS, they also produce significantly higher levels of IFNβ (Broad et al., 2007). Together, the aforementioned studies suggest the intriguing possibility that TLR stimulation prior to stroke may reprogram ischemia-induced TLR activation (Fig.). Specifically, administration of LPS or CpG may activate TLR4 and TLR9, respectively, causing a small inflammatory response, with an initial rise in TNFα. Cells would then regulate their inflammatory response through expression of negative feedback inhibitors of the TLR4–NFκB signaling axis that remain present when cells are subsequently exposed to endogenous TLR ligands generated from ischemia-injured tissue. Within this new cellular environment, stimulated TLRs such as TLR2 and TLR4 would be unable to activate NFκB-inducing pathways. Because of this, stroke-induced TLR2 signaling may be blocked completely leading to reduced injury, and stroke-induced TLR4 signaling would shift from NFκB induction to IRF3 induction (Fig.). Suppression of NFκB induction would be expected to protect the brain, as mice lacking the p50 subunit of NFκB suffer less cerebral ischemic damage than wild type mice (Schneider et al., 1999). Enhancement of IRF signaling would also be expected to protect the brain, as IFNβ, a downstream product of IRF3 induction, has been shown to act as an acute neuroprotectant (Liu et al., 2002; Veldhuis et al., 2003a). IFNβ, best known for its anti-viral effects, has potent anti-inflammatory activities as well. Several studies have shown that IFNβ can stabilize the blood–brain barrier, potentially by reducing matrix metalloprotease production by activated glia (Veldhuis et al., 2003b; Kraus et al., 2004; Liuzzi et al., 2004). Similarly, it has been shown to inhibit monocyte migration across human brainderived endothelial cells (Seguin et al., 2003) and reduce cellular infiltration into damaged brain regions (Veldhuis et al., 2003b). On a cellular level, IFNβ has been shown to reduce reactive oxygen species (Lopez-Collazo et al., 1998; Stewart et al., 1998; Hua et al., 2002), suppress inflammatory cytokine production and induce IL-1Ra (Bosca et al., 2000; Palmer et al., 2004), promote nerve growth factor production by astrocytes (Boutros et al., 1997) and protect neurons from toxicity induced by activated microglia (Jin et al., 2007). In addition, systemic administration of IFNβ has been shown to reduce infarct damage in rat and rabbit models of ischemic stroke (Liu et al., 2002; Veldhuis et al., 2003a). Therefore, in the setting of LPS preconditioning, upregulation of this cytokine following stroke would be expected to contribute to neuroprotection. IFNβ may not be the only neuroprotective molecule downstream of IRF signaling. TLR3 signals exclusively through the TRIFdependent pathway and stimulation of TLR3 in human astrocyte cultures induces the expression of several neuroprotective molecules such as brainderived neurotrophic factor, neurotrophin 4, pleiotrophin, and TGFβ2 (Bsibsi et al., 2006), all of which have been implicated in endogenous neuroprotection (Yeh et al., 1998; Endres et al., 2000; Zhang et al., 2005). Astrocytic TLR3 stimulation also results in production of the anti-inflammatory cytokine IL-10 (Bsibsi et al., 2006). Conditioned media from these cultures enhance neuronal survival and suppress astrocyte growth in slice cultures. Interestingly, LPS stimulation of macrophages has been shown to upregulate TLR3 expression (Nhu et al., 2006), inviting the possibility that LPS preconditioning may upregulate TLR3 in the brain, further enhancing

stroke-induced IRF signaling. We suggest that pretreatment with TLR ligands reprograms the brain's response to ischemia and alters endogenous stroke-induced TLR signaling by suppression of the NFκB-inducing pathway and upregulation of the IRF-inducing pathway. Reprogramming causes a finely controlled shift in the balance of proinflammatory and antiinflammatory cytokines, and represents an endogenously orchestrated mechanism that protects the organism from additional damage. We further suggest that reprogramming of endogenous TLR signaling, with the subsequent generation of neuroprotective type I IFNs, is a unifying property of the neuroprotected phenotype. The brain has evolved numerous mechanisms that allow it to withstand the shortage of energy and the oxidative stress caused by ischemia. This tolerant state can be induced by prior exposure to LPS or CpG, or by prior exposure to other non-damaging (i.e. sub-threshold) noxious stimuli. For example, mild exposure to ischemia, excitotoxic stimuli, or inflammatory mediators can precondition the brain to better tolerate a subsequent injurious ischemic event. These mild preconditioning exposures herald impending danger and, as such, induce endogenous protective strategies in anticipation of injury. Though the final outcome of tolerance induction is the same—protection of brain tissue from ischemic injury—the effector mechanisms employed by the brain are as diverse as the preconditioning stimuli that induced them. In fact, the phenotype of neuroprotection may be specifically tailored by the nature of the preconditioning stimulus (Stenzel-Poore et al., 2007). For example, preconditioning events that deprive the brain of oxygen or glucose for a short time lead to conservation of energy regulation and mitochondrial integrity during the injurious ischemic episode (Stenzel-Poore et al., 2003; McFalls et al., 2006). Further, as we have described above, preconditioning events that invoke a small inflammatory response lead to altered inflammatory responses to damaging ischemia (Rosenzweig et al., 2004, 2007). It should be emphasized that although significant overlap exists in the cellular processes induced by these diverse stimuli, the pathways that dominate each response are distinct. The first demonstration that a short period of oxygen deprivation could protect the brain from a subsequent extended period of hypoxia occurred in 1943 (Noble, 1943). Since then, hundreds of studies have been undertaken to better understand the underlying mechanisms of "ischemic preconditioning." Though several endogenously protective pathways are induced by the initiating ischemic event, one particular theme is emerging—that of mitochondrial maintenance and energy conservation (Dirnagl and Meisel, 2008). The priming ischemic episode appears to induce cellular pathways that protect mitochondria against stroke induced deficits in the electron transport chain (Dave et al., 2001). These pathways protect mitochondrial membrane potential (Wu et al., 2004), preserve mitochondrial cytochrome c (Zhan et al., 2002), increase mitochondrial sequestration of Ca+ and increase Ca+-ATPase activity. In addition, ischemic preconditioning appears to suppress molecules that regulate ion channels, leading to channel arrest—i.e. reduction in ion permeability through the plasma membrane—which has been shown to reduce the amount of ATP required to maintain ionic homeostasis (Buck and Hochachka, 1993; Stenzel-Poore et al., 2003). Finally, a decrease in the overall cellular metabolic rate limits the stressful effects of oxygen deprivation. The pre preconditioning stimulus suppresses the expression of genes involved in protein turnover, proteasomal degradation, and energy metabolism (Stenzel-Poore et al., 2003). Although ischemic preconditioning has also been shown to help maintain protein structure and function and to suppress the damaging inflammatory response to

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 447

(Schmidt et al, 1995; Vlassara et al, 1994; Bierhaus et al, 1998; Baynes, 2003; Thornalley, 1998; Brownlee, 2000). Glycation of macromolecules was originally thought to mark senescent proteins for subsequent degradation by macrophages. Receptors binding AGEs were regarded as scavenger receptors involved in AGE disposal and cell regeneration, and defective clearance of such modified proteins was believed to be important in aging and diseases with accelerated AGE-formation, such as diabetes or atherosclerosis (Vlassara et al,1994, 1985). However, when the receptor for AGEs (RAGE) was cloned and first characterized (Neeper et al,1992; Schmidt et al,1992, 1994) it turned out that binding of AGEs to RAGE did not accelerate their clearance and degradation. Rather, ligand–receptor interaction induced sustained post-receptor signaling, including activation of p21ras, MAP kinases, and the NF-κB pathway (Lander et al, 1997; Basta et al, 2002; Bucciarelli et al, 2002). Thus, the concept of RAGE as a scavenger/clearance receptor has to be revised and

RAGE is a member of the immunoglobulin superfamily of cell surface molecules (Schmidt et al, 1993; Sugaya, 1994). The gene is localized on chromosome 6 near the HLA locus in the vicinity of the MHCIII complex in humans and mice, in close proximity to the homeobox gene HOX12 and the human counterpart of the mouse mammary tumor gene int-3 (Malherbe et al, 1999). The receptor is composed of three immunoglobulin-like regions: one "V"- type domain and two "C"-type-domains, a short transmembrane domain, and a 43-amino acid cytoplasmic tail (Neeper et al, 1992; Schmidt et al, 1994; Lander et al, 1997). While the "V-type" domain confers ligand binding, the cytoplasmic tail is critical for intracellular signaling. Shortly after RAGE was recognized as a receptor for AGEs, it became evident that a number of other ligands also interacted with the receptor (Bucciarelli et al, 2002; Schmidt et al, 2001; Du Yan et al, 1997; Yan et al, 1996, 2000). Structural analysis of ligand–RAGE interaction revealed that the receptor recognized three-dimensional structures, such as β-sheets and fibrils, rather than specific amino acid sequences (i.e., primary structure) (Bucciarelli et al, 2002; Schmidt et al, 2001). In addition to AGEs, RAGE binds amyloid-β peptide (accumulating in Alzheimer's disease) (Du Yan et al, 1997; Yan et al, 2000) and amyloid A (accumulating in systemic amyloidosis). Further, ligands of RAGE are S100/calgranulins, a family of closely related calcium-binding polypeptides that accumulate extracellularly at sites of chronic inflammation (Hofmann et al, 1999; Marenholz et al, 2004). Another proinflammatory ligand of RAGE is the DNA binding protein HMGB1 (amphoterin), which is released by cells undergoing necrosis (Hori et al, 1995; Wang et al, 1999; Anderson and Tracey, 2003; Treutiger et al, 2003). Besides binding ligands actively participating in chronic inflammatory and immune responses, RAGE also interacts with surface molecules on bacteria (Chapman et al, 2002), prions (Sasaki et al, 2002), and leukocytes (Chavakis et al, 2003). Thus, RAGE is much more than a receptor for AGEs; it has a broad repertoire of ligands, which share the propensity to accumulate in tissues during aging, chronic degenerative diseases, inflammation and the host response (Tretiger et al,2003). Therefore, RAGE should be considered a pattern recognition receptor (PRR) (Schmidt et al, 2001; Chavakis et al, 2003; Liliensiek et al, 2004; Gordon, 2002), and potential similarities to members of the family of Toll-like receptors should be considered (Akira et al, 2001).

extended.

**7.1 RAGE: Structure and ligand recognition** 

stroke, it is increasingly clear that sustaining mitochondrial integrity and conserving energy are important mechanisms driving endogenous ischemic tolerance. Several studies have shown that the priming ischemic event induces HSP70 within the brain (Truettner et al., 2002). In addition to its role in stabilizing protein structure, HSP70 acts as an endogenous ligand of TLR4. In fact, extracellular HSP70 has been shown to induce endotoxin tolerance (Aneja et al., 2006). Hence TLRs may be stimulated in the course of ischemic preconditioning, resulting in a reprogrammed TLR response to subsequent injurious ischemia. One of the molecular consequences of reprogrammed TLR signaling is an increase in IFNβ. Notably, IFNβ has been shown to aid in the maintenance of mitochondrial integrity. For example, treatment of astrocytes with IFNβ prevents neuronal mitochondrial respiratory chain damage (Stewart et al., 1998) and reduce IFNβ induced nitric oxide synthase (Stewart et al., 1997). Thus reprogrammed TLR signaling may help shape the phenotype of ischemia-induced tolerance The phenomenon of inflammation-induced crosstolerance to ischemia is not limited to LPS, but extends to TNFα as well. Nawashiro et al. (1997) were the first to demonstrate that intracisternal administration of TNFα protects the brain from subsequent ischemic challenge. This protection is correlated to a decrease in CD11b immunoreactivity, suggesting a decrease in the inflammatory response to ischemia in the setting of preconditioning. Consistent with this observation, TNFα pretreatment of astrocytes and endothelial cells, through its signaling intermediate ceramide, produces a state of hypo-responsiveness as pretreated cells fail to upregulate ICAM-1 during subsequent hypoxia (Ginis et al., 2002). The decrease in ICAM-1 does not reflect global cellular suppression, but instead signifies a reprogrammed genomic response to stroke, as the hypoxiainduced expression of cytoprotective MnSOD is not affected by preconditioning. Evidence for a reprogrammed genomic response to ischemia is supported by the observation that TNFα preconditioning prevents hypoxia-induced phosphorylation of the proinflammatory transcription factor component NFκBp65, thereby preventing its interaction with the transcriptional activator p300. Taken together, these data indicate that pretreatment with TNFα reprograms the cellular environment and hence alters inflammatory reactions in response to ischemia. Just as TNFα can induce tolerance to subsequent ischemic exposure, it can induce tolerance to subsequent LPS exposure (Porter et al., 1998; Ferlito et al., 2001; Murphey and Traber, 2001). Hence TNFα preconditioning has the potential to induce a state of cross-tolerance to TLR ligands, and thereby reprogram the TLR response to stroke. IFNβ has been shown to cause many of the effects observed in TNFα-induced ischemic tolerance, such as suppression of inflammatory cytokine production, including TNFα itself, and reduction of cellular infiltration into ischemic brain regions (Veldhuis et al., 2003a). Together, these studies suggest that multiple preconditioning stimuli may cause a reprogrammed TLR response to stroke. IFNβ, produced secondary to this reprogrammed response, may aid in maintaining mitochondrial stability and in dampening the inflammatory responses to injurious ischemia.

### **7. The receptor for Advanced Glycation End Products (RAGE)**

Advanced glycation end products (AGEs) are nonenzymatical adducts of proteins, lipids, and nucleic acids which form in a time-dependent manner in a pro-oxidant environment, especially when target molecules turnover slowly and the level of aldoses is elevated (Schmidt et al, 1995; Vlassara et al, 1994; Bierhaus et al, 1998; Baynes, 2003; Thornalley, 1998; Brownlee, 2000). Glycation of macromolecules was originally thought to mark senescent proteins for subsequent degradation by macrophages. Receptors binding AGEs were regarded as scavenger receptors involved in AGE disposal and cell regeneration, and defective clearance of such modified proteins was believed to be important in aging and diseases with accelerated AGE-formation, such as diabetes or atherosclerosis (Vlassara et al,1994, 1985). However, when the receptor for AGEs (RAGE) was cloned and first characterized (Neeper et al,1992; Schmidt et al,1992, 1994) it turned out that binding of AGEs to RAGE did not accelerate their clearance and degradation. Rather, ligand–receptor interaction induced sustained post-receptor signaling, including activation of p21ras, MAP kinases, and the NF-κB pathway (Lander et al, 1997; Basta et al, 2002; Bucciarelli et al, 2002). Thus, the concept of RAGE as a scavenger/clearance receptor has to be revised and extended.

### **7.1 RAGE: Structure and ligand recognition**

446 Advances in the Preclinical Study of Ischemic Stroke

stroke, it is increasingly clear that sustaining mitochondrial integrity and conserving energy are important mechanisms driving endogenous ischemic tolerance. Several studies have shown that the priming ischemic event induces HSP70 within the brain (Truettner et al., 2002). In addition to its role in stabilizing protein structure, HSP70 acts as an endogenous ligand of TLR4. In fact, extracellular HSP70 has been shown to induce endotoxin tolerance (Aneja et al., 2006). Hence TLRs may be stimulated in the course of ischemic preconditioning, resulting in a reprogrammed TLR response to subsequent injurious ischemia. One of the molecular consequences of reprogrammed TLR signaling is an increase in IFNβ. Notably, IFNβ has been shown to aid in the maintenance of mitochondrial integrity. For example, treatment of astrocytes with IFNβ prevents neuronal mitochondrial respiratory chain damage (Stewart et al., 1998) and reduce IFNβ induced nitric oxide synthase (Stewart et al., 1997). Thus reprogrammed TLR signaling may help shape the phenotype of ischemia-induced tolerance The phenomenon of inflammation-induced crosstolerance to ischemia is not limited to LPS, but extends to TNFα as well. Nawashiro et al. (1997) were the first to demonstrate that intracisternal administration of TNFα protects the brain from subsequent ischemic challenge. This protection is correlated to a decrease in CD11b immunoreactivity, suggesting a decrease in the inflammatory response to ischemia in the setting of preconditioning. Consistent with this observation, TNFα pretreatment of astrocytes and endothelial cells, through its signaling intermediate ceramide, produces a state of hypo-responsiveness as pretreated cells fail to upregulate ICAM-1 during subsequent hypoxia (Ginis et al., 2002). The decrease in ICAM-1 does not reflect global cellular suppression, but instead signifies a reprogrammed genomic response to stroke, as the hypoxiainduced expression of cytoprotective MnSOD is not affected by preconditioning. Evidence for a reprogrammed genomic response to ischemia is supported by the observation that TNFα preconditioning prevents hypoxia-induced phosphorylation of the proinflammatory transcription factor component NFκBp65, thereby preventing its interaction with the transcriptional activator p300. Taken together, these data indicate that pretreatment with TNFα reprograms the cellular environment and hence alters inflammatory reactions in response to ischemia. Just as TNFα can induce tolerance to subsequent ischemic exposure, it can induce tolerance to subsequent LPS exposure (Porter et al., 1998; Ferlito et al., 2001; Murphey and Traber, 2001). Hence TNFα preconditioning has the potential to induce a state of cross-tolerance to TLR ligands, and thereby reprogram the TLR response to stroke. IFNβ has been shown to cause many of the effects observed in TNFα-induced ischemic tolerance, such as suppression of inflammatory cytokine production, including TNFα itself, and reduction of cellular infiltration into ischemic brain regions (Veldhuis et al., 2003a). Together, these studies suggest that multiple preconditioning stimuli may cause a reprogrammed TLR response to stroke. IFNβ, produced secondary to this reprogrammed response, may aid in maintaining mitochondrial

stability and in dampening the inflammatory responses to injurious ischemia.

Advanced glycation end products (AGEs) are nonenzymatical adducts of proteins, lipids, and nucleic acids which form in a time-dependent manner in a pro-oxidant environment, especially when target molecules turnover slowly and the level of aldoses is elevated

**7. The receptor for Advanced Glycation End Products (RAGE)** 

RAGE is a member of the immunoglobulin superfamily of cell surface molecules (Schmidt et al, 1993; Sugaya, 1994). The gene is localized on chromosome 6 near the HLA locus in the vicinity of the MHCIII complex in humans and mice, in close proximity to the homeobox gene HOX12 and the human counterpart of the mouse mammary tumor gene int-3 (Malherbe et al, 1999). The receptor is composed of three immunoglobulin-like regions: one "V"- type domain and two "C"-type-domains, a short transmembrane domain, and a 43-amino acid cytoplasmic tail (Neeper et al, 1992; Schmidt et al, 1994; Lander et al, 1997). While the "V-type" domain confers ligand binding, the cytoplasmic tail is critical for intracellular signaling. Shortly after RAGE was recognized as a receptor for AGEs, it became evident that a number of other ligands also interacted with the receptor (Bucciarelli et al, 2002; Schmidt et al, 2001; Du Yan et al, 1997; Yan et al, 1996, 2000). Structural analysis of ligand–RAGE interaction revealed that the receptor recognized three-dimensional structures, such as β-sheets and fibrils, rather than specific amino acid sequences (i.e., primary structure) (Bucciarelli et al, 2002; Schmidt et al, 2001). In addition to AGEs, RAGE binds amyloid-β peptide (accumulating in Alzheimer's disease) (Du Yan et al, 1997; Yan et al, 2000) and amyloid A (accumulating in systemic amyloidosis). Further, ligands of RAGE are S100/calgranulins, a family of closely related calcium-binding polypeptides that accumulate extracellularly at sites of chronic inflammation (Hofmann et al, 1999; Marenholz et al, 2004). Another proinflammatory ligand of RAGE is the DNA binding protein HMGB1 (amphoterin), which is released by cells undergoing necrosis (Hori et al, 1995; Wang et al, 1999; Anderson and Tracey, 2003; Treutiger et al, 2003). Besides binding ligands actively participating in chronic inflammatory and immune responses, RAGE also interacts with surface molecules on bacteria (Chapman et al, 2002), prions (Sasaki et al, 2002), and leukocytes (Chavakis et al, 2003). Thus, RAGE is much more than a receptor for AGEs; it has a broad repertoire of ligands, which share the propensity to accumulate in tissues during aging, chronic degenerative diseases, inflammation and the host response (Tretiger et al,2003). Therefore, RAGE should be considered a pattern recognition receptor (PRR) (Schmidt et al, 2001; Chavakis et al, 2003; Liliensiek et al, 2004; Gordon, 2002), and potential similarities to members of the family of Toll-like receptors should be considered (Akira et al, 2001).

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 449

RAGE transcription is controlled by several transcription factors, including SP-1, AP-2, NFκB, and NF-IL6 (Li and Schmidt, 1997). RAGE expression occurs in both a constitutive and inducible manner, depending on the cell type and developmental stage (Hori et al,1995; Brett et al,1993). Whereas RAGE is constitutively expressed during embryonic development, its expression is downregulated in adult life. However, known exceptions are skin and lung, which constitutively express RAGE throughout life. Most other cells, including monocytes/macrophages, endothelial cells, smooth musclecells, fibroblasts, and neuronal cells, do not express significant amounts of RAGE under physiological conditions but can be induced to express RAGE in situations where either ligands accumulate and/or transcription factors regulating RAGE are activated (Basta et al, 2002; Bucciarelli et al, 2002; Hanford et al, 2004; Akira et al, 2001; Li et al,2004; Sorci et al, 2004a, 2004b; Cortizo et al, 2003; Shanmugam et al, 2003; Ishihara et al, 2003). Due to its ability to sustain cellular activation, RAGE has the potential to function as a master switch capable of converting a transient proinflammatory response, evoked by an inflammatory stimulus into sustained cellular dysfunction (Schmidt et al, 2001; Bierhaus et al, 2001). The majority of cellular stressors induce both the formation of reactive oxygen species (ROS) and transient activation of NF-κB (Yeh et al, 2001; Taguchi et al, 2000; Huttunen et al, 19999; Huang et al, 2001; Wautier et al, 2001). In addition, inflammatory cells directly release RAGE ligands, such as S100/calgranulins and HMGB-1 (Kokkola et al, 2005). The myeloperoxidase system of human phagocytes generates N" -(carboxymethyl)lysine, a highly reactive AGE and RAGE-ligand, at sites of inflammation (Anderson et al, 1999; Kislinger et al, 1999). High glucose concentrations promote AGE formation inside and outside cells (Brownlee, 2000; Schiekofer et al, 2003). Such time-dependent formation of AGE might also play a role in the expression of binding sites for amyloid peptides (Yan et al, 2000). In turn, RAGE has been shown to mediate transport of pathophysiologically relevant concentrations of amyloid-β peptide into the CNS (Mackic et al, 1998). Thus, stimuli initially inducing oxidant stress and NF-κB activation have the potential to activate RAGE and thereby sustain NF-κB-dependent gene expression. Activation of NF-κB results in increased RAGE expression and increases the number of ligand binding sites, thereby prolonging NF-κB activation (Schmidt et al, 2001; Bierhaus et al, 2001). Frequently, the biology of RAGE coincides with settings in which ligands of the receptor accumulate, especially in a proinflammatory environment such as diabetes mellitus, atherosclerosis, neurodegenerative disorders, rheumatoid arthritis, chronic renal disease, and inflammatory bowl disease (Basta et al, 2002; Schmidt et al, 2001; Lalla et al, 2001; Wendt et al, 2003; Bierhaus et al, 2004; Sakaguchi et al, 2003; Kislinger et al, 2001; Drinda et al, 2004; Chen et al, 2004; Goosa et al, 2001). To better understand the role of RAGE in these pathophysiological situations, interaction of ligands with cell surface RAGE was intercepted using soluble RAGE (sRAGE). Soluble RAGE is a truncated form of the receptor comprising the extracellular domain and thereby functions as a decoy that prevents ligands from interacting with cell surface receptor. Application of sRAGE in vitro and in vivo resulted in an effective blockade of RAGE, according to a decoy mechanism, in a range of animal models (Hudson et al, 2003; Lue et al, 2001; Arancio et al, 2004; Constien et al, 2001). sRAGE prevented development of micro- and macrovascular diseases in rodents, suggesting a key role for RAGE in the development of chronic vascular disorders. Moreover, sRAGE efficiently reduced late complications of experimental diabetes in both autoimmune (Chen et al, 2004) and streptozotocin induced diabetes (Wendt et al, 2003; Bierhaus et al,

**7.4 RAGE and neuroprotection** 

### **7.2 RAGE-mediated NF-κB activation**

Engagement of RAGE results in intracellular signaling which leads to activation of the proinflammatory transcription factor NF-κB, the latter rapidly activated as part of the first line of cellular defense (Bierhaus et al, 2001). In resting cells, NF-κB resides in the cytoplasm in its inactive form bound to the inhibitor molecule IκBα (Barnes and Karin, 1997). Upon activation, IκBα is rapidly phosphorylated and degraded, resulting in release and translocation of NF-κB (preferentially the NF-κB-heterodimer p50/p65) into the nucleus. Subsequent to nuclear translocation, NF-κB binds to decameric DNA sequences and activates transcription of NF-κB regulated target genes, such as cytokines, adhesion molecules, prothrombotic and vasoconstrictive gene products, RAGE itself, and IκBα (Barnes and Karin, 1997; Li and Schmidt, 1997; Bierhaus et al, 2000). A number of antiapoptotic genes, including Bcl-XL, Bcl-2, and the Bcl-2 homologues A1, are also under control of NF-κB. NF-κB activation therefore provides a rapid and sensitive cellular response in the absence of new protein synthesis, which promotes cellular survival. One unique feature of RAGE-mediated NF-κB activation is the prolonged time course which appears to overwhelm endogenous autoregulatory feedback inhibition loops (Bierhaus et al, 2001). NF-κB activation subsequent to ligation of RAGE is initiated by the degradation of IκBα and IκBβ, followed by new synthesis of NF-κBp65 in the presence of newly synthesized IκBβ. De novo synthesis of p65 mRNA results in a constantly growing pool of excess transcriptionally active NF-κBp65. In contrast, the amount of newly synthesized IκBα is not sufficient to retain NF-κBp65 in the cytoplasm. In addition, newly synthesized IκBβ has been shown to be hyperphosphorylated, thereby sequestering newly synthesized NF-κB from IκBα (Thompson et al, 1995; Johnson et al, 1996)? Thus, new synthesis of IκBβ might further promote RAGEdependent sustained NF-κB activation. Since, in turn, RAGE expression is induced by NF-κB (Li and Schmidt, 1997), sustained activation of NF-κB results in upregulation of the receptor and further ensures maintenance and amplification of the signal.

### **7.3 AGEs and RAGE affect cellular defense mechanisms**

Besides activating proinflammatory responses, RAGE downregulates cellular defense mechanisms. Ligation of RAGE by AGEs results in the suppression of reduced glutathione (GSH) and ascorbic acid levels and thereby contributes to increased intracellular oxidant stress (Lander et al, 1997; Bierhaus et al, 1997). Depletion of glutathione accounts for diminished glyoxalase-1 recycling and decreased in situ activity of glyoxalase-1 (Thornalley,1998). Glyoxalase-1, in turn, is required to catalyze the conversion of reactive, acyclic alpha-oxoaldehydes into the corresponding alpha-hydroxyacids (Degenhard 1998; Thornalley et al,1999). Since alpha-oxoaldehydes, such as methylglyoxal, represent the largest pool of reactive intracellular AGEs, glyoxalase-1 has an important role in reduction of the cellular AGE load. Consistent with this concept, in vitro experiments with cultivated endothelial cells have demonstrated that glyoxalase-1 overexpression prevents intracellular AGE formation (Shinohara et al,1998). Studies in the model organism Caenorhabditis elegans have recently confirmed that overexpression of glyoxalase-1 not only prevents AGE formation, but also protects the animals from deleterious effects of oxidant stress, as evidenced by increased longevity (Morcos et al,2004). These observations imply that engagement of RAGE not only results in increased cellular activation, but also in reduction of AGE detoxifying mechanisms.

### **7.4 RAGE and neuroprotection**

448 Advances in the Preclinical Study of Ischemic Stroke

Engagement of RAGE results in intracellular signaling which leads to activation of the proinflammatory transcription factor NF-κB, the latter rapidly activated as part of the first line of cellular defense (Bierhaus et al, 2001). In resting cells, NF-κB resides in the cytoplasm in its inactive form bound to the inhibitor molecule IκBα (Barnes and Karin, 1997). Upon activation, IκBα is rapidly phosphorylated and degraded, resulting in release and translocation of NF-κB (preferentially the NF-κB-heterodimer p50/p65) into the nucleus. Subsequent to nuclear translocation, NF-κB binds to decameric DNA sequences and activates transcription of NF-κB regulated target genes, such as cytokines, adhesion molecules, prothrombotic and vasoconstrictive gene products, RAGE itself, and IκBα (Barnes and Karin, 1997; Li and Schmidt, 1997; Bierhaus et al, 2000). A number of antiapoptotic genes, including Bcl-XL, Bcl-2, and the Bcl-2 homologues A1, are also under control of NF-κB. NF-κB activation therefore provides a rapid and sensitive cellular response in the absence of new protein synthesis, which promotes cellular survival. One unique feature of RAGE-mediated NF-κB activation is the prolonged time course which appears to overwhelm endogenous autoregulatory feedback inhibition loops (Bierhaus et al, 2001). NF-κB activation subsequent to ligation of RAGE is initiated by the degradation of IκBα and IκBβ, followed by new synthesis of NF-κBp65 in the presence of newly synthesized IκBβ. De novo synthesis of p65 mRNA results in a constantly growing pool of excess transcriptionally active NF-κBp65. In contrast, the amount of newly synthesized IκBα is not sufficient to retain NF-κBp65 in the cytoplasm. In addition, newly synthesized IκBβ has been shown to be hyperphosphorylated, thereby sequestering newly synthesized NF-κB from IκBα (Thompson et al, 1995; Johnson et al, 1996)? Thus, new synthesis of IκBβ might further promote RAGEdependent sustained NF-κB activation. Since, in turn, RAGE expression is induced by NF-κB (Li and Schmidt, 1997), sustained activation of NF-κB results in upregulation of the receptor and further ensures maintenance and amplification of

Besides activating proinflammatory responses, RAGE downregulates cellular defense mechanisms. Ligation of RAGE by AGEs results in the suppression of reduced glutathione (GSH) and ascorbic acid levels and thereby contributes to increased intracellular oxidant stress (Lander et al, 1997; Bierhaus et al, 1997). Depletion of glutathione accounts for diminished glyoxalase-1 recycling and decreased in situ activity of glyoxalase-1 (Thornalley,1998). Glyoxalase-1, in turn, is required to catalyze the conversion of reactive, acyclic alpha-oxoaldehydes into the corresponding alpha-hydroxyacids (Degenhard 1998; Thornalley et al,1999). Since alpha-oxoaldehydes, such as methylglyoxal, represent the largest pool of reactive intracellular AGEs, glyoxalase-1 has an important role in reduction of the cellular AGE load. Consistent with this concept, in vitro experiments with cultivated endothelial cells have demonstrated that glyoxalase-1 overexpression prevents intracellular AGE formation (Shinohara et al,1998). Studies in the model organism Caenorhabditis elegans have recently confirmed that overexpression of glyoxalase-1 not only prevents AGE formation, but also protects the animals from deleterious effects of oxidant stress, as evidenced by increased longevity (Morcos et al,2004). These observations imply that engagement of RAGE not only results in increased cellular activation, but also in reduction

**7.2 RAGE-mediated NF-κB activation** 

the signal.

of AGE detoxifying mechanisms.

**7.3 AGEs and RAGE affect cellular defense mechanisms** 

RAGE transcription is controlled by several transcription factors, including SP-1, AP-2, NFκB, and NF-IL6 (Li and Schmidt, 1997). RAGE expression occurs in both a constitutive and inducible manner, depending on the cell type and developmental stage (Hori et al,1995; Brett et al,1993). Whereas RAGE is constitutively expressed during embryonic development, its expression is downregulated in adult life. However, known exceptions are skin and lung, which constitutively express RAGE throughout life. Most other cells, including monocytes/macrophages, endothelial cells, smooth musclecells, fibroblasts, and neuronal cells, do not express significant amounts of RAGE under physiological conditions but can be induced to express RAGE in situations where either ligands accumulate and/or transcription factors regulating RAGE are activated (Basta et al, 2002; Bucciarelli et al, 2002; Hanford et al, 2004; Akira et al, 2001; Li et al,2004; Sorci et al, 2004a, 2004b; Cortizo et al, 2003; Shanmugam et al, 2003; Ishihara et al, 2003). Due to its ability to sustain cellular activation, RAGE has the potential to function as a master switch capable of converting a transient proinflammatory response, evoked by an inflammatory stimulus into sustained cellular dysfunction (Schmidt et al, 2001; Bierhaus et al, 2001). The majority of cellular stressors induce both the formation of reactive oxygen species (ROS) and transient activation of NF-κB (Yeh et al, 2001; Taguchi et al, 2000; Huttunen et al, 19999; Huang et al, 2001; Wautier et al, 2001). In addition, inflammatory cells directly release RAGE ligands, such as S100/calgranulins and HMGB-1 (Kokkola et al, 2005). The myeloperoxidase system of human phagocytes generates N" -(carboxymethyl)lysine, a highly reactive AGE and RAGE-ligand, at sites of inflammation (Anderson et al, 1999; Kislinger et al, 1999). High glucose concentrations promote AGE formation inside and outside cells (Brownlee, 2000; Schiekofer et al, 2003). Such time-dependent formation of AGE might also play a role in the expression of binding sites for amyloid peptides (Yan et al, 2000). In turn, RAGE has been shown to mediate transport of pathophysiologically relevant concentrations of amyloid-β peptide into the CNS (Mackic et al, 1998). Thus, stimuli initially inducing oxidant stress and NF-κB activation have the potential to activate RAGE and thereby sustain NF-κB-dependent gene expression. Activation of NF-κB results in increased RAGE expression and increases the number of ligand binding sites, thereby prolonging NF-κB activation (Schmidt et al, 2001; Bierhaus et al, 2001). Frequently, the biology of RAGE coincides with settings in which ligands of the receptor accumulate, especially in a proinflammatory environment such as diabetes mellitus, atherosclerosis, neurodegenerative disorders, rheumatoid arthritis, chronic renal disease, and inflammatory bowl disease (Basta et al, 2002; Schmidt et al, 2001; Lalla et al, 2001; Wendt et al, 2003; Bierhaus et al, 2004; Sakaguchi et al, 2003; Kislinger et al, 2001; Drinda et al, 2004; Chen et al, 2004; Goosa et al, 2001). To better understand the role of RAGE in these pathophysiological situations, interaction of ligands with cell surface RAGE was intercepted using soluble RAGE (sRAGE). Soluble RAGE is a truncated form of the receptor comprising the extracellular domain and thereby functions as a decoy that prevents ligands from interacting with cell surface receptor. Application of sRAGE in vitro and in vivo resulted in an effective blockade of RAGE, according to a decoy mechanism, in a range of animal models (Hudson et al, 2003; Lue et al, 2001; Arancio et al, 2004; Constien et al, 2001). sRAGE prevented development of micro- and macrovascular diseases in rodents, suggesting a key role for RAGE in the development of chronic vascular disorders. Moreover, sRAGE efficiently reduced late complications of experimental diabetes in both autoimmune (Chen et al, 2004) and streptozotocin induced diabetes (Wendt et al, 2003; Bierhaus et al,

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 451

promote cell survival through increased expression of the anti-apoptotic protein Bcl-2. However, whereas nanomolar concentrations of S100B induced trophic effects in RAGEexpressing cells, micromolar concentrations caused apoptosis in a manner that appeared to depend on oxidant stress. For both of these outcomes, the cytoplasmic domain of RAGE was required, as cells expressing a dominant-negative mutant (i.e., lacking the cytosolic tail) are unresponsive to these stimuli. The neurite outgrowth-promoting role of RAGE was recently confirmed in vivo in a unilateral sciatic nerve crush model, in which blockade of RAGE, either by sRAGE or by blocking F(ab')2 fragments of antibodies (raised to either RAGE or to S100/calgranulins or amphoterin) reduced functional regeneration of the peripheral nerve (Rong et al, 2004a). Similar results were observed in transgenic mice overexpressing dominant negative RAGE (Rong et al, 2004b). However, RAGE−/− mice demonstrate neither obvious neuronal deficits nor overt behavior abnormalities, indicating that RAGE may contribute to neuronal development, but that there are redundant systems that substitute for this receptor in its absence. Furthermore, it will be interesting to see if future experiments in RAGE−/− mice confirm a role for RAGE in the repair of peripheral nerve injury. In terms of a contribution for RAGE in development, expression of the receptor in vivo appears to mirror developmental processes. After being highly expressed during embryonic development, RAGE is downregulated in most organs during normal life (Kokkola et al, 2005). Upon aging, RAGE expression increases again, although it is not known whether this is due to accumulation of RAGE ligands (which upregulate receptor expression) or whether this represents a compensatory mechanism protecting aging cells from cell death. Another line of evidence for a role of RAGE in the regulation of differentiation comes from recently published studies showing that non-small cell lung carcinomas are characterized by downregulation of RAGE (Bartling et al, 2004). One reason for this might be that loss of HMGB1(amphoterin)/RAGE-mediated regulation of tumor cell migration and invasive processes results in more aggressive tumor behavior (Huttunen et al, 2002). A COOH-terminal motif in HMGB1 (amino acids 150–183) has recently been identified as responsible for RAGE binding. This portion of HMGB1 efficiently inhibits RAGE-mediated extension of cellular processes and transendothelial migration of tumor cells. This observation leads us to propose that loss of RAGE might promote tumor growth, at least in settings affecting the lung, one of the few tissues in which RAGE is constitutively expressed at high levels. Since this observation contrasts with a previous finding in which sRAGE suppressed tumor growth and metastasis (Taguchi et al, 2000), the latter observations might be due to the ability of sRAGE to intercept the interaction of RAGE

Cell death from ischemia involves a complex biological cascade. Initially, energy failure is followed by glutamate overload and Ca2+ influx into the cell. These processes initiate a series of events, including the generation of free radicals, apoptosis, an inflammatory response and generation of growth factors. Many of these processes are the direct result of the up- or downregulation of specific gene families. Thus, a desirable neuroprotectant would, in theory, be one that antagonises multiple injury mechanisms. The studies described above demonstrate an emerging role for endogenous neuroprotectant in ischemic damage and

ligands with other receptors.

**8. Conclusion and prospective** 

2004), restored delayed wound healing (Goosa et al, 2001), protected rodent from tumor metastases and growth of primary tumors (Taguchi et al, 2000), and improved the outcome of experimental colitis (Hofmann et al, 1999). sRAGE and anti-RAGE F(ab')2-fragments suppressed abnormal findings associated with Alzheimer's-like pathology in transgenic rodent models (Lue et al, 2001; Arancio et al, 2004) and reduced the transport of amyloid-βpeptide across the blood-brain barrier (Mackic et al, 1998) . Since most of the data obtained with sRAGE were confirmed by application of neutralizing antibodies to the receptor and/or transfection with plasmids overexpressing dominant negative RAGE, the receptor has been suggested as a potentially effective therapeutic target (Hudson et al, 2003). At the same time, it seemed unlikely that RAGE could mediate so many deleterious effects in such diverse models of disease. Since RAGE has properties of a PRR, binding to a variety of ligands, the promising effects observed with sRAGE might not only result from intercepting the interaction of ligands with cell surface RAGE, but possibly with other receptors. For example, S100 proteins and HMGB1 certainly do not exclusively bind to RAGE. These ligands also recognize other cellular structures (Robinson et al, 2002; Erlandsson et al, 2004). In order to test the potential impact of RAGE blockade and to further define a potential role of RAGE in diabetic complications and chronic inflammatory disease, homozygous RAGEdeficient mice (RAGE−/− mice) and mice with tissue-specific RAGE expression (tie2-RAGE and tie2-RAGE°—RAGE−/−) have been made (Constien et al, 2001). These mice are viable and display normal reproductive fitness without any striking phenotype (Wendt et al, 2003; Bierhaus et al, 2004; Sakaguchi et al, 2003). Induction of diabetes in these mice confirmed that RAGE contributes, at least in part, to the development of diabetic complications. Diabetic nephropathy, characterized by renal enlargement, glomerular hypertrophy, albuminuria, and mesangial expansion, was significantly increased in diabetic mice overexpressing RAGE in the vasculature, but was reduced in RAGE−/−mice (Yamamoto et al, 2001). Similar changes were observed in diabetic neuropathy. Whereas diabetic mice overexpressing RAGE showed an increase in functional deficits, such as delayed motor nerve conduction velocity (Yajima et al, 2004), RAGE−/− mice were partially protected from diabetes-induced loss of neural function (Bierhaus et al, 2004). Neointimal expansion in RAGE−/− mice was significantly suppressed compared with that observed in wildtype littermates using a femoral artery denudation protocol to induce arterial injury (Sakaguchi et al, 2003). Remarkably, in each of these models (diabetic nephropathy, neuropathy, arterial restenosis, etc.), protection from development of pathology was more profound in wild-type mice treated with sRAGE than in RAGE−/− mice. In diabetic neuropathy, for example, administration of sRAGE to diabetic wild-type animals completely restored pain perception, whereas diabetic RAGE−/− mice were only partly protected from loss of pain perception. These observations suggest that ligands sequestered by sRAGE are likely to interact with cellular structures different from RAGE and are also involved in perturbation of pain perception. The absence of a developmental phenotype in RAGE−/− mice and the possibility that RAGE might impact on multiple chronic disease states have largely focussed attention away from physiologic roles of the receptor. So far, only a few reports have suggested that RAGE expression might contribute to developmental paradigms, based on in vitro studies. For example, in axonal sprouting which accompanies neuronal development, RAGE–HMGB1 interaction may contribute (Fages et al, 2000; Hittinen et al, 2000). Huttunen et al. further demonstrated that activation of RAGE by HMGB1 (amphoterin) and S100B can

2004), restored delayed wound healing (Goosa et al, 2001), protected rodent from tumor metastases and growth of primary tumors (Taguchi et al, 2000), and improved the outcome of experimental colitis (Hofmann et al, 1999). sRAGE and anti-RAGE F(ab')2-fragments suppressed abnormal findings associated with Alzheimer's-like pathology in transgenic rodent models (Lue et al, 2001; Arancio et al, 2004) and reduced the transport of amyloid-βpeptide across the blood-brain barrier (Mackic et al, 1998) . Since most of the data obtained with sRAGE were confirmed by application of neutralizing antibodies to the receptor and/or transfection with plasmids overexpressing dominant negative RAGE, the receptor has been suggested as a potentially effective therapeutic target (Hudson et al, 2003). At the same time, it seemed unlikely that RAGE could mediate so many deleterious effects in such diverse models of disease. Since RAGE has properties of a PRR, binding to a variety of ligands, the promising effects observed with sRAGE might not only result from intercepting the interaction of ligands with cell surface RAGE, but possibly with other receptors. For example, S100 proteins and HMGB1 certainly do not exclusively bind to RAGE. These ligands also recognize other cellular structures (Robinson et al, 2002; Erlandsson et al, 2004). In order to test the potential impact of RAGE blockade and to further define a potential role of RAGE in diabetic complications and chronic inflammatory disease, homozygous RAGEdeficient mice (RAGE−/− mice) and mice with tissue-specific RAGE expression (tie2-RAGE and tie2-RAGE°—RAGE−/−) have been made (Constien et al, 2001). These mice are viable and display normal reproductive fitness without any striking phenotype (Wendt et al, 2003; Bierhaus et al, 2004; Sakaguchi et al, 2003). Induction of diabetes in these mice confirmed that RAGE contributes, at least in part, to the development of diabetic complications. Diabetic nephropathy, characterized by renal enlargement, glomerular hypertrophy, albuminuria, and mesangial expansion, was significantly increased in diabetic mice overexpressing RAGE in the vasculature, but was reduced in RAGE−/−mice (Yamamoto et al, 2001). Similar changes were observed in diabetic neuropathy. Whereas diabetic mice overexpressing RAGE showed an increase in functional deficits, such as delayed motor nerve conduction velocity (Yajima et al, 2004), RAGE−/− mice were partially protected from diabetes-induced loss of neural function (Bierhaus et al, 2004). Neointimal expansion in RAGE−/− mice was significantly suppressed compared with that observed in wildtype littermates using a femoral artery denudation protocol to induce arterial injury (Sakaguchi et al, 2003). Remarkably, in each of these models (diabetic nephropathy, neuropathy, arterial restenosis, etc.), protection from development of pathology was more profound in wild-type mice treated with sRAGE than in RAGE−/− mice. In diabetic neuropathy, for example, administration of sRAGE to diabetic wild-type animals completely restored pain perception, whereas diabetic RAGE−/− mice were only partly protected from loss of pain perception. These observations suggest that ligands sequestered by sRAGE are likely to interact with cellular structures different from RAGE and are also involved in perturbation of pain perception. The absence of a developmental phenotype in RAGE−/− mice and the possibility that RAGE might impact on multiple chronic disease states have largely focussed attention away from physiologic roles of the receptor. So far, only a few reports have suggested that RAGE expression might contribute to developmental paradigms, based on in vitro studies. For example, in axonal sprouting which accompanies neuronal development, RAGE–HMGB1 interaction may contribute (Fages et al, 2000; Hittinen et al, 2000). Huttunen et al. further demonstrated that activation of RAGE by HMGB1 (amphoterin) and S100B can promote cell survival through increased expression of the anti-apoptotic protein Bcl-2. However, whereas nanomolar concentrations of S100B induced trophic effects in RAGEexpressing cells, micromolar concentrations caused apoptosis in a manner that appeared to depend on oxidant stress. For both of these outcomes, the cytoplasmic domain of RAGE was required, as cells expressing a dominant-negative mutant (i.e., lacking the cytosolic tail) are unresponsive to these stimuli. The neurite outgrowth-promoting role of RAGE was recently confirmed in vivo in a unilateral sciatic nerve crush model, in which blockade of RAGE, either by sRAGE or by blocking F(ab')2 fragments of antibodies (raised to either RAGE or to S100/calgranulins or amphoterin) reduced functional regeneration of the peripheral nerve (Rong et al, 2004a). Similar results were observed in transgenic mice overexpressing dominant negative RAGE (Rong et al, 2004b). However, RAGE−/− mice demonstrate neither obvious neuronal deficits nor overt behavior abnormalities, indicating that RAGE may contribute to neuronal development, but that there are redundant systems that substitute for this receptor in its absence. Furthermore, it will be interesting to see if future experiments in RAGE−/− mice confirm a role for RAGE in the repair of peripheral nerve injury. In terms of a contribution for RAGE in development, expression of the receptor in vivo appears to mirror developmental processes. After being highly expressed during embryonic development, RAGE is downregulated in most organs during normal life (Kokkola et al, 2005). Upon aging, RAGE expression increases again, although it is not known whether this is due to accumulation of RAGE ligands (which upregulate receptor expression) or whether this represents a compensatory mechanism protecting aging cells from cell death. Another line of evidence for a role of RAGE in the regulation of differentiation comes from recently published studies showing that non-small cell lung carcinomas are characterized by downregulation of RAGE (Bartling et al, 2004). One reason for this might be that loss of HMGB1(amphoterin)/RAGE-mediated regulation of tumor cell migration and invasive processes results in more aggressive tumor behavior (Huttunen et al, 2002). A COOH-terminal motif in HMGB1 (amino acids 150–183) has recently been identified as responsible for RAGE binding. This portion of HMGB1 efficiently inhibits RAGE-mediated extension of cellular processes and transendothelial migration of tumor cells. This observation leads us to propose that loss of RAGE might promote tumor growth, at least in settings affecting the lung, one of the few tissues in which RAGE is constitutively expressed at high levels. Since this observation contrasts with a previous finding in which sRAGE suppressed tumor growth and metastasis (Taguchi et al, 2000), the latter observations might be due to the ability of sRAGE to intercept the interaction of RAGE

### **8. Conclusion and prospective**

ligands with other receptors.

Cell death from ischemia involves a complex biological cascade. Initially, energy failure is followed by glutamate overload and Ca2+ influx into the cell. These processes initiate a series of events, including the generation of free radicals, apoptosis, an inflammatory response and generation of growth factors. Many of these processes are the direct result of the up- or downregulation of specific gene families. Thus, a desirable neuroprotectant would, in theory, be one that antagonises multiple injury mechanisms. The studies described above demonstrate an emerging role for endogenous neuroprotectant in ischemic damage and

Endogenous Agents That Contribute to Generate or Prevent Ischemic Damage 453

TLR signaling may be a unifying principle of tolerance to cerebral ischemia. Recent studies have also demonstrated an increased expression of the cell-surface RAGE in dying neurons after hypoxic-ischemic insults and human cerebral ischemia, and suggested that the RAGEligand interaction causes neuronal cytotoxicity. RAGE also has a circulating truncated variant isoform, soluble RAGE (sRAGE), which corresponds to its extracellular domain only. Exogenously administered sRAGE has been successfully used to antagonize advanced glycation end products (AGE)-RAGE-mediated vascular damage. Accordingly, sRAGE may compete with cell-surface RAGE for the ligand, thus functioning as a decoy and possibly exerting a cytoprotective effect. Most of the data available so far point to the RAGE/NF-κB axis as an attractive target for future clinical interventions in several chronic disease states. However, until physiologic properties of RAGE have been clearly deciphered, it is most prudent to adopt a cautious approach when future therapeutic strategies involving longterm blockade of RAGE or its ligands are considered. Another important issue to be addressed concerns how studies performed in rodent models will translate to human disease. Alternatively, if RAGE antagonists are eventually used in humans, it will be fascinating to understand the impact of long-term blockade of RAGE in critically ill patients, in view of the likely complex role of RAGE, and other receptors interacting with RAGE ligands, in regulating physiologic and pathophysiologic processes in a wide range of

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ischemic prophylaxis. Among these, erythropoietin (Epo), has a dominant role for neuroprotection, neurogenesis and act as a neurotrophic factor in the central nervous system. These functions make erythropoietin a good candidate for treating disease associated with neuronal cell death. However, our understanding of the underlying mechanisms is far from being complete and a number of open questions remain to be answered: 1) What is the exact route and mechanism through which Epo passes through the BBB? 2) Which cellular mechanisms govern the immunomodulatory effects of Epo in glial cells? 3) Does Epo activate the same or diverse intracellular signaling pathways in the different cells that express EpoR in the brain, neurons, glial, and endothelial cells? Nevertheless, since the discovery of Epo expression in the brain less than 15 years ago, a tremendous achievement in the understanding of its action in the CNS has been accomplished. Today, Epo is a prominent member of a growing list of hematopoietic and angiogenic factors found to be expressed and acting as protective factors in the CNS. Because of the observed increased death rate, rtPA-treated patients should be excluded from acute poststroke EPO application. In cerebral ischemia, albumin is mainly involved in the improvement of blood microcirculation; however, direct neuroprotection cannot be overlooked. Different in vitro and in vivo studies indicate that albumin has direct neuroprotective effects by acting on astrocytes, microglia and neurons. Altogether albumin can alter brain function by many direct and indirect mechanisms and detailed study of these actions will reveal the role of this multifunctional protein in brain functions. Furthermore, the evidence of de novo synthesis of albumin in microglial cells could encourage the neurologist to investigate newer roles of this multifunctional protein in many neurodegenerative diseases. The prothrombotic, proinflammatory state of stroke results in a promotion of thrombin formation and fibrin deposition at the vascular wall, as well as in the formation of platelet-leukocyte coaggregates, leading to severe disturbance of the microcirculation, capillary leakage and tissue damage. The ability of ATIII to inhibit the actions of both factor Xa and thrombin gives it the potential to block, in part or fully, a wide rage of proinflammatory events. Heparin and heparan sulfate glycoproteins (HSPGs) appear to function as receptors for ATIII on endothelium and leukocytes and can lead to the reduced expression of procoagulatory tissue factor and proinflammatory cytokines as well as heterologous receptor regulatory processes. ATIII has been shown in vitro to increase prostacyclin responses and to inhibit a variety of cell responses including endotoxininduced nuclear translocation of NF-kB, a key step in the generation of the inflammatory response.

Here, we also discussed the critical role of Toll-like receptors in mediating cerebral ischemic injury and suggested endogenous mechanisms that, when induced, redirect this role from detrimental to beneficial. In fact, many diverse neuroprotective paradigms may redirect TLR signaling as one mechanism of endogenous protection. Paradoxically, TLR ligands administered systemically induce a state of tolerance to subsequent ischemic injury. Herein we suggest that stimulation of TLRs prior to ischemia reprograms TLR signaling that occurs following ischemic injury. Such reprogramming leads to suppressed expression of proinflammatory molecules and enhanced expression of numerous anti-inflammatory mediators that collectively confer robust neuroprotection. Research findings indicate that numerous preconditioning stimuli lead to TLR activation, an event that occurs prior to ischemia and ultimately leads to TLR reprogramming. Thus genomic reprogramming of TLR signaling may be a unifying principle of tolerance to cerebral ischemia. Recent studies have also demonstrated an increased expression of the cell-surface RAGE in dying neurons after hypoxic-ischemic insults and human cerebral ischemia, and suggested that the RAGEligand interaction causes neuronal cytotoxicity. RAGE also has a circulating truncated variant isoform, soluble RAGE (sRAGE), which corresponds to its extracellular domain only. Exogenously administered sRAGE has been successfully used to antagonize advanced glycation end products (AGE)-RAGE-mediated vascular damage. Accordingly, sRAGE may compete with cell-surface RAGE for the ligand, thus functioning as a decoy and possibly exerting a cytoprotective effect. Most of the data available so far point to the RAGE/NF-κB axis as an attractive target for future clinical interventions in several chronic disease states. However, until physiologic properties of RAGE have been clearly deciphered, it is most prudent to adopt a cautious approach when future therapeutic strategies involving longterm blockade of RAGE or its ligands are considered. Another important issue to be addressed concerns how studies performed in rodent models will translate to human disease. Alternatively, if RAGE antagonists are eventually used in humans, it will be fascinating to understand the impact of long-term blockade of RAGE in critically ill patients, in view of the likely complex role of RAGE, and other receptors interacting with RAGE ligands, in regulating physiologic and pathophysiologic processes in a wide range of situations.

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Here, we also discussed the critical role of Toll-like receptors in mediating cerebral ischemic injury and suggested endogenous mechanisms that, when induced, redirect this role from detrimental to beneficial. In fact, many diverse neuroprotective paradigms may redirect TLR signaling as one mechanism of endogenous protection. Paradoxically, TLR ligands administered systemically induce a state of tolerance to subsequent ischemic injury. Herein we suggest that stimulation of TLRs prior to ischemia reprograms TLR signaling that occurs following ischemic injury. Such reprogramming leads to suppressed expression of proinflammatory molecules and enhanced expression of numerous anti-inflammatory mediators that collectively confer robust neuroprotection. Research findings indicate that numerous preconditioning stimuli lead to TLR activation, an event that occurs prior to ischemia and ultimately leads to TLR reprogramming. Thus genomic reprogramming of

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**20** 

*Tsurumai, Nagoya,* 

*1China, 2Japan* 

**Time-Window of Progesterone** 

Weiyan Cai1, Masahiro Sokabe2 and Ling Chen1,2

**Neuroprotection After Stroke and Its** 

**Underlying Molecular Mechanisms** 

*1Department of Physiology, Nanjing Medical University, Jiangsu,* 

*2Department of Physiology, Nagoya University Graduate School of Medicine,* 

Evidence exists for a gender difference in the vulnerability to either stroke or traumatic brain injury (TBI) in humans. For example, pre-menopausal women with the high serum levels of ovarian hormones estrogen (E2) and progesterone (P4) have a lower risk of stroke (Kannel et al., 1994; Sacco et al., 1997) and a better outcome following stroke (Thorvaldsen et al., 1995) or TBI (Groswasser et al., 1998) relative to men of the same age. After menopause, incidence of stroke in women increases abruptly (Wenger et al., 1993) coincident with decreases in the circulating levels of the ovarian steroid hormones, estrogen (E2) and progesterone (P4). Although clinical trial for TBI with P4 treatment has been well tolerated and giving improved outcomes (Wright et al., 2007; Stein et al., 2008), clinical trial with P4 treatment after cerebral stroke has yet to be initiated. There is increasing evidence that P4 exerts a potent neuroprotective effect against ischemia-induced brain injury in experimental models (Chen et al., 1999; Kumon et al., 2000; Morali et al., 2005; Sayeed et al., 2006) when administered either before insult or after the onset of reperfusion (Murphy et al., 2002; Sayeed et al., 2007). Furthermore, the administration of P4 promotes functional recovery after cerebral ischemia (Gibson & Murphy, 2004; Sayeed et al., 2007). Important enough, a single injection of P4 (4 mg/kg) conducted even 2 h after transient focal brain ischemia reduced cortical infarct volumes (Jiang et al., 1996). Our recent study (Cai et al., 2008) has demonstrated that in male rats a single injection of P4 (4 mg/kg) at 1 h or 48 h prior to an experimental stroke shows protective effects against the ischemia-induced neuronal death and the deficits in spatial cognition and LTP induction. However, to date no systematic study has conducted concerning the effects of P4 against brain injury beyond 6 h following the onset of ischemia (Gibson et al., 2008). Therefore, the present study focused on the effective time-window of neuroprotection by P4 treatment, which would give useful

Effects of P4 on the brain generally involve three principle mechanisms, including regulation of gene expression, activation of intracellular signal cascades and modulation of

**1. Introduction** 

information in treating stroke.


### **Time-Window of Progesterone Neuroprotection After Stroke and Its Underlying Molecular Mechanisms**

Weiyan Cai1, Masahiro Sokabe2 and Ling Chen1,2 *1Department of Physiology, Nanjing Medical University, Jiangsu, 2Department of Physiology, Nagoya University Graduate School of Medicine, Tsurumai, Nagoya, 1China, 2Japan* 

### **1. Introduction**

478 Advances in the Preclinical Study of Ischemic Stroke

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focal cerebral ischemia. Biochem Biophys Res Comm 359:574–579.

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4 in the brain in a rabbit experimental subarachnoid haemorrhage model. Inflamm

Nietfeld W, Trendelenburg G (2007) TLR2 has a detrimental role in mouse transient

Evidence exists for a gender difference in the vulnerability to either stroke or traumatic brain injury (TBI) in humans. For example, pre-menopausal women with the high serum levels of ovarian hormones estrogen (E2) and progesterone (P4) have a lower risk of stroke (Kannel et al., 1994; Sacco et al., 1997) and a better outcome following stroke (Thorvaldsen et al., 1995) or TBI (Groswasser et al., 1998) relative to men of the same age. After menopause, incidence of stroke in women increases abruptly (Wenger et al., 1993) coincident with decreases in the circulating levels of the ovarian steroid hormones, estrogen (E2) and progesterone (P4). Although clinical trial for TBI with P4 treatment has been well tolerated and giving improved outcomes (Wright et al., 2007; Stein et al., 2008), clinical trial with P4 treatment after cerebral stroke has yet to be initiated. There is increasing evidence that P4 exerts a potent neuroprotective effect against ischemia-induced brain injury in experimental models (Chen et al., 1999; Kumon et al., 2000; Morali et al., 2005; Sayeed et al., 2006) when administered either before insult or after the onset of reperfusion (Murphy et al., 2002; Sayeed et al., 2007). Furthermore, the administration of P4 promotes functional recovery after cerebral ischemia (Gibson & Murphy, 2004; Sayeed et al., 2007). Important enough, a single injection of P4 (4 mg/kg) conducted even 2 h after transient focal brain ischemia reduced cortical infarct volumes (Jiang et al., 1996). Our recent study (Cai et al., 2008) has demonstrated that in male rats a single injection of P4 (4 mg/kg) at 1 h or 48 h prior to an experimental stroke shows protective effects against the ischemia-induced neuronal death and the deficits in spatial cognition and LTP induction. However, to date no systematic study has conducted concerning the effects of P4 against brain injury beyond 6 h following the onset of ischemia (Gibson et al., 2008). Therefore, the present study focused on the effective time-window of neuroprotection by P4 treatment, which would give useful information in treating stroke.

Effects of P4 on the brain generally involve three principle mechanisms, including regulation of gene expression, activation of intracellular signal cascades and modulation of

Time-Window of Progesterone Neuroprotection

**2.2 Preparation of focal cerebral ischemia model** 

identically, except that MCAs were not occluded.

the same dosage of P4 increases ERK1/2 phosphorylation.

= 5µl/rat). Control rats were given an equal volume of vehicle.

**2.3 Drug administration** 

**2.4 Histological examination 2.4.1 Infarct volume measuring** 

groups (P > 0.05).

After Stroke and Its Underlying Molecular Mechanisms 481

Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Rats were anesthetized with a mixture of 70% N2O and 30% O2 containing 2.5% isoflurane, and were maintained by the inhalation of 1.5% isoflurane during the operation. Briefly, a heatblunted black monofilament surgical suture (4/0 G) was inserted into the internal carotid artery to occlude the origin of MCA. Adequacy of vascular occlusion and reperfusion was monitored in the front parietal cortex of the occluded side with a multi-channel laser Doppler flow-meter (PF5050 Q4, Perimed, Jarfalla, Sweden). Body and head temperatures were controlled at 37±0.5°C using a water pads. Arterial blood pressure and gases were monitored through a femoral catheter. After 60 min of occlusion, the filament was withdrawn to allow for reperfusion. Sham-operated (sham-op) animals were treated

P4 was dissolved in dimethylsulfoxide (DMSO), then in sesame oil to a final concentration of 1% DMSO. P4 (4 mg/kg) was intraperitonealy (i.p.) injected. Two injections of P4 with 8 h interval were given starting at 1, 24, 48, 72 or 96 h after the initiation of MCAO (post-MCAO). We selected this low dosage because P4 at this dosage is reported to significantly reduce the ischemic damage and regulate anti-apoptotic gene expression following TBI in rats (Stein, 2008). In addition, our study (Cai et al., 2008) determined that the treatment with

To analyze the molecular mechanisms underlying the P4-actions, the P4R antagonist RU486 (3 mg/kg) and the 5α-reductase inhibitor finasteride (20 mg/kg) (Finn et al., 2006) were given by intraperitoneal injection (i.p.) at 30 min before each administration of P4. The MEK inhibitor U0126 (0.5 nmol) and the PI3K inhibitor LY294002 (0.3 nmol) were injected into the cerebroventricle (i.c.v.) at 30 min before each injections of P4. For i.c.v. implantation, rats were anaesthetized with ketamine (80 mg/kg i.p.). A guide cannula (10 mm length, 22 gauge) aiming above the right lateral ventricle was implanted. The inhibitors or vehicle were injected with a stepper-motorized micro-syringe (Stoelting, Wood Dale, IL, USA) at a rate of 0.5 μl/min. The drugs were prepared freshly on the day of experiment (final volume

Brains were removed on 7th day post-MCAO, sectioned into 5 equidistant slices (2.0-mmthick), and incubated in a 2% 2,3,5-triphenyle-tetrazoliumchloride (TTC) solution (15 min) to visualize infarcted tissue. Measurements were performed by manually outlining the margins of the infarcted areas. Unstained areas of brain sections were defined as infarcted using the image analysis software NIH-Image 3.12. Briefly, the infarcted area on the ipsilateral side was indirectly measured by subtracting the noninfarcted area in the ipsilateral hemisphere from the total nonischaemic area of the contralateral (nonischaemic) hemisphere. Hemispheric infarcted areas were calculated separately on each coronal slice and scored from 1 to 5, and each such area was defined as a percentage of the affected hemisphere. The infarction volume did not differ significantly across the samples in MCAO-

neurotransmitter systems. P4 has been well known to affect transcription processes through the action on the classical nuclear progesterone's receptor (P4R) followed by multiple interactions with DNA and sequence-specific transcription factors (Beato et al., 1995; Guerra-Araiza et al., 2003). The activation of P4R regulates the expression of anti-apoptotic proteins such as bcl-2, and pro-apoptotic genes including bax and bad and caspase-3 (Schlesinger and Saito, 2006). On the transcriptional level, P4 reduces both the nuclear concentration of NFκB and expression of NFκB target genes. P4 has been found to influence the activity of many signaling pathways so-called "nongenomic mechanisms" *via* a membrane-associated P4R (mP4R) that lacks functional DNA-binding domain (Guerra-Araiza et al., 2009). Increasing evidence indicates that P4R activates Src-ERK signaling pathway which serves as an indicator of growth factor activity in mammalian breast cancer cells (Boonyaratanakornkit et al., 2008; Faivre and Lange, 2007). Cai et al. (2008) has demonstrated that P4 triggers P4R-mediated long-lasting (> 48 h) phosphorylation of ERK1/2 and enhances the translocation of phosphorelated ERK2 into the nucleus. In addition, rapid effects of P4 is suggested to be mediated by membrane-associated P4 binding protein 25-Dx (Meffre et al., 2005) to increase the level of phosphorylated Akt in neuronal cells (Singh et al., 2001). The membrane-associated P4R component 1 (PGRMC1) has been reported to elevate the level of Akt phosphorylation in breast cancer (Neubauer et al., 2008). P4 increases the phosphorylation of ERK and Akt, and the expression of the regulatory (p85) subunits of phosphoinositide-3 kinase (PI3K) in the brain (Guerra-Araiza et al., 2009). Furthermore, the P4's metabolite allopregnanolone (ALLO) potentiates the GABAergic synapse activity (Ardeshiri et al., 2006). Finally, much attention has recently been attracted to the antagonizing effects of P4 on sigma-1 (σ1) receptor (Maurice et al., 2006; Monnet & Maurice, 2006).

The objective of the present study was to determine the P4-neuroprotective effect and its effective therapeutic time-window after transient cerebral ischemia. To this end, male animals subjected to 60 min middle cerebral artery occlusion (MCAO) were given a pair of intraperitoneal injections of P4 (4 mg/kg) separated by 8 h starting at 1, 24, 48, 72 or 96 h after the initiation of cerebral ischemia by middle cerebral artery occlusion (MCAO), and the size of brain infarct, loss of pyramidal neurons in the hippocampal CA1 and cognitive performance of the animals were assessed on 7th day after MCAO. Using pharmacologic tools and western blot analysis, molecular mechanisms underlying the P4-neuroprotective effects against ischemia-induced cerebral injury were also investigated.

### **2. Materials and methods**

### **2.1 Experimental animals**

The present studies were approved by Animal Care and Ethical Committee of Nanjing Medical University. All procedures were in accordance with the guidelines of Institute for Laboratory Animal Research of Nanjing Medical University. Male Sprague-Dawley rats (200-250g, Oriental Bio Service Inc., Nanjing, China) before experiments were used throughout the study. We chose to use only adult male rats in the present study to avoide influence of the E2 effects (Nilsen and Brinton, 2003). Animal rooms were maintained on a 12:12 light-dark cycle starting at AM 7:00 and kept at a temperature of 22-23°C. The animals were permitted free access to food and tap water. All efforts were made to minimize animal suffering and to reduce the number of animals used.

### **2.2 Preparation of focal cerebral ischemia model**

Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Rats were anesthetized with a mixture of 70% N2O and 30% O2 containing 2.5% isoflurane, and were maintained by the inhalation of 1.5% isoflurane during the operation. Briefly, a heatblunted black monofilament surgical suture (4/0 G) was inserted into the internal carotid artery to occlude the origin of MCA. Adequacy of vascular occlusion and reperfusion was monitored in the front parietal cortex of the occluded side with a multi-channel laser Doppler flow-meter (PF5050 Q4, Perimed, Jarfalla, Sweden). Body and head temperatures were controlled at 37±0.5°C using a water pads. Arterial blood pressure and gases were monitored through a femoral catheter. After 60 min of occlusion, the filament was withdrawn to allow for reperfusion. Sham-operated (sham-op) animals were treated identically, except that MCAs were not occluded.

### **2.3 Drug administration**

480 Advances in the Preclinical Study of Ischemic Stroke

neurotransmitter systems. P4 has been well known to affect transcription processes through the action on the classical nuclear progesterone's receptor (P4R) followed by multiple interactions with DNA and sequence-specific transcription factors (Beato et al., 1995; Guerra-Araiza et al., 2003). The activation of P4R regulates the expression of anti-apoptotic proteins such as bcl-2, and pro-apoptotic genes including bax and bad and caspase-3 (Schlesinger and Saito, 2006). On the transcriptional level, P4 reduces both the nuclear concentration of NFκB and expression of NFκB target genes. P4 has been found to influence the activity of many signaling pathways so-called "nongenomic mechanisms" *via* a membrane-associated P4R (mP4R) that lacks functional DNA-binding domain (Guerra-Araiza et al., 2009). Increasing evidence indicates that P4R activates Src-ERK signaling pathway which serves as an indicator of growth factor activity in mammalian breast cancer cells (Boonyaratanakornkit et al., 2008; Faivre and Lange, 2007). Cai et al. (2008) has demonstrated that P4 triggers P4R-mediated long-lasting (> 48 h) phosphorylation of ERK1/2 and enhances the translocation of phosphorelated ERK2 into the nucleus. In addition, rapid effects of P4 is suggested to be mediated by membrane-associated P4 binding protein 25-Dx (Meffre et al., 2005) to increase the level of phosphorylated Akt in neuronal cells (Singh et al., 2001). The membrane-associated P4R component 1 (PGRMC1) has been reported to elevate the level of Akt phosphorylation in breast cancer (Neubauer et al., 2008). P4 increases the phosphorylation of ERK and Akt, and the expression of the regulatory (p85) subunits of phosphoinositide-3 kinase (PI3K) in the brain (Guerra-Araiza et al., 2009). Furthermore, the P4's metabolite allopregnanolone (ALLO) potentiates the GABAergic synapse activity (Ardeshiri et al., 2006). Finally, much attention has recently been attracted to the antagonizing effects of P4 on sigma-1 (σ1) receptor (Maurice et al., 2006;

The objective of the present study was to determine the P4-neuroprotective effect and its effective therapeutic time-window after transient cerebral ischemia. To this end, male animals subjected to 60 min middle cerebral artery occlusion (MCAO) were given a pair of intraperitoneal injections of P4 (4 mg/kg) separated by 8 h starting at 1, 24, 48, 72 or 96 h after the initiation of cerebral ischemia by middle cerebral artery occlusion (MCAO), and the size of brain infarct, loss of pyramidal neurons in the hippocampal CA1 and cognitive performance of the animals were assessed on 7th day after MCAO. Using pharmacologic tools and western blot analysis, molecular mechanisms underlying the P4-neuroprotective

The present studies were approved by Animal Care and Ethical Committee of Nanjing Medical University. All procedures were in accordance with the guidelines of Institute for Laboratory Animal Research of Nanjing Medical University. Male Sprague-Dawley rats (200-250g, Oriental Bio Service Inc., Nanjing, China) before experiments were used throughout the study. We chose to use only adult male rats in the present study to avoide influence of the E2 effects (Nilsen and Brinton, 2003). Animal rooms were maintained on a 12:12 light-dark cycle starting at AM 7:00 and kept at a temperature of 22-23°C. The animals were permitted free access to food and tap water. All efforts were made to minimize animal

effects against ischemia-induced cerebral injury were also investigated.

suffering and to reduce the number of animals used.

Monnet & Maurice, 2006).

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

P4 was dissolved in dimethylsulfoxide (DMSO), then in sesame oil to a final concentration of 1% DMSO. P4 (4 mg/kg) was intraperitonealy (i.p.) injected. Two injections of P4 with 8 h interval were given starting at 1, 24, 48, 72 or 96 h after the initiation of MCAO (post-MCAO). We selected this low dosage because P4 at this dosage is reported to significantly reduce the ischemic damage and regulate anti-apoptotic gene expression following TBI in rats (Stein, 2008). In addition, our study (Cai et al., 2008) determined that the treatment with the same dosage of P4 increases ERK1/2 phosphorylation.

To analyze the molecular mechanisms underlying the P4-actions, the P4R antagonist RU486 (3 mg/kg) and the 5α-reductase inhibitor finasteride (20 mg/kg) (Finn et al., 2006) were given by intraperitoneal injection (i.p.) at 30 min before each administration of P4. The MEK inhibitor U0126 (0.5 nmol) and the PI3K inhibitor LY294002 (0.3 nmol) were injected into the cerebroventricle (i.c.v.) at 30 min before each injections of P4. For i.c.v. implantation, rats were anaesthetized with ketamine (80 mg/kg i.p.). A guide cannula (10 mm length, 22 gauge) aiming above the right lateral ventricle was implanted. The inhibitors or vehicle were injected with a stepper-motorized micro-syringe (Stoelting, Wood Dale, IL, USA) at a rate of 0.5 μl/min. The drugs were prepared freshly on the day of experiment (final volume = 5µl/rat). Control rats were given an equal volume of vehicle.

### **2.4 Histological examination**

### **2.4.1 Infarct volume measuring**

Brains were removed on 7th day post-MCAO, sectioned into 5 equidistant slices (2.0-mmthick), and incubated in a 2% 2,3,5-triphenyle-tetrazoliumchloride (TTC) solution (15 min) to visualize infarcted tissue. Measurements were performed by manually outlining the margins of the infarcted areas. Unstained areas of brain sections were defined as infarcted using the image analysis software NIH-Image 3.12. Briefly, the infarcted area on the ipsilateral side was indirectly measured by subtracting the noninfarcted area in the ipsilateral hemisphere from the total nonischaemic area of the contralateral (nonischaemic) hemisphere. Hemispheric infarcted areas were calculated separately on each coronal slice and scored from 1 to 5, and each such area was defined as a percentage of the affected hemisphere. The infarction volume did not differ significantly across the samples in MCAOgroups (P > 0.05).

Time-Window of Progesterone Neuroprotection

with the image analysis software package, NIH Image.

P<0.05 were considered statistically significant.

**2.7 Data analysis/statistics** 

**3. Results** 

**cognitive impairment** 

After Stroke and Its Underlying Molecular Mechanisms 483

incubated with an HRP-labeled secondary antibody, and developed using the ECL detection Kit (Amersham Biosciences, Piscataway, NJ). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce Chemical Co, Rockford IL) for 5 min, re-blocked for 60 min with 5% nonfat dried milk at room temperature, then incubated with anti-total ERK1/2 (diluted 1:5000, Cell Signaling, Beverly, MA). In each experiment, levels of both ERK1/2 and phosphorelated ERK1/2 (phospho-ERK1/2) were measured in the hippocampus of ischemic hemisphere in MCAO-rats and sham-op rats (control). For each animal, phospho-ERK1/2 was normalized by respective ERK1/2 protein. Each experimental group contained 12 rats. The Western blot bands were scanned and analyzed

Data were retrieved and processed with the software Microcal Origin 6.1. The group data are expressed as the means ± standard error (SE). For comparison between two groups the 2-sided student t-test was used. For comparison between more than 2 groups one-way analysis of variance (ANOVA) followed by the Bonferroni's post hoc test was performed. Statistical analysis was performed using the software State7 (STATA Corporation, USA). For the analysis of Morris water maze test, statistical differences were determined by an ANOVA with repeated measures, followed by the Bonferroni post hoc test. Statistical analysis was performed using the State7 software (Stata Corporation, USA). Differences at

**3.1 Effective time-window of P4 against ischemic brain infarct and motor dysfunction**  To examine the effects of P4 on ischemia-induced brain infarction, a pair of injections (i.p.) of P4 (4 mg/kg) with an 8 h interval were given starting at 1, 24, 48, 72 or 96 h post-MCAO (Figure 1A). On 7th day post-MCAO the results of TTC staining showed that the 60 min MCAO caused approximately 34% brain infarction mainly in the striatum and the frontoparietal cortex (Figure 1B). In comparison with vehicle-treated MCAO-rats, infarct volumes were significantly decreased by the administration of P4 at 1 and 24 h (P<0.01, n=12) or 48 and 72 h post-MCAO (P<0.05, n=12), but not at 96 h (P>0.05, n=12). Similarly, the performance of rota rod test on 7th day post-MCAO perfectly restored in MCAO-rats treated with P4 at 1, 24, 48 (P<0.01, n=12) and 72 h post-MCAO (P<0.05, n=12; Figure 1C) compared to vehicle-treated MCAO-rats. By contrast, P4 when administered at 96 h post-MCAO had no effect on the ischemia-induced motor impairment (P>0.05, n=12). The results indicate that the administration of P4 after stroke exerts a powerful neuroprotection against

ischemia-induced brain damages with a wide effective time-window up to 72 h.

**3.2 Effective time-window of P4 against ischemic death of neuronal cells and** 

Consistent with the previous report (Cai et al., 2008), the number of hippocampal CA1 pyramidal neurons in ischemic hemisphere decreased to approximately 50% of sham-op hemisphere on 7th day post-MCAO (P<0.01, n=8; Figure 2B). To examine the effects of P4 on ischemia-induced death of pyramidal neurons and impairment of spatial memory, a pair of injections (i.p.) of P4 (4 mg/kg) with an 8 h interval was given at 1, 24, 48, 72 or 96 h

### **2.4.2 Pyramidal cells counting**

Rats were deeply anesthetized with pentobarbital (50 mg/kg), transcardially perfused with 4% paraformaldehyde at 7th day post-MCAO. The brains were removed, post-fixed for 24 h, and then processed for paraffin embedding. Coronal sections (4-μm-thick) including the dorsal hippocampus were cut and stained with toluidine blue. Healthy pyramidal cells showing a round cell body with a plainly stained nucleus were counted by eye using a conventional light microscope (PD70) with a 100×objective. The number of surviving CA1 pyramidal cells per 1 mm length along the extent of pyramidal layer were counted as neuronal density (cells/mm) (Cai et al., 2008). We also made supplemental examinations on several slices stained with trypan blue that stains dead cells, and obtained essentially the same result as that determined by eye with hematoxylin and eosin (HE) stained slices.

### **2.5 Behavioral analysis**

### **2.5.1 Rota rod test**

The Rota rod test was used to assess the sensorimotor coordination of rodent on 7th day post-MCAO (see Figure 1A) using an accelerating treadmill (TSE Systems, Germany; 3 cm diameter). For Rota rod training sessions, animals were habituated to the Rota rod and trained to remain on the rotating drum (constant speed 6 rpm) for a minimum of 90 s to provide a preoperative baseline. Animals not achieving baseline criteria were excluded from further study. In the testing sessions, animals were placed on the Rota rod, and the rotational speed was set to accelerate from 6 to 19 rpm over 180 s. The latency time to fall (time on rod), namely the time when the animal first fell off the drum, was recorded.

### **2.5.2 Morris Water Maze (MWM) test**

Morris water maze test was performed from 4th day post-MCAO for consecutive 4 days (see Figure 2A) using a swimming pool (diameter: 180 cm; height: 30 cm) filled with water (20°C) to a depth of 15 cm. A transparent plexiglass platform (7 cm in diameter) was submerged with the top located 1 cm below the water surface. Swimming paths were analyzed by a computer system with a video camera (AXIS-90 Target/2; Neuroscience). After reaching the platform, rat was allowed to remain on it for 30 sec. If the rat did not find the platform within 90 sec, the rat was put on the platform for 30 sec. The escape-latency to reach hiddenplatform was measured from three trials to provide a single value for each rat.

### **2.6 Western blot analysis**

Rats were decapitated under deep anesthesia with ethyl ether. The hippocampus in ischemic hemisphere was taken quickly, then homogenized in a lysis buffer containing 50 mM TriseHCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). Protein concentration was determined with BCA Protein Assay Kit (Pierce, Rochford, IL, USA). Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyphorylated difluoride (PVDF) membrane. The membranes were incubated with 5% bovine serum albumin or 5% nonfat dried milk in tris-buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature, and then were incubated with a mouse monoclonal anti-phospho-ERK1/2 antibody (diluted 1:2500, Cell Signaling, Beverly, MA) at 4°C overnight. After being washed with TBST for three times, the membranes were incubated with an HRP-labeled secondary antibody, and developed using the ECL detection Kit (Amersham Biosciences, Piscataway, NJ). Following visualization, the blots were stripped by incubation in stripping buffer (Restore, Pierce Chemical Co, Rockford IL) for 5 min, re-blocked for 60 min with 5% nonfat dried milk at room temperature, then incubated with anti-total ERK1/2 (diluted 1:5000, Cell Signaling, Beverly, MA). In each experiment, levels of both ERK1/2 and phosphorelated ERK1/2 (phospho-ERK1/2) were measured in the hippocampus of ischemic hemisphere in MCAO-rats and sham-op rats (control). For each animal, phospho-ERK1/2 was normalized by respective ERK1/2 protein. Each experimental group contained 12 rats. The Western blot bands were scanned and analyzed with the image analysis software package, NIH Image.

### **2.7 Data analysis/statistics**

Data were retrieved and processed with the software Microcal Origin 6.1. The group data are expressed as the means ± standard error (SE). For comparison between two groups the 2-sided student t-test was used. For comparison between more than 2 groups one-way analysis of variance (ANOVA) followed by the Bonferroni's post hoc test was performed. Statistical analysis was performed using the software State7 (STATA Corporation, USA). For the analysis of Morris water maze test, statistical differences were determined by an ANOVA with repeated measures, followed by the Bonferroni post hoc test. Statistical analysis was performed using the State7 software (Stata Corporation, USA). Differences at P<0.05 were considered statistically significant.

### **3. Results**

482 Advances in the Preclinical Study of Ischemic Stroke

Rats were deeply anesthetized with pentobarbital (50 mg/kg), transcardially perfused with 4% paraformaldehyde at 7th day post-MCAO. The brains were removed, post-fixed for 24 h, and then processed for paraffin embedding. Coronal sections (4-μm-thick) including the dorsal hippocampus were cut and stained with toluidine blue. Healthy pyramidal cells showing a round cell body with a plainly stained nucleus were counted by eye using a conventional light microscope (PD70) with a 100×objective. The number of surviving CA1 pyramidal cells per 1 mm length along the extent of pyramidal layer were counted as neuronal density (cells/mm) (Cai et al., 2008). We also made supplemental examinations on several slices stained with trypan blue that stains dead cells, and obtained essentially the same result as that determined by eye with hematoxylin and eosin (HE) stained slices.

The Rota rod test was used to assess the sensorimotor coordination of rodent on 7th day post-MCAO (see Figure 1A) using an accelerating treadmill (TSE Systems, Germany; 3 cm diameter). For Rota rod training sessions, animals were habituated to the Rota rod and trained to remain on the rotating drum (constant speed 6 rpm) for a minimum of 90 s to provide a preoperative baseline. Animals not achieving baseline criteria were excluded from further study. In the testing sessions, animals were placed on the Rota rod, and the rotational speed was set to accelerate from 6 to 19 rpm over 180 s. The latency time to fall

Morris water maze test was performed from 4th day post-MCAO for consecutive 4 days (see Figure 2A) using a swimming pool (diameter: 180 cm; height: 30 cm) filled with water (20°C) to a depth of 15 cm. A transparent plexiglass platform (7 cm in diameter) was submerged with the top located 1 cm below the water surface. Swimming paths were analyzed by a computer system with a video camera (AXIS-90 Target/2; Neuroscience). After reaching the platform, rat was allowed to remain on it for 30 sec. If the rat did not find the platform within 90 sec, the rat was put on the platform for 30 sec. The escape-latency to reach hidden-

Rats were decapitated under deep anesthesia with ethyl ether. The hippocampus in ischemic hemisphere was taken quickly, then homogenized in a lysis buffer containing 50 mM TriseHCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). Protein concentration was determined with BCA Protein Assay Kit (Pierce, Rochford, IL, USA). Total proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyphorylated difluoride (PVDF) membrane. The membranes were incubated with 5% bovine serum albumin or 5% nonfat dried milk in tris-buffered saline containing 0.1% Tween 20 (TBST) for 60 min at room temperature, and then were incubated with a mouse monoclonal anti-phospho-ERK1/2 antibody (diluted 1:2500, Cell Signaling, Beverly, MA) at 4°C overnight. After being washed with TBST for three times, the membranes were

(time on rod), namely the time when the animal first fell off the drum, was recorded.

platform was measured from three trials to provide a single value for each rat.

**2.4.2 Pyramidal cells counting** 

**2.5 Behavioral analysis 2.5.1 Rota rod test** 

**2.5.2 Morris Water Maze (MWM) test** 

**2.6 Western blot analysis** 

### **3.1 Effective time-window of P4 against ischemic brain infarct and motor dysfunction**

To examine the effects of P4 on ischemia-induced brain infarction, a pair of injections (i.p.) of P4 (4 mg/kg) with an 8 h interval were given starting at 1, 24, 48, 72 or 96 h post-MCAO (Figure 1A). On 7th day post-MCAO the results of TTC staining showed that the 60 min MCAO caused approximately 34% brain infarction mainly in the striatum and the frontoparietal cortex (Figure 1B). In comparison with vehicle-treated MCAO-rats, infarct volumes were significantly decreased by the administration of P4 at 1 and 24 h (P<0.01, n=12) or 48 and 72 h post-MCAO (P<0.05, n=12), but not at 96 h (P>0.05, n=12). Similarly, the performance of rota rod test on 7th day post-MCAO perfectly restored in MCAO-rats treated with P4 at 1, 24, 48 (P<0.01, n=12) and 72 h post-MCAO (P<0.05, n=12; Figure 1C) compared to vehicle-treated MCAO-rats. By contrast, P4 when administered at 96 h post-MCAO had no effect on the ischemia-induced motor impairment (P>0.05, n=12). The results indicate that the administration of P4 after stroke exerts a powerful neuroprotection against ischemia-induced brain damages with a wide effective time-window up to 72 h.

### **3.2 Effective time-window of P4 against ischemic death of neuronal cells and cognitive impairment**

Consistent with the previous report (Cai et al., 2008), the number of hippocampal CA1 pyramidal neurons in ischemic hemisphere decreased to approximately 50% of sham-op hemisphere on 7th day post-MCAO (P<0.01, n=8; Figure 2B). To examine the effects of P4 on ischemia-induced death of pyramidal neurons and impairment of spatial memory, a pair of injections (i.p.) of P4 (4 mg/kg) with an 8 h interval was given at 1, 24, 48, 72 or 96 h

Time-Window of Progesterone Neuroprotection

After Stroke and Its Underlying Molecular Mechanisms 485

Fig. 2. Effects of P4 on ischemia-induced neuronal cell death and cognitive impairment. (**A**) Time chart of experimental procedure in Figure 2B&C. Two injections (i.p.) of P4 (4 mg/kg) with 8 h interval (black arrows) were given starting at 1, 24, 48, 72 or 96 h post-MCAO. (**B**) Time-window of P4-effect against MCAO-induced neuronal cell death.

Representative pictures of hippocampal CA1 region in sham-op rats, MCAO-rats, P4-treated MCAO-rats (upper panels). Scale bar=100μm. Bar graph shows density of surviving neurons in the hippocampal CA1 on 7th day post-MCAO. Horizontal hollow bar: P4 administration. (**C**) Time-window of P4-effect against MCAO-induced deficits in spatial memory. Typical

Fig. 1. Effects of P4 on ischemia-induced brain infarct and motor dysfunction. (**A**) Time chart of experimental procedure in Figure 1B&C. Two injections (i.p.) of P4 (4 mg/kg) with 8 h interval (black arrows) were given starting at 1, 24, 48, 72 or 96 h post-MCAO. (**B**) Timewindow of P4-effect against MCAO-induced brain infarct. Representative pictures of TTC-staining in sham-op rats, MCAO-rats, P4-treated MCAO-rats (upper panels). Bar graph shows the size of brain infarct that was expressed as percentage of the non-infarcted hemisphere on 7th day post-MCAO. Horizontal hollow bar: P4 administration. (**C**) Time-window of P4-effect against ischemic motor dysfunction. Bar graph shows time on rod in sham-op (open bar) and MCAO-rats (hatched bars) on 7th day post-MCAO. \*\*P<0.01 vs. sham-op rats; #P<0.05 and ##P<0.01 vs. MCAO-rats.

post-MCAO. The number of dead pyramidal cells was significantly reduced by the treatment with P4 at 1, 24 and 48 h (P<0.01, n=8) or 72 h post-MCAO (P<0.05, n=8) compared to vehicle-treated MCAO-rats. However, the administration of P4 at 96 h post-MCAO exerted no significant effect in reducing the number of ischemia-induced loss of

Fig. 1. Effects of P4 on ischemia-induced brain infarct and motor dysfunction. (**A**) Time chart of experimental procedure in Figure 1B&C. Two injections (i.p.) of P4 (4 mg/kg) with 8 h interval (black arrows) were given starting at 1, 24, 48, 72 or 96 h post-MCAO. (**B**) Timewindow of P4-effect against MCAO-induced brain infarct. Representative pictures of TTC-staining in sham-op rats, MCAO-rats, P4-treated MCAO-rats (upper panels). Bar graph

shows the size of brain infarct that was expressed as percentage of the non-infarcted hemisphere on 7th day post-MCAO. Horizontal hollow bar: P4 administration.

\*\*P<0.01 vs. sham-op rats; #P<0.05 and ##P<0.01 vs. MCAO-rats.

(**C**) Time-window of P4-effect against ischemic motor dysfunction. Bar graph shows time on rod in sham-op (open bar) and MCAO-rats (hatched bars) on 7th day post-MCAO.

post-MCAO. The number of dead pyramidal cells was significantly reduced by the treatment with P4 at 1, 24 and 48 h (P<0.01, n=8) or 72 h post-MCAO (P<0.05, n=8) compared to vehicle-treated MCAO-rats. However, the administration of P4 at 96 h post-MCAO exerted no significant effect in reducing the number of ischemia-induced loss of

Fig. 2. Effects of P4 on ischemia-induced neuronal cell death and cognitive impairment. (**A**) Time chart of experimental procedure in Figure 2B&C. Two injections (i.p.) of P4 (4 mg/kg) with 8 h interval (black arrows) were given starting at 1, 24, 48, 72 or 96 h post-MCAO. (**B**) Time-window of P4-effect against MCAO-induced neuronal cell death. Representative pictures of hippocampal CA1 region in sham-op rats, MCAO-rats, P4-treated MCAO-rats (upper panels). Scale bar=100μm. Bar graph shows density of surviving neurons in the hippocampal CA1 on 7th day post-MCAO. Horizontal hollow bar: P4 administration. (**C**) Time-window of P4-effect against MCAO-induced deficits in spatial memory. Typical

Time-Window of Progesterone Neuroprotection

mediated mechanism.

After Stroke and Its Underlying Molecular Mechanisms 487

affect the neuroprotection by P4 at 24, 48 or 72 h post-MCAO (P>0.05, n=8). The results indicate that the neuroprotection by P4 at 1 h post-MCAO is, if not all, caused by a protective action of its metabolite ALLO against ischemia-induced brain damage.

**3.4 P4-neuroprotection at 24 and 48 h post-MCAO is mediated by P4R activation**  The neuroprotection by P4 administered at 48 h pre-MCAO has been known to depend on P4R function (Faivre and Lange, 2007). To test this possibility in our case, the nuclear P4R blocker RU486 (3 mg/kg, i.p.) was given at 30 min prior to each P4 injection. The results showed that the pre-treatment with RU486 abolished the neuroprotective effects of P4 at 24 and 48 h post-MCAO against ischemia-induced neuronal death (P<0.01, n=8; Figure 4A) and spatial memory impairment (P<0.01, n=8; Figure 4B), whereas it failed to affect the neuroprotection by P4 at 1 or 72 h post-MCAO (P>0.05, n=8). Meanwhile, in the absence of P4 the administration of RU486 at 24 or 48 h post-MCAO had no effect on either neuronal death (P>0.05, n=8) or spatial cognitive function (P>0.05, n=8). These results suggest that the neuroprotective effect of P4 administered at 24 and 48 h post-MCAO involves the P4R-

finasteride. \*P<0.05 and \*P<0.01 vs. P4-treated MCAO-rats at 1 h post-MCAO.

Fig. 4. Effects of RU486, a R4R antagonist, on the neuroprotection of P4 against

MCAO-induced neuronal cell death (**A**) and cognitive impairment (**B**). Horizontal hollow bar: P4 administration. Animals were treated with RU486 at 30 min before every time P4-injection. Note that the P4-neuroprotection at 24 and 48 h post-MCAO is blocked by RU486. \*P<0.05 and \*P<0.01 vs. P4-treated MCAO-rats at 24 and 48 h post-MCAO.

**3.5 P4-neuroprotection at 24 and 48 h post-MCAO depends on P4R-ERK signaling**  As P4R-mediated ERK1/2 activation protects the ischemic brain damage (Cai et al., 2008), the experiment was designed to explore the involvement of ERK1/2 in the P4R-dependent neuroprotection after MCAO. Expectedly, the ERK kinase (MEK) inhibitor U0126 (0.3 nmol, i.c.v.) blocked the neuroprotection by P4 at 24 and 48 h against MCAO-induced neuronal death (P<0.01, n=8; Figure 5A) and spatial memory impairment (P4 at 24 h post-MCAO, P<0.01, n=8; P4 at 48 h post-MCAO, P<0.05, n=8; Figure 5B), whereas it failed to affect the

trials of ''Morris'' water maze test (left panel) show latency (sec) to reach the hidden-platform against training time (day, day 4-7 post-MCAO) in sham-op rats, MCAO-rats, P4 (1h post-MCAO)-treated MCAO-rats. Bar graph shows the mean latency (±SEM) to reach the hiddenplatform on 4th day post-training. \*\*P<0.01 vs. sham-op rats; #P<0.05 and ##P<0.01 vs. MCAO-rats.

pyramidal cells (P>0.05, n=8). The P4 administration per se caused no observable change in CA1 pyramidal neurons on 7th day after sham-op. Spatial learning and memory function was examined by the Morris water maze test from 4th day post-MCAO for consecutive 4 days (Figure 2A). In comparison with sham-op rats, the escape-latency to reach the hiddenplatform on 7th day post-MCAO increased approximately 2-fold (P<0.01, n=8; Figure 2C). The behavior of acquisition performance coincided with the histological changes; the prolongation of escape-latency was perfectly improved by the treatment with P4 at 1, 24 and 48 h post-MCAO (P<0.01, n=8), while was partially reduced by the injection of P4 at 72 h post-MCAO (P<0.05, n=8). By contrast, the administration of P4 at 96 h post-MCAO failed to affect the prolonged escape-latency (P>0.05, n=8). Both the histological and behavioral examinations here strongly suggest that the effective time-window of the neuroprotection by the P4 treatment is spanning from 1 h to 72 h post-MCAO. In the following sections, we describe the results on the analyses of the molecular mechanisms underlying the P4 affording neuroprotective effects on ischemia-induced death of pyramidal cells.

### **3.3 P4-neuroprotection at 1 h post-MCAO is mediated by its metabolite ALLO**

A recent study (Ciriza et al., 2006) has revealed that the neuroprotection by P4 after ischemic brain injury is abolished by finasteride, a 5α-reductase inhibitor that inhibits the conversion of P4 to allopregnanolone (ALLO). To determine whether P4 exerts neuroprotection through its metabolite ALLO, finasteride (20 mg/kg i.p.) was given at 30 min prior to every P4 injection. The results showed that the pre-treatment with finasteride partially attenuated the neuroprotection of P4 at 1 h post-MCAO against MCAO-induced neuronal death (P<0.05, n=8; Figure 3A) and prolongation of escape-latency (P<0.05, n=8; Figure 3B), but it did not

Fig. 3. Effects of finasteride, a 5α-reductase inhibitor, on the neuroprotection of P4 against MCAO-induced neuronal cell death (**A**) and cognitive impairment (**B**). Horizontal hollow bar: P4 administration. Animals were treated with finasteride at 30 min before every P4-injection. Note that the P4-neuroprotection at 1 h post-MCAO is partially blocked by

trials of ''Morris'' water maze test (left panel) show latency (sec) to reach the hidden-platform against training time (day, day 4-7 post-MCAO) in sham-op rats, MCAO-rats, P4 (1h post-MCAO)-treated MCAO-rats. Bar graph shows the mean latency (±SEM) to reach the hiddenplatform on 4th day post-training. \*\*P<0.01 vs. sham-op rats; #P<0.05 and ##P<0.01 vs.

pyramidal cells (P>0.05, n=8). The P4 administration per se caused no observable change in CA1 pyramidal neurons on 7th day after sham-op. Spatial learning and memory function was examined by the Morris water maze test from 4th day post-MCAO for consecutive 4 days (Figure 2A). In comparison with sham-op rats, the escape-latency to reach the hiddenplatform on 7th day post-MCAO increased approximately 2-fold (P<0.01, n=8; Figure 2C). The behavior of acquisition performance coincided with the histological changes; the prolongation of escape-latency was perfectly improved by the treatment with P4 at 1, 24 and 48 h post-MCAO (P<0.01, n=8), while was partially reduced by the injection of P4 at 72 h post-MCAO (P<0.05, n=8). By contrast, the administration of P4 at 96 h post-MCAO failed to affect the prolonged escape-latency (P>0.05, n=8). Both the histological and behavioral examinations here strongly suggest that the effective time-window of the neuroprotection by the P4 treatment is spanning from 1 h to 72 h post-MCAO. In the following sections, we describe the results on the analyses of the molecular mechanisms underlying the P4

affording neuroprotective effects on ischemia-induced death of pyramidal cells.

**3.3 P4-neuroprotection at 1 h post-MCAO is mediated by its metabolite ALLO** 

A recent study (Ciriza et al., 2006) has revealed that the neuroprotection by P4 after ischemic brain injury is abolished by finasteride, a 5α-reductase inhibitor that inhibits the conversion of P4 to allopregnanolone (ALLO). To determine whether P4 exerts neuroprotection through its metabolite ALLO, finasteride (20 mg/kg i.p.) was given at 30 min prior to every P4 injection. The results showed that the pre-treatment with finasteride partially attenuated the neuroprotection of P4 at 1 h post-MCAO against MCAO-induced neuronal death (P<0.05, n=8; Figure 3A) and prolongation of escape-latency (P<0.05, n=8; Figure 3B), but it did not

Fig. 3. Effects of finasteride, a 5α-reductase inhibitor, on the neuroprotection of P4 against MCAO-induced neuronal cell death (**A**) and cognitive impairment (**B**). Horizontal hollow bar: P4 administration. Animals were treated with finasteride at 30 min before every P4-injection. Note that the P4-neuroprotection at 1 h post-MCAO is partially blocked by

MCAO-rats.

finasteride. \*P<0.05 and \*P<0.01 vs. P4-treated MCAO-rats at 1 h post-MCAO. affect the neuroprotection by P4 at 24, 48 or 72 h post-MCAO (P>0.05, n=8). The results indicate that the neuroprotection by P4 at 1 h post-MCAO is, if not all, caused by a protective action of its metabolite ALLO against ischemia-induced brain damage.

### **3.4 P4-neuroprotection at 24 and 48 h post-MCAO is mediated by P4R activation**

The neuroprotection by P4 administered at 48 h pre-MCAO has been known to depend on P4R function (Faivre and Lange, 2007). To test this possibility in our case, the nuclear P4R blocker RU486 (3 mg/kg, i.p.) was given at 30 min prior to each P4 injection. The results showed that the pre-treatment with RU486 abolished the neuroprotective effects of P4 at 24 and 48 h post-MCAO against ischemia-induced neuronal death (P<0.01, n=8; Figure 4A) and spatial memory impairment (P<0.01, n=8; Figure 4B), whereas it failed to affect the neuroprotection by P4 at 1 or 72 h post-MCAO (P>0.05, n=8). Meanwhile, in the absence of P4 the administration of RU486 at 24 or 48 h post-MCAO had no effect on either neuronal death (P>0.05, n=8) or spatial cognitive function (P>0.05, n=8). These results suggest that the neuroprotective effect of P4 administered at 24 and 48 h post-MCAO involves the P4Rmediated mechanism.

Fig. 4. Effects of RU486, a R4R antagonist, on the neuroprotection of P4 against MCAO-induced neuronal cell death (**A**) and cognitive impairment (**B**). Horizontal hollow bar: P4 administration. Animals were treated with RU486 at 30 min before every time P4-injection. Note that the P4-neuroprotection at 24 and 48 h post-MCAO is blocked by RU486. \*P<0.05 and \*P<0.01 vs. P4-treated MCAO-rats at 24 and 48 h post-MCAO.

### **3.5 P4-neuroprotection at 24 and 48 h post-MCAO depends on P4R-ERK signaling**

As P4R-mediated ERK1/2 activation protects the ischemic brain damage (Cai et al., 2008), the experiment was designed to explore the involvement of ERK1/2 in the P4R-dependent neuroprotection after MCAO. Expectedly, the ERK kinase (MEK) inhibitor U0126 (0.3 nmol, i.c.v.) blocked the neuroprotection by P4 at 24 and 48 h against MCAO-induced neuronal death (P<0.01, n=8; Figure 5A) and spatial memory impairment (P4 at 24 h post-MCAO, P<0.01, n=8; P4 at 48 h post-MCAO, P<0.05, n=8; Figure 5B), whereas it failed to affect the

Time-Window of Progesterone Neuroprotection

P4 injection.

**4. Discussion** 

After Stroke and Its Underlying Molecular Mechanisms 489

phospho-ERK1/2 at 1, 24, 48 and 72 h post-MCAO. Representative western blots represent ERK1/2 phosphorylation immunoreactivity obtained from whole-cell lysates. Level of phospho-ERK1/2 is expressed as a percentage of phospho-ERK1/2 in sham-op rats. (**D**) Effect of P4 on phospho-ERK1/2 at 1, 24, 48 and 72 h post-MCAO. P4 was given at 30 min before harvested hippocampus. Values of phospho-ERK1/2 were normalized by phospho-ERK1/2 in sham-op rats. \*P<0.05 and \*\*P<0.01 vs. sham-op group; #<0.05 vs. MCAO-rats treated with P4 at 24 h post-MCAO. Horizontal axis: Time of post-MCAO p4 injection.

Emerging evidence indicates that transient cerebral ischemia promotes the dephosphorylation of ERK1/2 (Jover-Mengual et al., 2007). To confirm this, kinetics of hippocampal ERK1/2 phosphorylation (phospho-ERK1/2) after MCAO was measured using Western blot analysis. In comparison with that before MCAO, the level of phospho-ERK1/2 was largely increased at 1 h post-MCAO (P<0.05, n=12; Figure 5C), followed by a persistent decrease at 24 h and 48 h post-MCAO (P<0.05, n=12), then returned to the basal level at 72 h post-MCAO (P>0.05, n=12). To investigate the effects of P4 on the changes in ERK1/2 phosphorylation after MCAO, the MCAO-rats were given a single injection of P4 at 1, 24, 48 or 72 h post-MCAO. Thirty minutes after the P4-injection hippocampal preparations were harvested to measure phospho-ERK1/2. As shown in Figure 5D, the treatment with P4 slightly attenuated the increased phospho-ERK1/2 at 1 h post-MCAO (P<0.05, n=12), perfectly rescued the reduction of phospho-ERK1/2 at 24 and 48 h post-MCAO (P<0.01, n=12), and elevated the level of phospho-ERK1/2 at 72 h post-MCAO (P<0.05, n=12). The P4R antagonist RU486 could block the protective effect of P4 on the reduction of phospho-ERK1/2 at 24 h post-MCAO (P<0.05 vs. P4-treated MCAO-rats, n=12). These observations clearly indicate that the P4R-dependent neuroprotection against ischemia-induced brain

damage is mediated, at least in part, through the regulation of ERK1/2 activity.

As P4 increases the level of Akt-phosphorylation, a partial process of PI3K signaling (Guerra-Araiza et al., 2009), the specific PI3K inhibitor LY294002 (0.3 nmol) was injected into the cereboventricle (i.c.v.) at 30 min prior to P4 injection to examine the involvement of PI3K-Akt signaling pathway in the P4-neuroprotection. The results showed that the pretreatment with LY294002 partially attenuated the neuroprotection of P4 at 24 and 48 h post-MCAO (P<0.05, n=8; Figure 6A) and completely abolished the neuroprotection of P4 at 72 h post-MCAO (P<0.01, n=8), whereas it had no effect on the neuroprotection of P4 at 1 h post-MCAO (P>0.05, n=8). Furthermore, the pre-treatment with LY294002 blocked the P4 improved impairment of spatial memory when administered at 24, 48 and 72 h post-MCAO (P<0.05, n=8; Figure 6B). The results indicate that the PI3K-Akt signaling is involved in the P4R-dependent and P4R-independent neuroprotections by P4, depending on the timing of

The present study provides evidence that the treatment with P4 after transient brain ischemia exerts a powerful neuroprotection with a wide effective time-window up to 72 h post-MCAO. The neuroprotective effects of P4 were mediated by different molecular mechanisms depending on the timing of P4 administration after ischemia. The neuroprotection by P4 at 1 h post-MCAO appeared to be caused through P4's metabolite

**3.6 P4-neuroprotection at 24−72 h post-MCAO requires PI3K signaling** 

P4R-independent neuroprotection exerted by P4 administered at 1 or 72 h post-MCAO (P>0.05, n=8). These results indicate that the P4R-mediated neuroprotection is highly coupled with ERK1/2 signaling pathway.

Fig. 5. Effects of U0126, a MEK inhibitor, on the neuroprotection of P4 against MCAOinduced neuronal cell death (**A**) and cognitive impairment (**B**). Horizontal hollow bar: P4 administration. Animals were treated with U0126 at 30 min before every time P4-injection. Note that the phospho-ERK1/2 at 24 and 48 h post-MCAO is blocked by U0126. \*P<0.05 and \*P<0.01 vs. P4-treated MCAO-rats at 24 and 48 h post-MCAO. (**C**) Kinetics of hippocampal

P4R-independent neuroprotection exerted by P4 administered at 1 or 72 h post-MCAO (P>0.05, n=8). These results indicate that the P4R-mediated neuroprotection is highly

Fig. 5. Effects of U0126, a MEK inhibitor, on the neuroprotection of P4 against MCAOinduced neuronal cell death (**A**) and cognitive impairment (**B**). Horizontal hollow bar: P4 administration. Animals were treated with U0126 at 30 min before every time P4-injection. Note that the phospho-ERK1/2 at 24 and 48 h post-MCAO is blocked by U0126. \*P<0.05 and \*P<0.01 vs. P4-treated MCAO-rats at 24 and 48 h post-MCAO. (**C**) Kinetics of hippocampal

coupled with ERK1/2 signaling pathway.

phospho-ERK1/2 at 1, 24, 48 and 72 h post-MCAO. Representative western blots represent ERK1/2 phosphorylation immunoreactivity obtained from whole-cell lysates. Level of phospho-ERK1/2 is expressed as a percentage of phospho-ERK1/2 in sham-op rats. (**D**) Effect of P4 on phospho-ERK1/2 at 1, 24, 48 and 72 h post-MCAO. P4 was given at 30 min before harvested hippocampus. Values of phospho-ERK1/2 were normalized by phospho-ERK1/2 in sham-op rats. \*P<0.05 and \*\*P<0.01 vs. sham-op group; #<0.05 vs. MCAO-rats treated with P4 at 24 h post-MCAO. Horizontal axis: Time of post-MCAO p4 injection.

Emerging evidence indicates that transient cerebral ischemia promotes the dephosphorylation of ERK1/2 (Jover-Mengual et al., 2007). To confirm this, kinetics of hippocampal ERK1/2 phosphorylation (phospho-ERK1/2) after MCAO was measured using Western blot analysis. In comparison with that before MCAO, the level of phospho-ERK1/2 was largely increased at 1 h post-MCAO (P<0.05, n=12; Figure 5C), followed by a persistent decrease at 24 h and 48 h post-MCAO (P<0.05, n=12), then returned to the basal level at 72 h post-MCAO (P>0.05, n=12). To investigate the effects of P4 on the changes in ERK1/2 phosphorylation after MCAO, the MCAO-rats were given a single injection of P4 at 1, 24, 48 or 72 h post-MCAO. Thirty minutes after the P4-injection hippocampal preparations were harvested to measure phospho-ERK1/2. As shown in Figure 5D, the treatment with P4 slightly attenuated the increased phospho-ERK1/2 at 1 h post-MCAO (P<0.05, n=12), perfectly rescued the reduction of phospho-ERK1/2 at 24 and 48 h post-MCAO (P<0.01, n=12), and elevated the level of phospho-ERK1/2 at 72 h post-MCAO (P<0.05, n=12). The P4R antagonist RU486 could block the protective effect of P4 on the reduction of phospho-ERK1/2 at 24 h post-MCAO (P<0.05 vs. P4-treated MCAO-rats, n=12). These observations clearly indicate that the P4R-dependent neuroprotection against ischemia-induced brain damage is mediated, at least in part, through the regulation of ERK1/2 activity.

### **3.6 P4-neuroprotection at 24−72 h post-MCAO requires PI3K signaling**

As P4 increases the level of Akt-phosphorylation, a partial process of PI3K signaling (Guerra-Araiza et al., 2009), the specific PI3K inhibitor LY294002 (0.3 nmol) was injected into the cereboventricle (i.c.v.) at 30 min prior to P4 injection to examine the involvement of PI3K-Akt signaling pathway in the P4-neuroprotection. The results showed that the pretreatment with LY294002 partially attenuated the neuroprotection of P4 at 24 and 48 h post-MCAO (P<0.05, n=8; Figure 6A) and completely abolished the neuroprotection of P4 at 72 h post-MCAO (P<0.01, n=8), whereas it had no effect on the neuroprotection of P4 at 1 h post-MCAO (P>0.05, n=8). Furthermore, the pre-treatment with LY294002 blocked the P4 improved impairment of spatial memory when administered at 24, 48 and 72 h post-MCAO (P<0.05, n=8; Figure 6B). The results indicate that the PI3K-Akt signaling is involved in the P4R-dependent and P4R-independent neuroprotections by P4, depending on the timing of P4 injection.

### **4. Discussion**

The present study provides evidence that the treatment with P4 after transient brain ischemia exerts a powerful neuroprotection with a wide effective time-window up to 72 h post-MCAO. The neuroprotective effects of P4 were mediated by different molecular mechanisms depending on the timing of P4 administration after ischemia. The neuroprotection by P4 at 1 h post-MCAO appeared to be caused through P4's metabolite

Time-Window of Progesterone Neuroprotection

effects on NMDAr-Ca2+ influx in an acute phase after stroke.

**4.2 P4R-dependent ERK activation at 24—48 h after stroke** 

dependent neuroprotection is closely coupled with ERK1/2 signaling.

After Stroke and Its Underlying Molecular Mechanisms 491

However, we noted that the treatment with finasteride could not completely block the P4 neuroprotection at 1 h post-MCAO (see Figure 3A). Excessive presynaptic glutamate releases after cerebral ischemia lead to neuronal death mainly by excessive calcium entry through N-methyl-D-aspartate receptor (NMDAr). Our recent study (Cai et al., 2008) has revealed that P4, as a potential σ1 receptor antagonist (Monnet and Maurice, 2006), reduces Ca2+ influx across NMDAr-channels to protect hippocampal neurons from ischemia-induced cell death. In addition, at 1 h after brain ischemia the activation of σ1 receptor by PRE-084, a σ1 receptor agonist, exacerbates ischemia-induced neuronal cell death in an NMDArdependent manner (Li et al., 2009). However, conflicting results have reported that the activation of σ1 receptor enhances presynaptic glutamate release in the hippocampal CA1 (Meyer et al., 2002), and promotes the Ca2+ influx across NMDAr-channels (Monnet et al., 2003) and the Ca2+ efflux from calcium pools via inositol 1,4,5-trisphosphate receptors (Su and Hayashi, 2003). This discrepancy may be due to the difference in experimental condition or timing of P4 action. Further studies are required to directly observe the P4

Our results revealed that the P4R-mediated ERK1/2 signaling was involved in the neuroprotection by P4 at 24—48 h post-MCAO. P4R ligand has been demonstrated to induce a transient (5—10 min after P4-application) activation of Src-Ras-ERK1/2 and a persistent (6—72 h) ERK1/2 activation (Faivre and Lange, 2007). Recently, Cai et al. (2008) provided in vivo evidence that P4 acts on P4R to trigger a long-lasting (> 48 hr) phosphorylation of ERK1/2, resulting in a promoted translocation of phosphorelated ERK2 into the nucleus. The translocation of ERK1/2 is a pivotal and necessary process for the activation of cAMP response element binding protein (CREB) (Nilsen and Brinton, 2003). The ERK1/2-CREB signaling has been implicated to play a critical role in the brain ischemic tolerance (Gonzalez-Zulueta et al., 2000) and neuronal cell survival (Singh, 2005; 2006). The CREB cascade can increase the expression of anti-apoptotic molecules such as Bcl-2 and Bcl-XL (Yao et al., 2005) and decrease the expression of pro-apoptotic molecules such as Bax, Bad and caspase-3 (Djebaili et al., 2004). Consistent with the results reported by Jover-Mengual et al. (2007), we in the present study showed a decreased activity of ERK1/2 at 24 and 48 h post-MCAO. More importantly, the activation of P4R at 24—48 h after ischemia could rescue the down-regulation of ERK1/2. Therefore, it is highly likely that the P4R-

On the other hand, western blot analysis showed a transient elevation of ERK1/2 phosphorylation at 1 h post-MCAO. The elevation of ERK1/2 activity immediately after stroke has also been observed in humans (Slevin et al., 2000) and a rat model of cerebral ischemia (Wang et al., 2003), in which increased intracellular Ca2+ levels ([Ca2+]i) after ischemia seem to lead hyper-activation of ERK1/2. Alessandrini et al. (1999) and Namura et al. (2001) provided evidence for a neuroprotective role of MEK inhibitor following transient ischemia as manifested by the reduction of infarct size and improvement of functional outcome. The neuroprotection by MEK-inhibition in ischemic brain is associated with an activation of potential anti-apoptotic pathway that suppresses caspase-3 activation and apoptosis (Wang et al., 2003). To our surprise, we observed that the treatment with P4 could attenuate the elevation of ERK1/2 phosphorylation at 1 h post-MCAO. Because the neuroprotection by P4 at 1 h post-MCAO was P4R-independent, it is proposed that P4

allopregnanolone (ALLO) because the protection was significantly attenuated by the 5αreductase inhibitor finasteride. The neuroprotection of P4 at 24 and 48 h post-MCAO appeared to be P4R-dependent through rescuing the down-regulation of ERK1/2 phosphorylation after stroke. The neuroprotective effects of P4 at 72 h post-MCAO required PI3K activation in a P4R-independent way.

Fig. 6. Involvement of PI3K in P4-neuroprotection. Animals were treated with the specific PI3K inhibitor LY294002 (LY) at 30 min before administration of P4. Horizontal hollow bar: P4 administration. Note that the P4-neuroprotection at 24−72 hr post-MCAO requires PI3K signaling. \*P<0.05 and \*\*P<0.01 vs. MCAO-rats treated with P4 at 24, 48 and 72 hr post-MCAO.

### **4.1 Anti-excitotoxic effect of P4's metabolite ALLO at 1 h after MCAO**

One recent report indicates that either P4 or ALLO when administered at 2 h post-MCAO is effective in reducing the infarct volume after focal brain ischemia, where ALLO shows more effective neuroprotection than its parent compound (Sayeed et al., 2007). Similarly, our results in the present study showed that the neuroprotection of P4 at 1 h post-MCAO was sensitive to the 5α-reductase inhibitor fenasteride. Thus, it is proposed that the acute neuroprotection of P4 within 1—2 h ischemia/reperfusion is caused by ALLO, a positive regulator of GABAA receptor (Belelli & Lambert, 2005). This notion is supported by an earlier study (Ardeshiri et al., 2006) showing that the GABAA receptor antagonist picrotoxin could prevent the neuroprotection afforded by P4. The P4 neuroprotection mainly focuses on some populations of neurons that are sensitive to excitotoxicty, including the pyramidal neurons in the hippocampus and cerebral cortex, Purkinje cells in the cerebellum, as well as the neurons in the dorsal striatum and the caudate nucleus (Monnet and Maurice, 2006; Schumacher et al., 2007). Immediately after ischemia, excessive presynaptic glutamate releases result in the accumulation of extracellular glutamate to reach concentrations that induce over-activation of glutamate receptors called excitotoxicity (Jabaudon et al., 2000; Phillis and O'Regan, 2003). The process of excitotoxicity has been demonstrated in several experimental models of cerebral ischemia (Butcher et al., 1990). Therefore, it is highly likely that P4 and ALLO at 1 h post-MCAO prevent the brain injury by suppressing overexcitation of pyramidal neurons through the activation of GABAA receptors.

allopregnanolone (ALLO) because the protection was significantly attenuated by the 5αreductase inhibitor finasteride. The neuroprotection of P4 at 24 and 48 h post-MCAO appeared to be P4R-dependent through rescuing the down-regulation of ERK1/2 phosphorylation after stroke. The neuroprotective effects of P4 at 72 h post-MCAO required

Fig. 6. Involvement of PI3K in P4-neuroprotection. Animals were treated with the specific PI3K inhibitor LY294002 (LY) at 30 min before administration of P4. Horizontal hollow bar: P4 administration. Note that the P4-neuroprotection at 24−72 hr post-MCAO requires PI3K signaling. \*P<0.05 and \*\*P<0.01 vs. MCAO-rats treated with P4 at 24, 48 and 72 hr post-

One recent report indicates that either P4 or ALLO when administered at 2 h post-MCAO is effective in reducing the infarct volume after focal brain ischemia, where ALLO shows more effective neuroprotection than its parent compound (Sayeed et al., 2007). Similarly, our results in the present study showed that the neuroprotection of P4 at 1 h post-MCAO was sensitive to the 5α-reductase inhibitor fenasteride. Thus, it is proposed that the acute neuroprotection of P4 within 1—2 h ischemia/reperfusion is caused by ALLO, a positive regulator of GABAA receptor (Belelli & Lambert, 2005). This notion is supported by an earlier study (Ardeshiri et al., 2006) showing that the GABAA receptor antagonist picrotoxin could prevent the neuroprotection afforded by P4. The P4 neuroprotection mainly focuses on some populations of neurons that are sensitive to excitotoxicty, including the pyramidal neurons in the hippocampus and cerebral cortex, Purkinje cells in the cerebellum, as well as the neurons in the dorsal striatum and the caudate nucleus (Monnet and Maurice, 2006; Schumacher et al., 2007). Immediately after ischemia, excessive presynaptic glutamate releases result in the accumulation of extracellular glutamate to reach concentrations that induce over-activation of glutamate receptors called excitotoxicity (Jabaudon et al., 2000; Phillis and O'Regan, 2003). The process of excitotoxicity has been demonstrated in several experimental models of cerebral ischemia (Butcher et al., 1990). Therefore, it is highly likely that P4 and ALLO at 1 h post-MCAO prevent the brain injury by suppressing over-

**4.1 Anti-excitotoxic effect of P4's metabolite ALLO at 1 h after MCAO** 

excitation of pyramidal neurons through the activation of GABAA receptors.

PI3K activation in a P4R-independent way.

MCAO.

However, we noted that the treatment with finasteride could not completely block the P4 neuroprotection at 1 h post-MCAO (see Figure 3A). Excessive presynaptic glutamate releases after cerebral ischemia lead to neuronal death mainly by excessive calcium entry through N-methyl-D-aspartate receptor (NMDAr). Our recent study (Cai et al., 2008) has revealed that P4, as a potential σ1 receptor antagonist (Monnet and Maurice, 2006), reduces Ca2+ influx across NMDAr-channels to protect hippocampal neurons from ischemia-induced cell death. In addition, at 1 h after brain ischemia the activation of σ1 receptor by PRE-084, a σ1 receptor agonist, exacerbates ischemia-induced neuronal cell death in an NMDArdependent manner (Li et al., 2009). However, conflicting results have reported that the activation of σ1 receptor enhances presynaptic glutamate release in the hippocampal CA1 (Meyer et al., 2002), and promotes the Ca2+ influx across NMDAr-channels (Monnet et al., 2003) and the Ca2+ efflux from calcium pools via inositol 1,4,5-trisphosphate receptors (Su and Hayashi, 2003). This discrepancy may be due to the difference in experimental condition or timing of P4 action. Further studies are required to directly observe the P4 effects on NMDAr-Ca2+ influx in an acute phase after stroke.

### **4.2 P4R-dependent ERK activation at 24—48 h after stroke**

Our results revealed that the P4R-mediated ERK1/2 signaling was involved in the neuroprotection by P4 at 24—48 h post-MCAO. P4R ligand has been demonstrated to induce a transient (5—10 min after P4-application) activation of Src-Ras-ERK1/2 and a persistent (6—72 h) ERK1/2 activation (Faivre and Lange, 2007). Recently, Cai et al. (2008) provided in vivo evidence that P4 acts on P4R to trigger a long-lasting (> 48 hr) phosphorylation of ERK1/2, resulting in a promoted translocation of phosphorelated ERK2 into the nucleus. The translocation of ERK1/2 is a pivotal and necessary process for the activation of cAMP response element binding protein (CREB) (Nilsen and Brinton, 2003). The ERK1/2-CREB signaling has been implicated to play a critical role in the brain ischemic tolerance (Gonzalez-Zulueta et al., 2000) and neuronal cell survival (Singh, 2005; 2006). The CREB cascade can increase the expression of anti-apoptotic molecules such as Bcl-2 and Bcl-XL (Yao et al., 2005) and decrease the expression of pro-apoptotic molecules such as Bax, Bad and caspase-3 (Djebaili et al., 2004). Consistent with the results reported by Jover-Mengual et al. (2007), we in the present study showed a decreased activity of ERK1/2 at 24 and 48 h post-MCAO. More importantly, the activation of P4R at 24—48 h after ischemia could rescue the down-regulation of ERK1/2. Therefore, it is highly likely that the P4Rdependent neuroprotection is closely coupled with ERK1/2 signaling.

On the other hand, western blot analysis showed a transient elevation of ERK1/2 phosphorylation at 1 h post-MCAO. The elevation of ERK1/2 activity immediately after stroke has also been observed in humans (Slevin et al., 2000) and a rat model of cerebral ischemia (Wang et al., 2003), in which increased intracellular Ca2+ levels ([Ca2+]i) after ischemia seem to lead hyper-activation of ERK1/2. Alessandrini et al. (1999) and Namura et al. (2001) provided evidence for a neuroprotective role of MEK inhibitor following transient ischemia as manifested by the reduction of infarct size and improvement of functional outcome. The neuroprotection by MEK-inhibition in ischemic brain is associated with an activation of potential anti-apoptotic pathway that suppresses caspase-3 activation and apoptosis (Wang et al., 2003). To our surprise, we observed that the treatment with P4 could attenuate the elevation of ERK1/2 phosphorylation at 1 h post-MCAO. Because the neuroprotection by P4 at 1 h post-MCAO was P4R-independent, it is proposed that P4

Time-Window of Progesterone Neuroprotection

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prevents ischemia-increased [Ca2+]i by antagonizing σ1 receptor, which may stabilize the ERK1/2 activation.

### **4.3 PI3K signaling is required for P4R-independent and P4R-dependnet neuroprotections**

P4 has been reported to enhance the phosphorylation of Akt/PKB (Singh et al., 2001; Kuolen et al., 2008) in the hippocampus and cerebellum (Guerra-Araiza et al., 2009). On the other hand, PI3K signaling is believed to suppress apoptotic cell death via its downstream effectors, such as Akt/PKB, to inhibit the Bcl2 family protein Bad (Noshita et al., 2001). Using the specific PI3K inhibitor LY294002, the present study provided in vivo evidence that the PI3K signaling is required for the P4-neuroprotection at 24—72 h post-MCAO against ischemia-induced brain damage. In human breast cancer cells, P4 induces rapid and transient activation of PI3K-Akt pathway in a P4R-dependent manner (Migliaccio et al., 1998; Castoria et al., 2001). It was reported that progestins rapidly activated PI3K-Akt pathway via P4R (Vallejo et al., 2005; Ballare et al., 2006). However, our results here showed that the PI3K-mediated neuroprotection by P4 administered at 72 h post-MCAO is P4Rindependent. P4 is reportedly to regulate PI3K signaling pathway through its metabolites (Guerra-Araiza et al., 2009), but our data determined that the neuroprotection of P4 at 24— 72 h post-MCAO was insensitive to the inhibition of 5α-reductase by fenasteride. P4-binding membrane protein 25-Dx (also known as PGRMC1) in the brain (Krebs et al., 2000; Sakamoto et al., 2004) is involved in the anti-apoptotic actions of P4 (Peluso et al., 2006, 2008). Further studies are needed to elucidate whether P4 cascades PI3K signaling after stroke through P4-binding 25-Dx mechanisms.

### **4.4 Clinical significance**

P4 treatment after ischemia at relatively a low dose (4 mg/kg) exerts powerful neuroprotective effects with a wide, at least up to 72 h post ischemia, effective time-window, which would provide a great benefit in treating stroke. The present study provides evidence that the P4 neuroprotection has a wide effective time-window that is realized by a time dependent multiple neuroprotective mechanisms after ischemia. The results shown here not only help to understand the correlation between the declined level of P4 and the abruptly increasing incidence of stroke following the menopause, but also provide a novel the therapeutic opportunity of P4 against the ischemic brain injury.

### **5. Acknowledgments**

This work was supported by grants for NSFC (30872725; 81071027; 31171440) to Chen L. We declare that there is no competing financial that could be construed as influencing the results or interpretation of the reported study.

### **6. References**

Alessandrini A, Namura S, Moskowitz MA & Bonventre JV. (1999). MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia. *Proc Natl Acad Sci USA,* vol. 96, pp. 12866−12869

prevents ischemia-increased [Ca2+]i by antagonizing σ1 receptor, which may stabilize the

P4 has been reported to enhance the phosphorylation of Akt/PKB (Singh et al., 2001; Kuolen et al., 2008) in the hippocampus and cerebellum (Guerra-Araiza et al., 2009). On the other hand, PI3K signaling is believed to suppress apoptotic cell death via its downstream effectors, such as Akt/PKB, to inhibit the Bcl2 family protein Bad (Noshita et al., 2001). Using the specific PI3K inhibitor LY294002, the present study provided in vivo evidence that the PI3K signaling is required for the P4-neuroprotection at 24—72 h post-MCAO against ischemia-induced brain damage. In human breast cancer cells, P4 induces rapid and transient activation of PI3K-Akt pathway in a P4R-dependent manner (Migliaccio et al., 1998; Castoria et al., 2001). It was reported that progestins rapidly activated PI3K-Akt pathway via P4R (Vallejo et al., 2005; Ballare et al., 2006). However, our results here showed that the PI3K-mediated neuroprotection by P4 administered at 72 h post-MCAO is P4Rindependent. P4 is reportedly to regulate PI3K signaling pathway through its metabolites (Guerra-Araiza et al., 2009), but our data determined that the neuroprotection of P4 at 24— 72 h post-MCAO was insensitive to the inhibition of 5α-reductase by fenasteride. P4-binding membrane protein 25-Dx (also known as PGRMC1) in the brain (Krebs et al., 2000; Sakamoto et al., 2004) is involved in the anti-apoptotic actions of P4 (Peluso et al., 2006, 2008). Further studies are needed to elucidate whether P4 cascades PI3K signaling after

P4 treatment after ischemia at relatively a low dose (4 mg/kg) exerts powerful neuroprotective effects with a wide, at least up to 72 h post ischemia, effective time-window, which would provide a great benefit in treating stroke. The present study provides evidence that the P4 neuroprotection has a wide effective time-window that is realized by a time dependent multiple neuroprotective mechanisms after ischemia. The results shown here not only help to understand the correlation between the declined level of P4 and the abruptly increasing incidence of stroke following the menopause, but also provide a novel the

This work was supported by grants for NSFC (30872725; 81071027; 31171440) to Chen L. We declare that there is no competing financial that could be construed as influencing the

Alessandrini A, Namura S, Moskowitz MA & Bonventre JV. (1999). MEK1 protein kinase

inhibition protects against damage resulting from focal cerebral ischemia. *Proc Natl* 

**4.3 PI3K signaling is required for P4R-independent and P4R-dependnet** 

ERK1/2 activation.

**neuroprotections** 

stroke through P4-binding 25-Dx mechanisms.

results or interpretation of the reported study.

*Acad Sci USA,* vol. 96, pp. 12866−12869

therapeutic opportunity of P4 against the ischemic brain injury.

**4.4 Clinical significance** 

**5. Acknowledgments** 

**6. References** 


Time-Window of Progesterone Neuroprotection

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**The Na+**

**1. Introduction** 

2005).

**/H<sup>+</sup>**

**21** 

*USA* 

 **Exchanger-1 as a New Molecular** 

Vishal Chanana1, Dandan Sun2 and Peter Ferrazzano1,3

Loss of ion homeostasis plays an important role in the pathogenesis of ischemic cell damage. Ischemia induces accumulation of intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i), and subsequent activation of proteases, phospholipases, and formation of oxygen and nitrogen free radicals. The Na+/H+ exchanger (NHEs) family is a group of secondary active membrane transport proteins that catalyze the electroneutral exchange of Na+ for H+ and is important in restoring intracellular pH (pHi) after ischemia-induced intracellular acidosis. Nine isoforms of NHE (NHE1-9) have been identified in mammalian tissues (Orlowski & Grinstein, 2004). These isoforms differ in their tissue expression, subcellular distribution, kinetic properties, inhibitor sensitivity, and physiological functions. NHE-1 is ubiquitously expressed on the plasma membrane of virtually all mammalian cell types (Sardet et al., 1989). NHE-2-4 are expressed on the plasma membrane, predominantly in the epithelia of the kidney and gastrointestinal tract (Orlowski & Grinstein, 2004). NHE-3 is the only isoform known to recycle between the plasma membrane and the endosomal compartment (D'Souza et al., 1998). NHE-5 expression is concentrated in neurons (Attaphitaya et al., 1999) and may modulate the pH of synaptic vesicles (Szaszi et al., 2002). NHE-6 and NHE-9 are expressed predominantly in endosomal vesicles (Nakamura et al., 2005) and NHE-7 localizes to the trans-Golgi network and associated endosomes (Numata & Orlowski, 2001). NHE-8 has been localized to the plasma membrane of renal proximal tubule epithelial cells, and to endosomal vesicles and the trans-Golgi network (Goyal et al., 2003; Nakamura et al.,

NHE-1 is the most extensively studied isoform, and the most abundant isoform in the CNS (Ma & Haddad, 1997; Orlowski et al., 1992). Research over the past two decades has expanded our understanding of the role of NHE-1 beyond that of simply maintenance of ion homeostasis and cell volume, to an emerging picture of a regulator of many cell functions. NHE-1 plays a role in regulation of cell proliferation, migration (Bussolino et al., 1989), and the microglial respiratory burst (Liu et al., 2010). NHE-1 protein consists of 815 amino acids with a calculated molecular weight of 85 kDa. However, NHE-1 has an apparent size of ~110 kDa due to its N- and O- linked glycosylation in the extracellular loop 1. NHE-1 has

**Target in Stroke Interventions** 

*1Waisman Center, University of Wisconsin, Madison, WI 2Dept. of Neurology, University of Pittsburgh, Pittsburgh, PA 3Dept. of Pediatrics, University of Wisconsin, Madison, WI* 


#### **The Na+ /H<sup>+</sup> Exchanger-1 as a New Molecular Target in Stroke Interventions**

Vishal Chanana1, Dandan Sun2 and Peter Ferrazzano1,3

*1Waisman Center, University of Wisconsin, Madison, WI 2Dept. of Neurology, University of Pittsburgh, Pittsburgh, PA 3Dept. of Pediatrics, University of Wisconsin, Madison, WI USA* 

### **1. Introduction**

496 Advances in the Preclinical Study of Ischemic Stroke

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MAP kinase (ERK-1/ERK-2), tyrosine kinase and VEGF in the human brain

towards a hypothesis that sigma-1 receptors are intracellular amplifiers for signal

incidence, case fatality, and mortality in the WHO MONICA project. *Stroke*, vol. 26,

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and anti-apoptotic gene expression in cerebral cortex following traumatic brain

Loss of ion homeostasis plays an important role in the pathogenesis of ischemic cell damage. Ischemia induces accumulation of intracellular Na+ ([Na+]i) and Ca2+ ([Ca2+]i), and subsequent activation of proteases, phospholipases, and formation of oxygen and nitrogen free radicals. The Na+/H+ exchanger (NHEs) family is a group of secondary active membrane transport proteins that catalyze the electroneutral exchange of Na+ for H+ and is important in restoring intracellular pH (pHi) after ischemia-induced intracellular acidosis. Nine isoforms of NHE (NHE1-9) have been identified in mammalian tissues (Orlowski & Grinstein, 2004). These isoforms differ in their tissue expression, subcellular distribution, kinetic properties, inhibitor sensitivity, and physiological functions. NHE-1 is ubiquitously expressed on the plasma membrane of virtually all mammalian cell types (Sardet et al., 1989). NHE-2-4 are expressed on the plasma membrane, predominantly in the epithelia of the kidney and gastrointestinal tract (Orlowski & Grinstein, 2004). NHE-3 is the only isoform known to recycle between the plasma membrane and the endosomal compartment (D'Souza et al., 1998). NHE-5 expression is concentrated in neurons (Attaphitaya et al., 1999) and may modulate the pH of synaptic vesicles (Szaszi et al., 2002). NHE-6 and NHE-9 are expressed predominantly in endosomal vesicles (Nakamura et al., 2005) and NHE-7 localizes to the trans-Golgi network and associated endosomes (Numata & Orlowski, 2001). NHE-8 has been localized to the plasma membrane of renal proximal tubule epithelial cells, and to endosomal vesicles and the trans-Golgi network (Goyal et al., 2003; Nakamura et al., 2005).

NHE-1 is the most extensively studied isoform, and the most abundant isoform in the CNS (Ma & Haddad, 1997; Orlowski et al., 1992). Research over the past two decades has expanded our understanding of the role of NHE-1 beyond that of simply maintenance of ion homeostasis and cell volume, to an emerging picture of a regulator of many cell functions. NHE-1 plays a role in regulation of cell proliferation, migration (Bussolino et al., 1989), and the microglial respiratory burst (Liu et al., 2010). NHE-1 protein consists of 815 amino acids with a calculated molecular weight of 85 kDa. However, NHE-1 has an apparent size of ~110 kDa due to its N- and O- linked glycosylation in the extracellular loop 1. NHE-1 has

The Na<sup>+</sup>

/H<sup>+</sup>

**2.2 Ionic homeostasis and brain cell function** 

and seizures (Bell et al., 1999; Gu et al., 2001).

rapid influx of Ca2+ and subsequent cell death.

**2.3.1 Global ischemia** 

(Phillis et al., 1999).

Exchanger-1 as a New Molecular Target in Stroke Interventions 499

Secondary active ion transport proteins are important in maintaining steady-state intracellular ion concentrations. NHE-1 plays an important role in regulation of many cellular processes in addition to pHi and cell volume regulation, such as cell growth, proliferation and differentiation, cell migration and adhesion, cellular immunity, and as cytoskeletal scaffolding for the assembly of intracellular signaling complexes (De Vito, 2006; Luo & Sun, 2007; Luo et al., 2005; Meima et al., 2007; Orlowski & Grinstein, 2004; Pedersen et al., 2006; Xue & Haddad, 2010). Due to their high metabolic rate and rapid changes in metabolic demand, neurons are exposed to frequent fluctuations in pHi, making efficient acid extrusion mechanisms essential for normal neuronal function. Neurons and astrocytes from mice deficient in the NHE-1 protein (NHE-1-/-) demonstrate decreased basal pHi and are unable to recover from an acid load (Luo et al., 2005). NHE-1 is the predominant NHE isoform in the CNS (Ma & Haddad, 1997; Orlowski et al., 1992), and as evidence of its importance in normal neurologic function, NHE-1-/- mice exhibit severe neurologic defects

**2.3 Role of NHE-1 in cellular dysfunction and cerebral injury during** *in vivo* **ischemia**  Results from *in vivo* experimental studies support the importance of ion transport proteins in ischemia-mediated loss of ion homeostasis. NHE-1 activity in astrocytes (Cengiz et al., 2010), neurons (Manhas et al., 2010), and microglia (Shi et al., 2011) is stimulated following cerebral ischemia. Excessive stimulation of NHE-1 leads to intracellular Na+ overload, and in turn causes a rise in intracellular Ca2+ due to increased Ca2+ influx via reversal of the Na/Ca exchanger. Thus, NHE-1 activity contributes to cerebral ischemic damage in part by disruption of intracellular Na+ and Ca2+ homeostasis, an event which is characterized by

Global cerebral ischemia entails diminution in cerebral blood flow (CBF) over the entire brain, and is encountered clinically in cardiac arrest. On restoration of CBF, a secondary reperfusion brain injury may occur due to altered ionic homeostasis, increases in ROS, cerebral edema, and inflammatory cascades (Schaller & Graf, 2004). The contribution of NHE-1 activity to global cerebral ischemia has been reported in a number of animal models. In a gerbil model of transient forebrain ischemia, NHE-1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as in glial cells 4 days following injury, and inhibition of NHE protected CA1 pyramidal neurons and attenuated the activation of astrocytes and microglia (Hwang et al., 2008). Yorkshire-Duroc pigs treated with cariporide (HOE 642), a potent and selective inhibitor of NHE-1, at the onset of a 90 min deep hypothermic circulatory arrest demonstrated improved neurologic recovery (Castellá et al., 2005). Similarly, inhibition of NHE-1 with N-[aminoiminomethyl]-1-methyl-1H-indole-2 carboxamide methanesulfonate (SM-20220) improved neurologic function in a gerbil model of transient global cerebral ischemia (Kuribayashi et al., 2000). Administration of the NHE-1 inhibitor ethylisopropylamiloride (EIPA) prior to bilateral carotid artery occlusion in gerbils resulted in decreased hippocampal neuronal cell death and improved neurologic function

two large functional domains, the highly conserved amphipathic N-terminal domain (~500 amino acids), which is responsible for cation translocation, and a less conserved hydrophilic cytoplasmic C-terminal domain (~315 amino acids), which is crucial for modulating NHE-1 activity (Putney et al., 2002). Activation of NHE-1 has been shown to be a pivotal event in cell damage induced by ischemia and reperfusion in the brain (Horikawa et al., 2001; Hwang et al., 2008; Luo et al., 2005), heart (Liu et al., 1997; Murphy et al., 1991; Wang et al., 2003), liver (Gores et al., 1989), and lungs (Rios et al., 2005). Here we will review recent findings implicating NHE-1 activation as a critical event in the pathogenesis of cellular dysfunction after cerebral ischemia, and the growing evidence supporting the use of NHE inhibitors as neuroprotective agents following cerebral ischemia.

#### **2. Na<sup>+</sup> /H<sup>+</sup> Exchanger isoform-1 (NHE-1) in cerebral ischemia**

Ischemia and reperfusion injury is a complex and incompletely understood phenomenon. Ischemia deprives the cell of the energy required for normal cell function and leads to loss of ionic homeostasis within the cell due to opening of ionotropic glutamate receptors (Nishizawa, 2001) and acid sensing non-glutamate-dependent channels (Xiong et al., 2004), as well as activation of ion transport proteins such as NHE-1, Na+/K+/Cl- cotransporter (NKCCl) (Chen et al., 2005), and the Na+/Ca2+ exchanger (NCXs) (Hoyt et al., 1998). Reperfusion triggers a cascade of intracellular events including release of reactive oxygen species (ROS) and inflammatory mediators, which exacerbate injury and promote cell death. Ischemia and reperfusion induces intracellular acidosis due to a shift from aerobic to anaerobic glycolysis, and leads to an increase in [Na+]i and [Ca2+]i by mechanisms that include the activation of acid responsive ion transporters (Yao & Haddad, 2004). Recent findings from our group and others highlight the important role of NHE-1 in pHi regulation after cerebral ischemia and reperfusion.

### **2.1 NHE-1 mediated intracellular pH regulation**

To regulate and maintain constant pHi, eukaryotic cells express plasma membrane ion transporters such as NHE-1 that protect cells from internal acidification by exchanging extracellular Na+ for intracellular H+ (Luo et al., 2005). At physiological pHi, NHE-1 is essentially inactive, despite the large inward Na+ gradient established by Na+/K+- ATPase. However, upon exposure to intracellular acidification, NHE-1 is rapidly activated and uses the electrochemical gradient of Na+ to pump H+ out of the cell and restore pHi. Upon restoration of pHi, NHE-1 activity returns to steady state levels (Pedersen, 2006). Extracellular acidification (low pHo) or removal of extracellular sodium suppresses this gradient-driven Na+/H+ exchange (Bobulescu et al., 2005). While NHE-1 serves to maintain homeostasis in the face of normal pHi fluctuations (which result from changes in metabolic activity), profound acidosis after anoxia can induce a NHE-1 mediated parodoxic alkalinization, a so-called "overshoot" of pHi restoration. We reported that post-anoxia alkalinization is ablated by pharmacological inhibition of NHE-1 and removal of extracellular sodium (Kintner et al., 2005). Protein kinase inhibitors attenuate this alkalinization, suggesting that activation of NHE-1 involves protein phosphorylation and multiple up-stream regulatory pathways such as extracellular signalregulated kinases (ERK 1/2), protein kinase A (PKA), and protein kinase C (PKC) (Kintner et al., 2005; Luo et al., 2007; Yao et al., 2001).

### **2.2 Ionic homeostasis and brain cell function**

498 Advances in the Preclinical Study of Ischemic Stroke

two large functional domains, the highly conserved amphipathic N-terminal domain (~500 amino acids), which is responsible for cation translocation, and a less conserved hydrophilic cytoplasmic C-terminal domain (~315 amino acids), which is crucial for modulating NHE-1 activity (Putney et al., 2002). Activation of NHE-1 has been shown to be a pivotal event in cell damage induced by ischemia and reperfusion in the brain (Horikawa et al., 2001; Hwang et al., 2008; Luo et al., 2005), heart (Liu et al., 1997; Murphy et al., 1991; Wang et al., 2003), liver (Gores et al., 1989), and lungs (Rios et al., 2005). Here we will review recent findings implicating NHE-1 activation as a critical event in the pathogenesis of cellular dysfunction after cerebral ischemia, and the growing evidence supporting the use of NHE

inhibitors as neuroprotective agents following cerebral ischemia.

 **Exchanger isoform-1 (NHE-1) in cerebral ischemia** 

Ischemia and reperfusion injury is a complex and incompletely understood phenomenon. Ischemia deprives the cell of the energy required for normal cell function and leads to loss of ionic homeostasis within the cell due to opening of ionotropic glutamate receptors (Nishizawa, 2001) and acid sensing non-glutamate-dependent channels (Xiong et al., 2004), as well as activation of ion transport proteins such as NHE-1, Na+/K+/Cl- cotransporter (NKCCl) (Chen et al., 2005), and the Na+/Ca2+ exchanger (NCXs) (Hoyt et al., 1998). Reperfusion triggers a cascade of intracellular events including release of reactive oxygen species (ROS) and inflammatory mediators, which exacerbate injury and promote cell death. Ischemia and reperfusion induces intracellular acidosis due to a shift from aerobic to anaerobic glycolysis, and leads to an increase in [Na+]i and [Ca2+]i by mechanisms that include the activation of acid responsive ion transporters (Yao & Haddad, 2004). Recent findings from our group and others highlight the important role of NHE-1 in pHi regulation

To regulate and maintain constant pHi, eukaryotic cells express plasma membrane ion transporters such as NHE-1 that protect cells from internal acidification by exchanging extracellular Na+ for intracellular H+ (Luo et al., 2005). At physiological pHi, NHE-1 is essentially inactive, despite the large inward Na+ gradient established by Na+/K+- ATPase. However, upon exposure to intracellular acidification, NHE-1 is rapidly activated and uses the electrochemical gradient of Na+ to pump H+ out of the cell and restore pHi. Upon restoration of pHi, NHE-1 activity returns to steady state levels (Pedersen, 2006). Extracellular acidification (low pHo) or removal of extracellular sodium suppresses this gradient-driven Na+/H+ exchange (Bobulescu et al., 2005). While NHE-1 serves to maintain homeostasis in the face of normal pHi fluctuations (which result from changes in metabolic activity), profound acidosis after anoxia can induce a NHE-1 mediated parodoxic alkalinization, a so-called "overshoot" of pHi restoration. We reported that post-anoxia alkalinization is ablated by pharmacological inhibition of NHE-1 and removal of extracellular sodium (Kintner et al., 2005). Protein kinase inhibitors attenuate this alkalinization, suggesting that activation of NHE-1 involves protein phosphorylation and multiple up-stream regulatory pathways such as extracellular signalregulated kinases (ERK 1/2), protein kinase A (PKA), and protein kinase C (PKC) (Kintner et

**2. Na<sup>+</sup>**

**/H<sup>+</sup>**

after cerebral ischemia and reperfusion.

al., 2005; Luo et al., 2007; Yao et al., 2001).

**2.1 NHE-1 mediated intracellular pH regulation** 

Secondary active ion transport proteins are important in maintaining steady-state intracellular ion concentrations. NHE-1 plays an important role in regulation of many cellular processes in addition to pHi and cell volume regulation, such as cell growth, proliferation and differentiation, cell migration and adhesion, cellular immunity, and as cytoskeletal scaffolding for the assembly of intracellular signaling complexes (De Vito, 2006; Luo & Sun, 2007; Luo et al., 2005; Meima et al., 2007; Orlowski & Grinstein, 2004; Pedersen et al., 2006; Xue & Haddad, 2010). Due to their high metabolic rate and rapid changes in metabolic demand, neurons are exposed to frequent fluctuations in pHi, making efficient acid extrusion mechanisms essential for normal neuronal function. Neurons and astrocytes from mice deficient in the NHE-1 protein (NHE-1-/-) demonstrate decreased basal pHi and are unable to recover from an acid load (Luo et al., 2005). NHE-1 is the predominant NHE isoform in the CNS (Ma & Haddad, 1997; Orlowski et al., 1992), and as evidence of its importance in normal neurologic function, NHE-1-/- mice exhibit severe neurologic defects and seizures (Bell et al., 1999; Gu et al., 2001).

### **2.3 Role of NHE-1 in cellular dysfunction and cerebral injury during** *in vivo* **ischemia**

Results from *in vivo* experimental studies support the importance of ion transport proteins in ischemia-mediated loss of ion homeostasis. NHE-1 activity in astrocytes (Cengiz et al., 2010), neurons (Manhas et al., 2010), and microglia (Shi et al., 2011) is stimulated following cerebral ischemia. Excessive stimulation of NHE-1 leads to intracellular Na+ overload, and in turn causes a rise in intracellular Ca2+ due to increased Ca2+ influx via reversal of the Na/Ca exchanger. Thus, NHE-1 activity contributes to cerebral ischemic damage in part by disruption of intracellular Na+ and Ca2+ homeostasis, an event which is characterized by rapid influx of Ca2+ and subsequent cell death.

### **2.3.1 Global ischemia**

Global cerebral ischemia entails diminution in cerebral blood flow (CBF) over the entire brain, and is encountered clinically in cardiac arrest. On restoration of CBF, a secondary reperfusion brain injury may occur due to altered ionic homeostasis, increases in ROS, cerebral edema, and inflammatory cascades (Schaller & Graf, 2004). The contribution of NHE-1 activity to global cerebral ischemia has been reported in a number of animal models. In a gerbil model of transient forebrain ischemia, NHE-1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as in glial cells 4 days following injury, and inhibition of NHE protected CA1 pyramidal neurons and attenuated the activation of astrocytes and microglia (Hwang et al., 2008). Yorkshire-Duroc pigs treated with cariporide (HOE 642), a potent and selective inhibitor of NHE-1, at the onset of a 90 min deep hypothermic circulatory arrest demonstrated improved neurologic recovery (Castellá et al., 2005). Similarly, inhibition of NHE-1 with N-[aminoiminomethyl]-1-methyl-1H-indole-2 carboxamide methanesulfonate (SM-20220) improved neurologic function in a gerbil model of transient global cerebral ischemia (Kuribayashi et al., 2000). Administration of the NHE-1 inhibitor ethylisopropylamiloride (EIPA) prior to bilateral carotid artery occlusion in gerbils resulted in decreased hippocampal neuronal cell death and improved neurologic function (Phillis et al., 1999).

The Na<sup>+</sup>

conditions.

**2.4.1 Cell cultures** 

/H<sup>+</sup>

Exchanger-1 as a New Molecular Target in Stroke Interventions 501

and hippocampus and improved performance on tests of motor learning and memory (Cengiz et al., 2010). These findings suggest that NHE-1 mediated disruption of ionic homeostasis can contribute to CA1 pyramidal neuronal injury after neonatal HI**.** Moreover, T2 weighted and Diffusion Tensor (DTI) MRI revealed that NHE-1 inhibition with HOE 642 after HI resulted in improved white matter injury in the corpus callosum, which correlated

Extensive *in vitro* studies have established that ischemia stimulates NHE-1 by reduction in pHi or via signaling pathways such as ERK-p90rsk, PKA or PKC (Dunbar & Caplan, 2001; Herrera et al., 1994; Kintner et al., 2007a; Li et al., 2004). The role of NHE-1 in cerebral ischemia has been mainly examined in two types of *in vitro* ischemic models, oxygen glucose deprivation/reoxygenation (OGD/REOX) or the hypoxic, acidic, ion-shifted Ringers's solution (HAIR). Superfused brain slices also represent a useful preparation to study acid-base disturbance that occurs in the mammalian brain during *in vitro* ischemic

NHE-1 activity is stimulated during *in vitro* ischemia and subsequent reoxygenation and contributes substantially to neuronal and glial cell injury. Acutely isolated CA1 neurons exhibit a tri-phasic response to 5 minutes of anoxia. During anoxia, an initial acidification progresses to alkalinization that is followed by further alkalinization on exposure to reoxygenation. This alkalinization is attenuated by reduction of external pH, removal of extracellular sodium, or inhibition of NHE-1 (Sheldon & Church, 2002; Yao et al., 2001). Additionally, inhibition of PKA can block post-anoxia alkalinization, suggesting cAMP-

We have demonstrated that NHE-1 is essential in pHi regulation using an internal acid load in cultured cortical neurons (Luo et al., 2005). Additionally, we found that activation of NHE-1 after OGD/REOX results in a significant increase in neuronal [Na+]i. This rise in [Na+]i following OGD is significantly attenuated in HOE 642-treated or NHE-1-/- neurons, and cell death is reduced (Luo et al., 2005). In a separate study, we demonstrated that NHE-1-mediated Na+ entry leads to reverse activation of the Na+/Ca2+ exchanger (NCXrev) and rise in [Ca2+]i, which contribute to the selective dendritic vulnerability to *in vitro* ischemia (Kintner et al., 2010). Taken together, our studies suggest that NHE-1 activity in neurons is significantly stimulated in response to the metabolic acidification associated with an ischemic insult. This ischemia-induced increase in NHE-1 activity causes intracellular Na+

In another series of studies, we examined the role of NHE-1 in ischemic astrocyte damage using OGD/REOX in cultured cortical astrocytes and found that NHE-1 is the primary pH regulatory mechanism after ischemia. Astrocyte NHE-1 activity is increased by ~ 1.8 fold during REOX (Kintner et al., 2004), and depends on ERK1/2 signaling pathways (Kintner et al., 2005). OGD/REOX results in a drop in pHi by 0.29 pH units (Kintner et al., 2004), and inhibition of NHE-1 results in a further decrease of pHi. Additionally, we observed that OGD/REOX triggers a ~5-fold increase in [Na+]i and 26% increase in astrocyte cell volume. This increase in [Na+]i and cell swelling are significantly reduced either with HOE 642 treatment or in NHE-1-/- astrocytes (Kintner et al., 2004). Using the HAIR model in

dependent signaling pathways for NHE-1 activation (Sheldon & Church, 2002).

and Ca2+ overload, and eventually leads to cell death.

with improvements in memory and learning (Cengiz et al., 2011).

**2.4 Role of NHE-1 in cellular dysfunction during** *in vitro* **ischemia** 

### **2.3.2 Focal ischemia**

Unlike global cerebral ischemia, focal cerebral ischemia entails reduction in regional CBF in a specific vascular territory and is usually encountered clinically as an "ischemic stroke" due to thromboembolic or vaso-occlusive disease. An abundance of *in vivo* studies support the importance of NHE-1 in focal ischemia. The NHE-1 inhibitor SM-20220 reduces infarct size in both transient and permanent focal ischemia models (Kuribayashi et al., 1999). Another NHE inhibitor, Sabiporide, reduces infarct size and edema volume when administered before or after ischemia (Park et al., 2005).

Our investigations into the role of NHE-1 in cerebral ischemia have used both genetic and pharmacologic inhibition of NHE-1 in a mouse transient middle cerebral artery occlusion model (MCAO). Mice treated with HOE 642, a potent and selective inhibitor of NHE-1, prior to MCAO demonstrated a 35% reduction in infarct volume compared to vehicle treated controls. NHE-1 heterozygous mice (NHE-1+/-), which demonstrate a ~ 70% reduction in NHE-1 protein expression, exhibited a similar reduction in infarct volumes, establishing the importance of NHE-1 over other NHE isoforms in the CNS (Luo et al., 2005; Wang et al., 2008). With T2-weighted and Diffusion Weighted MRI, we further confirmed that NHE-1+/+ mice treated with HOE 642 immediately prior to reperfusion or 60 minutes postreperfusiion, exhibited a significant reduction in infarct volume compared to NHE-1+/+ vehicle control mice. NHE-1+/- mice demonstrated a significant reduction in infarct volume on T2 MRI at 72 hours after injury (Ferrazzano et al., 2011). These findings suggest that elevated NHE-1 activity contributes to neuronal injury following ischemia and reperfusion. We subsequently revealed that focal cerebral ischemia triggers a transient stimulation of the extracellular signal-regulated kinase/p90 ribosomal S6 kinase (ERK/p90RSK) pathway that contributes to ischemic damage in part via phosphorylation of NHE-1 protein (Manhas et al., 2010). The NHE-1-mediated [Na+ ]i overload causes reverse function of the Na+/Ca2+ exchanger, elevating [Ca2+]i and enhancing the p38 mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (Liu et al., 2010). NHE-1 activity also plays a detrimental role in mitochondrial Ca2+ overload and mitochondrial dysfunction after ischemia as evidenced by attenuation of ischemia-induced cytochrome C release from mitochondria after NHE-1 inhibition (Wang et al., 2008). Interestingly, when NHE-1 activity is blocked either pharmacologically or by genetic knockdown, microglia activation and proinflammatory cytokine formation is significantly reduced in ischemic brains after MCAO (Shi et al., 2011). Taken together, these results strongly support that NHE-1 is activated after cerebral ischemia and worsens ischemic brain injury.

### **2.3.3 Hypoxia/Ischemia**

Hypoxia-ischemia (HI) is a common cause of brain injury in neonates (Ferriero, 2004). We recently investigated the role of NHE-1 using a mouse model of neonatal hypoxia-ischemia as described by Vannucci (Vannucci & Vannucci, 2005). In these studies, post-natal day 9 mice (P9) underwent unilateral carotid artery ligation and subsequent exposure to 55 minutes of 8% O2. Following carotid ligation, mice were treated with HOE 642 either immediately before or 10 minutes following exposure to hypoxia (Cengiz et al., 2010). Following HI, vehicle-treated control brains exhibited astrogliosis in the ipsilateral hippocampus, and reactive astrocytes expressed an abundant level of NHE-1. Inhibition of NHE-1 before or after HI resulted in decreased neurodegeneration in striatum, thalamus and hippocampus and improved performance on tests of motor learning and memory (Cengiz et al., 2010). These findings suggest that NHE-1 mediated disruption of ionic homeostasis can contribute to CA1 pyramidal neuronal injury after neonatal HI**.** Moreover, T2 weighted and Diffusion Tensor (DTI) MRI revealed that NHE-1 inhibition with HOE 642 after HI resulted in improved white matter injury in the corpus callosum, which correlated with improvements in memory and learning (Cengiz et al., 2011).

### **2.4 Role of NHE-1 in cellular dysfunction during** *in vitro* **ischemia**

Extensive *in vitro* studies have established that ischemia stimulates NHE-1 by reduction in pHi or via signaling pathways such as ERK-p90rsk, PKA or PKC (Dunbar & Caplan, 2001; Herrera et al., 1994; Kintner et al., 2007a; Li et al., 2004). The role of NHE-1 in cerebral ischemia has been mainly examined in two types of *in vitro* ischemic models, oxygen glucose deprivation/reoxygenation (OGD/REOX) or the hypoxic, acidic, ion-shifted Ringers's solution (HAIR). Superfused brain slices also represent a useful preparation to study acid-base disturbance that occurs in the mammalian brain during *in vitro* ischemic conditions.

### **2.4.1 Cell cultures**

500 Advances in the Preclinical Study of Ischemic Stroke

Unlike global cerebral ischemia, focal cerebral ischemia entails reduction in regional CBF in a specific vascular territory and is usually encountered clinically as an "ischemic stroke" due to thromboembolic or vaso-occlusive disease. An abundance of *in vivo* studies support the importance of NHE-1 in focal ischemia. The NHE-1 inhibitor SM-20220 reduces infarct size in both transient and permanent focal ischemia models (Kuribayashi et al., 1999). Another NHE inhibitor, Sabiporide, reduces infarct size and edema volume when administered

Our investigations into the role of NHE-1 in cerebral ischemia have used both genetic and pharmacologic inhibition of NHE-1 in a mouse transient middle cerebral artery occlusion model (MCAO). Mice treated with HOE 642, a potent and selective inhibitor of NHE-1, prior to MCAO demonstrated a 35% reduction in infarct volume compared to vehicle treated controls. NHE-1 heterozygous mice (NHE-1+/-), which demonstrate a ~ 70% reduction in NHE-1 protein expression, exhibited a similar reduction in infarct volumes, establishing the importance of NHE-1 over other NHE isoforms in the CNS (Luo et al., 2005; Wang et al., 2008). With T2-weighted and Diffusion Weighted MRI, we further confirmed that NHE-1+/+ mice treated with HOE 642 immediately prior to reperfusion or 60 minutes postreperfusiion, exhibited a significant reduction in infarct volume compared to NHE-1+/+ vehicle control mice. NHE-1+/- mice demonstrated a significant reduction in infarct volume on T2 MRI at 72 hours after injury (Ferrazzano et al., 2011). These findings suggest that elevated NHE-1 activity contributes to neuronal injury following ischemia and reperfusion. We subsequently revealed that focal cerebral ischemia triggers a transient stimulation of the extracellular signal-regulated kinase/p90 ribosomal S6 kinase (ERK/p90RSK) pathway that contributes to ischemic damage in part via phosphorylation of NHE-1 protein (Manhas et al., 2010). The NHE-1-mediated [Na+ ]i overload causes reverse function of the Na+/Ca2+ exchanger, elevating [Ca2+]i and enhancing the p38 mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (Liu et al., 2010). NHE-1 activity also plays a detrimental role in mitochondrial Ca2+ overload and mitochondrial dysfunction after ischemia as evidenced by attenuation of ischemia-induced cytochrome C release from mitochondria after NHE-1 inhibition (Wang et al., 2008). Interestingly, when NHE-1 activity is blocked either pharmacologically or by genetic knockdown, microglia activation and proinflammatory cytokine formation is significantly reduced in ischemic brains after MCAO (Shi et al., 2011). Taken together, these results strongly support that NHE-1 is activated after cerebral ischemia and worsens ischemic brain

Hypoxia-ischemia (HI) is a common cause of brain injury in neonates (Ferriero, 2004). We recently investigated the role of NHE-1 using a mouse model of neonatal hypoxia-ischemia as described by Vannucci (Vannucci & Vannucci, 2005). In these studies, post-natal day 9 mice (P9) underwent unilateral carotid artery ligation and subsequent exposure to 55 minutes of 8% O2. Following carotid ligation, mice were treated with HOE 642 either immediately before or 10 minutes following exposure to hypoxia (Cengiz et al., 2010). Following HI, vehicle-treated control brains exhibited astrogliosis in the ipsilateral hippocampus, and reactive astrocytes expressed an abundant level of NHE-1. Inhibition of NHE-1 before or after HI resulted in decreased neurodegeneration in striatum, thalamus

**2.3.2 Focal ischemia** 

injury.

**2.3.3 Hypoxia/Ischemia** 

before or after ischemia (Park et al., 2005).

NHE-1 activity is stimulated during *in vitro* ischemia and subsequent reoxygenation and contributes substantially to neuronal and glial cell injury. Acutely isolated CA1 neurons exhibit a tri-phasic response to 5 minutes of anoxia. During anoxia, an initial acidification progresses to alkalinization that is followed by further alkalinization on exposure to reoxygenation. This alkalinization is attenuated by reduction of external pH, removal of extracellular sodium, or inhibition of NHE-1 (Sheldon & Church, 2002; Yao et al., 2001). Additionally, inhibition of PKA can block post-anoxia alkalinization, suggesting cAMPdependent signaling pathways for NHE-1 activation (Sheldon & Church, 2002).

We have demonstrated that NHE-1 is essential in pHi regulation using an internal acid load in cultured cortical neurons (Luo et al., 2005). Additionally, we found that activation of NHE-1 after OGD/REOX results in a significant increase in neuronal [Na+]i. This rise in [Na+]i following OGD is significantly attenuated in HOE 642-treated or NHE-1-/- neurons, and cell death is reduced (Luo et al., 2005). In a separate study, we demonstrated that NHE-1-mediated Na+ entry leads to reverse activation of the Na+/Ca2+ exchanger (NCXrev) and rise in [Ca2+]i, which contribute to the selective dendritic vulnerability to *in vitro* ischemia (Kintner et al., 2010). Taken together, our studies suggest that NHE-1 activity in neurons is significantly stimulated in response to the metabolic acidification associated with an ischemic insult. This ischemia-induced increase in NHE-1 activity causes intracellular Na+ and Ca2+ overload, and eventually leads to cell death.

In another series of studies, we examined the role of NHE-1 in ischemic astrocyte damage using OGD/REOX in cultured cortical astrocytes and found that NHE-1 is the primary pH regulatory mechanism after ischemia. Astrocyte NHE-1 activity is increased by ~ 1.8 fold during REOX (Kintner et al., 2004), and depends on ERK1/2 signaling pathways (Kintner et al., 2005). OGD/REOX results in a drop in pHi by 0.29 pH units (Kintner et al., 2004), and inhibition of NHE-1 results in a further decrease of pHi. Additionally, we observed that OGD/REOX triggers a ~5-fold increase in [Na+]i and 26% increase in astrocyte cell volume. This increase in [Na+]i and cell swelling are significantly reduced either with HOE 642 treatment or in NHE-1-/- astrocytes (Kintner et al., 2004). Using the HAIR model in

The Na<sup>+</sup>

/H<sup>+</sup>

**2.5.2 Transgenic approach** 

functional outcomes are still lacking.

2005).

**3. Conclusion** 

Exchanger-1 as a New Molecular Target in Stroke Interventions 503

1995). Both classes are more specific for NHE-1 than NHE-3, with the amiloride compounds demonstrating ~102-fold increased specificity and the HOE compounds ~103- to 105-fold more NHE-1 specificity. The HOE compounds are viewed as the most promising agents for treatment of ischemia-reperfusion injury due to their selectivity for NHE-1, and excellent solubility, resorption, and bioavailability profiles (Scholz et al., 1999; Baumgarth et al., 1997; Xue & Haddad, 2010). HOE compounds are competitive inhibitors of Na+ binding at the extracellular cation-binding site (Baumgarth et al., 1997; Counillon et al., 1993; Kinsella & Aronson, 1981; Mahnensmith & Aronson, 1985), while the amiloride derivatives also act non-competitively (Warnock et al., 1988). More recently, several new molecules have been designed as potential NHE blockers based on the bicyclic template, including SM-20220, SM-20550, BMS-284640, T-162559, and TY-12533, which have also shown promising results in *in vivo* studies of cerebral ischemia (Kitayama et al., 2001). The IC50 for the human NHE-1 are as follows: Amiloride = 10.7 µM, Cariporide = 0.08 µM, T-165229 = 13 nM. Importantly, the NHE inhibitors HOE 642 and SM-20220 not only reduce cell death and edema, but also improve neurological function in *in vivo* ischemia models, and have demonstrated benefits

when administered after ischemia (Kintner et al., 2007b; Kuribayashi et al., 2000).

While pharmacological studies indicate that NHE-1 plays a central role in cerebral ischemiareperfusion injury, the use of pharmacologic inhibitors to study ion transport function raises questions regarding dosing, absorption, species specific T1/2, and non-specific effects. For this reason, confirmation by an alternative method using NHE-1 knockdown mice is warranted. NHE-1-/- mice exhibit neurologic abnormalities, seizures, ataxia, and growth retardation, and do not survive into adulthood (Bell et al., 1999; Gu et al., 2001). Therefore, NHE-1-/- mice are useful for cultures of NHE-1 null neurons, astrocytes and microglia, but cannot be used for *in vivo* studies. NHE-1+/- mice express <50% of NHE-1 protein levels, and are useful for *in vivo* studies of the role of NHE function after cerebral ischemia. A marked decrease of infarct volume, microglial activation and proinflammatory cytokine formation is found in NHE-1+/- mice after MCAO (Luo et al., 2005). NHE-1 +/- and NHE-1 -/- cortical neurons and astrocytes demonstrate decreased cell death after OGD/REOX (Luo et al.,

The fact that NHE-1 inhibitors applied during or after cerebral ischemia protect the brain against ischemic damage is now well established in animal studies. Despite the uniformity of results from animal models, a number of challenges remain before NHE-1 inhibitors can be translated into clinical use. Questions regarding safety, optimal dose, and timing of administration remain to be addressed, and large animal studies demonstrating improved

NHE-1 plays a pivotal role in maintaining tissue ionic homeostasis under normal physiological conditions. However, excessive stimulation of NHE-1 appears to be a major contributor to cellular damage in ischemic conditions. The proposed mechanism for injury induced by NHE-1 activation includes accumulation of [Na+]i, subsequent [Ca2+]i overload via reverse activation of the Na+/Ca2+ exchanger, and eventual cell death. Additionally, activation of MAPKs, and release of excitatory amino acids and ROS also contribute to cell

astrocytes, we found a similar increase in [Na+]i which could be abolished by the NHE-1 inhibitor HOE 642 (Kintner et al., 2007b). It has been reported that the expected rise in [Ca2+]i after HAIR exposure is inhibited by NHE-1 inhibition with HOE 694 (Bondarenko et al., 2005). Taken together, these results indicate that NHE-1 activity raises [Na+]i which fosters reversal of the Na+/Ca+ exchanger leading to increased intracellular Ca2+ and astrocyte cell death.

More recently, new evidence supports a role of NHE-1 in microglial pHi regulation. Microglia activation by lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or OGD/REOX triggers a concurrent stimulation of NHE-1 and NADPH oxidase (Liu et al., 2010). The elevation in NHE-1-mediated H+ extrusion prevents intracellular acidosis, allowing for sustained NADPH oxidase function (Liu et al., 2010). Moreover, the coupling of NHE-1 activation with NCXrev activates [Na+]i and [Ca2+]i dependent signaling, which promotes the microglial respiratory burst and production of proinflammatory cytokines (Liu et al., 2010).

### **2.4.2 Brain slice**

Few studies have used brain slice preparations to examine acid-base homeostatic disturbances during ischemia. In hippocampal slices, hypoxia induces a significant drop in both pHi and pHo, and a brief alkaline peak is also occasionally observed (Fujiwara et al., 1992; Melzian et al., 1996; Roberts & Chih, 1997). In slice preparations from various brain regions, hypoxia causes acidosis with an approximately 0.8-1.2 pHi unit drop (Ballanyi et al, 1996; Knopfel et al., 1998; Pirttila & Kauppinen, 1994). Cytosolic calcium changes are observed during ischemia in cortical brain slices that can be only partially inhibited by combined blockade of ion channels (Bickler and Hansen, 1994). Only one report shows a direct involvement of NHE mediated pHi regulation in slice preparations. In brainstem slices from neonatal rats exposed to 10 minutes of anoxia, intracellular pH drops by 0.1-0.3 pH units in neurons. Inhibition of NHE with amiloride increases this anoxia-induced intracellular acidification (Chambers-Kersh et al., 2000).

### **2.5 NHE-1 inhibitors and potential therapies**

Despite decades of research, the effective treatment and prevention of cerebral ischemic injury remains challenging. Inhibition of NHE-1 with either pharmacological agents or genetic ablation has been demonstrated to significantly reduce brain damage after ischemic insult, in both *in vitro* and *in vivo* models. These encouraging findings suggest the potential use of NHE inhibitors as neuroprotective therapies after cerebral ischemia.

### **2.5.1 Pharmacological approach**

Two major classes of pharmacological agents are currently used to inhibit NHE-1 activity (Putney et al., 2002). The first class of drugs includes amiloride and its 5' alkyl-substituted derivatives (Counillon et al., 1993; Yu et al., 1993), such as ethylisopropylamiloride (EIPA), dimethylamiloride (DMA), 5-*N* (methylpropyl)amiloride (MPA), 5-(N-methyl-N-isobutyl) amiloride (MIBA), and 5-(N, N-hexamethylene) amiloride (HMA). These agents are more effective inhibitors of NHE-1 than amiloride but have relatively weak selectivity toward NHE-1. The simultaneous replacement of the pyrazine ring by a phenyl and of the 6-chloro by sulfomethyl leads to another class of inhibitors that includes the benzoylguanidines and derivatives such as HOE 694 (Counillon et al., 1993) and HOE 642 (cariporide) (Scholz et al., 1995). Both classes are more specific for NHE-1 than NHE-3, with the amiloride compounds demonstrating ~102-fold increased specificity and the HOE compounds ~103- to 105-fold more NHE-1 specificity. The HOE compounds are viewed as the most promising agents for treatment of ischemia-reperfusion injury due to their selectivity for NHE-1, and excellent solubility, resorption, and bioavailability profiles (Scholz et al., 1999; Baumgarth et al., 1997; Xue & Haddad, 2010). HOE compounds are competitive inhibitors of Na+ binding at the extracellular cation-binding site (Baumgarth et al., 1997; Counillon et al., 1993; Kinsella & Aronson, 1981; Mahnensmith & Aronson, 1985), while the amiloride derivatives also act non-competitively (Warnock et al., 1988). More recently, several new molecules have been designed as potential NHE blockers based on the bicyclic template, including SM-20220, SM-20550, BMS-284640, T-162559, and TY-12533, which have also shown promising results in *in vivo* studies of cerebral ischemia (Kitayama et al., 2001). The IC50 for the human NHE-1 are as follows: Amiloride = 10.7 µM, Cariporide = 0.08 µM, T-165229 = 13 nM. Importantly, the NHE inhibitors HOE 642 and SM-20220 not only reduce cell death and edema, but also improve neurological function in *in vivo* ischemia models, and have demonstrated benefits when administered after ischemia (Kintner et al., 2007b; Kuribayashi et al., 2000).

### **2.5.2 Transgenic approach**

502 Advances in the Preclinical Study of Ischemic Stroke

astrocytes, we found a similar increase in [Na+]i which could be abolished by the NHE-1 inhibitor HOE 642 (Kintner et al., 2007b). It has been reported that the expected rise in [Ca2+]i after HAIR exposure is inhibited by NHE-1 inhibition with HOE 694 (Bondarenko et al., 2005). Taken together, these results indicate that NHE-1 activity raises [Na+]i which fosters reversal of the Na+/Ca+ exchanger leading to increased intracellular Ca2+ and

More recently, new evidence supports a role of NHE-1 in microglial pHi regulation. Microglia activation by lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or OGD/REOX triggers a concurrent stimulation of NHE-1 and NADPH oxidase (Liu et al., 2010). The elevation in NHE-1-mediated H+ extrusion prevents intracellular acidosis, allowing for sustained NADPH oxidase function (Liu et al., 2010). Moreover, the coupling of NHE-1 activation with NCXrev activates [Na+]i and [Ca2+]i dependent signaling, which promotes the microglial respiratory burst and production of proinflammatory cytokines

Few studies have used brain slice preparations to examine acid-base homeostatic disturbances during ischemia. In hippocampal slices, hypoxia induces a significant drop in both pHi and pHo, and a brief alkaline peak is also occasionally observed (Fujiwara et al., 1992; Melzian et al., 1996; Roberts & Chih, 1997). In slice preparations from various brain regions, hypoxia causes acidosis with an approximately 0.8-1.2 pHi unit drop (Ballanyi et al, 1996; Knopfel et al., 1998; Pirttila & Kauppinen, 1994). Cytosolic calcium changes are observed during ischemia in cortical brain slices that can be only partially inhibited by combined blockade of ion channels (Bickler and Hansen, 1994). Only one report shows a direct involvement of NHE mediated pHi regulation in slice preparations. In brainstem slices from neonatal rats exposed to 10 minutes of anoxia, intracellular pH drops by 0.1-0.3 pH units in neurons. Inhibition of NHE with amiloride increases this anoxia-induced

Despite decades of research, the effective treatment and prevention of cerebral ischemic injury remains challenging. Inhibition of NHE-1 with either pharmacological agents or genetic ablation has been demonstrated to significantly reduce brain damage after ischemic insult, in both *in vitro* and *in vivo* models. These encouraging findings suggest the potential

Two major classes of pharmacological agents are currently used to inhibit NHE-1 activity (Putney et al., 2002). The first class of drugs includes amiloride and its 5' alkyl-substituted derivatives (Counillon et al., 1993; Yu et al., 1993), such as ethylisopropylamiloride (EIPA), dimethylamiloride (DMA), 5-*N* (methylpropyl)amiloride (MPA), 5-(N-methyl-N-isobutyl) amiloride (MIBA), and 5-(N, N-hexamethylene) amiloride (HMA). These agents are more effective inhibitors of NHE-1 than amiloride but have relatively weak selectivity toward NHE-1. The simultaneous replacement of the pyrazine ring by a phenyl and of the 6-chloro by sulfomethyl leads to another class of inhibitors that includes the benzoylguanidines and derivatives such as HOE 694 (Counillon et al., 1993) and HOE 642 (cariporide) (Scholz et al.,

use of NHE inhibitors as neuroprotective therapies after cerebral ischemia.

intracellular acidification (Chambers-Kersh et al., 2000).

**2.5 NHE-1 inhibitors and potential therapies** 

**2.5.1 Pharmacological approach** 

astrocyte cell death.

(Liu et al., 2010).

**2.4.2 Brain slice** 

While pharmacological studies indicate that NHE-1 plays a central role in cerebral ischemiareperfusion injury, the use of pharmacologic inhibitors to study ion transport function raises questions regarding dosing, absorption, species specific T1/2, and non-specific effects. For this reason, confirmation by an alternative method using NHE-1 knockdown mice is warranted. NHE-1-/- mice exhibit neurologic abnormalities, seizures, ataxia, and growth retardation, and do not survive into adulthood (Bell et al., 1999; Gu et al., 2001). Therefore, NHE-1-/- mice are useful for cultures of NHE-1 null neurons, astrocytes and microglia, but cannot be used for *in vivo* studies. NHE-1+/- mice express <50% of NHE-1 protein levels, and are useful for *in vivo* studies of the role of NHE function after cerebral ischemia. A marked decrease of infarct volume, microglial activation and proinflammatory cytokine formation is found in NHE-1+/- mice after MCAO (Luo et al., 2005). NHE-1 +/- and NHE-1 -/- cortical neurons and astrocytes demonstrate decreased cell death after OGD/REOX (Luo et al., 2005).

The fact that NHE-1 inhibitors applied during or after cerebral ischemia protect the brain against ischemic damage is now well established in animal studies. Despite the uniformity of results from animal models, a number of challenges remain before NHE-1 inhibitors can be translated into clinical use. Questions regarding safety, optimal dose, and timing of administration remain to be addressed, and large animal studies demonstrating improved functional outcomes are still lacking.

### **3. Conclusion**

NHE-1 plays a pivotal role in maintaining tissue ionic homeostasis under normal physiological conditions. However, excessive stimulation of NHE-1 appears to be a major contributor to cellular damage in ischemic conditions. The proposed mechanism for injury induced by NHE-1 activation includes accumulation of [Na+]i, subsequent [Ca2+]i overload via reverse activation of the Na+/Ca2+ exchanger, and eventual cell death. Additionally, activation of MAPKs, and release of excitatory amino acids and ROS also contribute to cell

The Na<sup>+</sup>

/H<sup>+</sup>

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characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting antiischemic properties. *Molecular* 

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pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death. *Journal of Clinical* 

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Fujiwara, N., Abe, T., Endoh, H., Warashina, A. & Shimoji, K. (1992). Changes in

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damage and death after ischemia. NHE-1 inhibitors have been demonstrated to be neuroprotective in both *in vitro* and *in vivo* ischemia models, making NHE-1 an attractive therapeutic target for cerebral ischemia. Thus, mechanisms of NHE-1 activation in ischemia continue to present an interesting focus for future research in this field.

### **4. Acknowledgment**

This work was supported by NIH grants R01NS48216 and R01NS38118 (D. Sun), 1UL1RR025011 from the Clinical and Translational Science Award (CTSA) program of the National Center for Research Resources (NCRR) (P. Ferrazzano), and P30HD03352 (Waisman Center).

### **5. References**


damage and death after ischemia. NHE-1 inhibitors have been demonstrated to be neuroprotective in both *in vitro* and *in vivo* ischemia models, making NHE-1 an attractive therapeutic target for cerebral ischemia. Thus, mechanisms of NHE-1 activation in ischemia

This work was supported by NIH grants R01NS48216 and R01NS38118 (D. Sun), 1UL1RR025011 from the Clinical and Translational Science Award (CTSA) program of the National Center for Research Resources (NCRR) (P. Ferrazzano), and P30HD03352

Attaphitaya, S., Park, K. & Melvin, J.E. (1999). Molecular cloning and functional expression

Ballanyi, K., Doutheil, J. & Brockhaus, J. (1996). Membrane potentials and microenvironment

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**22** 

*1Italy 2UK* 

**PPAR Agonism as New Pharmacological** 

*2Centre for Translational Medicine and Therapeutics, Queen Mary University of London,* 

Despite increasing knowledge of the biochemical mechanisms that occur in the brain following an ischemic insult and the availability of several diverse animal models of stroke, there are still no drugs that can be given to stroke patients soon after the onset of symptoms to minimize the subsequent neurological damage. To date, the thrombolytic compound recombinant tissue Plasminogen Activator (rt-PA) remains the only approved drug for the treatment of stroke. At present, intravenous administration of rt-PA is the only proven effective treatment to re-establish cerebral blood flow in the case of acute vessel occlusion, but unfortunately, only few patients with acute ischemic stroke are qualified to receive this drug. The failure of rt-PA to achieve rapid reperfusion in many patients and its bleeding risk have prompted the development of fibrinolytic agents with greater fibrin specificity and better risk-benefit profiles, such as tenecteplase or desmoteplase, which are now under active investigation. Early restoration of blood flow remains the treatment of choice for limiting brain injury following stroke, but a second fundamental goal of intervention is to protect neurons by interrupting or slowing the ischemic cascade. Current research is being done to develop neuroprotective agents that are able to block amino acid pathways and decrease neurotransmitter activity of injured tissue. Drugs blocking voltage-dependent calcium channels were effective in stroke rodent models but the results of clinical trials have been often discouraging. Overactivation of the N-methyl-D-aspartate receptor (NMDAR) is crucial for neuronal death after stroke. Several compounds that interfere with glutamate receptor activation have been developed and tested, in particular noncompetitive NMDA antagonists. However, their clinical use is limited by intolerable side effects, including some psycomimetic symptoms, as these blockers may also impair some key brain functions mediated by the same receptor. Accumulating evidence strongly suggests that apoptosis contributes to neuronal cell death in stroke injury and currently several caspase inhibitors are under investigation, but to date the efficacy of antiapoptotic agents in human stroke patients has not yet been tested. Anti-inflammatory approaches to stroke treatment intended

**1. Introduction** 

**Approach to the Management** 

Elisa Benetti1, Nimesh Patel2 and Massimo Collino1

**of Acute Ischemic Stroke** 

*University of Turin, Turin,* 

*1Dipartimento di Scienza e Tecnologia del Farmaco,* 

*The William Harvey Research Institute, London,* 

Yu, F.H., Shull, G.E. & Orlowski, J. (1993). Functional properties of the rat Na/H exchanger NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary cells. *Journal of Biological Chemistry,* 268, 25536–25541.

### **PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke**

Elisa Benetti1, Nimesh Patel2 and Massimo Collino1

*1Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin, Turin, 2Centre for Translational Medicine and Therapeutics, Queen Mary University of London, The William Harvey Research Institute, London, 1Italy 2UK* 

### **1. Introduction**

510 Advances in the Preclinical Study of Ischemic Stroke

Yu, F.H., Shull, G.E. & Orlowski, J. (1993). Functional properties of the rat Na/H exchanger

cells. *Journal of Biological Chemistry,* 268, 25536–25541.

NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary

Despite increasing knowledge of the biochemical mechanisms that occur in the brain following an ischemic insult and the availability of several diverse animal models of stroke, there are still no drugs that can be given to stroke patients soon after the onset of symptoms to minimize the subsequent neurological damage. To date, the thrombolytic compound recombinant tissue Plasminogen Activator (rt-PA) remains the only approved drug for the treatment of stroke. At present, intravenous administration of rt-PA is the only proven effective treatment to re-establish cerebral blood flow in the case of acute vessel occlusion, but unfortunately, only few patients with acute ischemic stroke are qualified to receive this drug. The failure of rt-PA to achieve rapid reperfusion in many patients and its bleeding risk have prompted the development of fibrinolytic agents with greater fibrin specificity and better risk-benefit profiles, such as tenecteplase or desmoteplase, which are now under active investigation. Early restoration of blood flow remains the treatment of choice for limiting brain injury following stroke, but a second fundamental goal of intervention is to protect neurons by interrupting or slowing the ischemic cascade. Current research is being done to develop neuroprotective agents that are able to block amino acid pathways and decrease neurotransmitter activity of injured tissue. Drugs blocking voltage-dependent calcium channels were effective in stroke rodent models but the results of clinical trials have been often discouraging. Overactivation of the N-methyl-D-aspartate receptor (NMDAR) is crucial for neuronal death after stroke. Several compounds that interfere with glutamate receptor activation have been developed and tested, in particular noncompetitive NMDA antagonists. However, their clinical use is limited by intolerable side effects, including some psycomimetic symptoms, as these blockers may also impair some key brain functions mediated by the same receptor. Accumulating evidence strongly suggests that apoptosis contributes to neuronal cell death in stroke injury and currently several caspase inhibitors are under investigation, but to date the efficacy of antiapoptotic agents in human stroke patients has not yet been tested. Anti-inflammatory approaches to stroke treatment intended

PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke 513

recognised as homologues for each other, it was not originally certain whether PPARβ from

All members of this superfamily share the typical domain organization of nuclear receptors (Figure 1). The N-terminal A/B domain contains a ligand-independent transactivation function. In the α and isotypes, the activity of this domain can be regulated by Mitogen-Activated Protein Kinase (MAPK) phosphorylation (Hu, Kim et al. 1996). The C domain is the DNA binding domain with its typical two zinc-finger-like motifs, as previously described for the steroid receptors, and the D domain is the co-factor docking domain (Schwabe, Neuhaus et al. 1990). The E/F domain is the ligand binding domain, it contains a ligand-dependent trans-activation function (AF)-2 (Fajas, Auboeuf et al. 1997), and is able to interact with transcriptional coactivators such as steroid receptor coactivator (SRC)-1 (Onate, Tsai et al. 1995) and CREB-binding protein (CBP) (Amri,

Fig. 1. Schematic representation of the domain organization of human PPAR isoforms. The A/B domain contains the Activation Function 1 (AF-1) which has a ligand-independent transcriptional activity. The C domain corresponds to the DNA Binding Domain (DBD). The D domain is the co-factor docking domain. The E/F domain contains the Ligand Binding Domain (LBD) and carries the Activation Function 2 (AF-2), which has a liganddependent transcriptional activity. The human chromosome regions in which disting genes encoding for PPAR isoforms are mapped, the percentage of amino acid sequence identity (in comparison with PPAR) and the amino acid number of different isoforms are reported

The highest PPARα expression has been found in the liver and in tissues with high fatty acid catabolism, such as the kidney, heart, skeletal muscle, and brown fat (Lefebvre, Chinetti et al. 2006). PPARα mainly regulates energy homeostasis, activating fatty acid catabolism and stimulating gluconeogenesis (Kersten, Seydoux et al. 1999). This increased fatty acid oxidation in response to PPARα activation with a selective agonist, WY14643, results in lower circulating triglyceride levels and reduction of lipid storage in liver, muscle, and adipose tissue (Chou, Haluzik et al. 2002), which is associated with improved insulin sensitivity (Kim, Haluzik et al. 2003). Consequently, fibrates (fenofibrate, bezafibrate,

Xenopus was identical to murine PPARδ, hence the terminology PPARβ/δ.

Bonino et al. 1995).

in the Table.

to block cell-mediated inflammation with different strategies such as humanized antibodies against ICAM-1, inhibitors of interleukin-1 beta or a interleukin-1 receptor antagonist. However, there have been no successful clinical trials of these anti-inflammatory agents so far.

The complexity of events in cerebral ischemia and the disappointing results from human clinical stroke trials using a single agent suggest that perhaps to treat the stroke a new pleiotropic approach is required. In the pharmacological perspective, the evaluation of drugs with multiple effects on the ischemic cascade may be more effective in reducing infarct size and improving outcome in respect to single target strategy, because the ischemic cascade is diverse and it is likely that many different mechanisms of ischemia induced cell death occur simultaneously. Therefore, the development of neuroprotective drugs with multiple effects on the ischemic cascade is potentially more appealing than drugs acting on only one component of the cascade, if the safety profile is reasonable and the preclinical assessment package fulfils recent recommendations. Most recent discoveries portray Peroxisome Proliferator-Activated Receptors (PPARs) as promising pharmacological targets for the treatment of acute ischemic stroke, thanks to their ability to simultaneously interfere with several mechanisms that underlie the pathophysiology of brain ischemia, thus leading to an interesting protective strategy to counteract the multiple deleterious effects of ischemic injury.

### **2. PPAR**

Peroxisome Proliferator-Activated Receptors (PPARs) are members of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcription factors. There are three PPAR subtypes: α, β/δ and γ, named also NR1C1, NR1C2 and NR1C3, respectively, according to the unified nomenclature of nuclear receptors (Nuclear Receptors Nomenclature Committee, 1999). The three isoforms are the products of distinct genes: the human PPAR gene was mapped on chromosome 22 in the general region 22q12–q13.1, the PPAR gene is located on chromosome 3 at position 3p25, whereas PPARβ/δ has been assigned to chromosome 6, at position 6p21.1–p21.2 (Sher, Yi et al. 1993; Greene, Blumberg et al. 1995; Yoshikawa, Brkanac et al. 1996). PPARs were originally identified by Isseman and Green (Issemann and Green 1990) after screening the rat liver cDNA library with a cDNA sequence located in the highly conserved C domain of NHRs. The name PPAR is derived from the fact that activation of PPAR, the first member of the PPAR family to be cloned, results in peroxisome proliferation in rodent hepatocytes (Desvergne and Wahli 1999). Activation of neither PPARβ/δ nor PPAR, however, elicits this response and, interestingly, the phenomenon of peroxisome proliferation does not occur in humans. The molecular basis for this difference between species is not yet clear. With respect to the PPAR isotype, alternative splicing and promoter use results in the formation of two further isoforms: PPAR1 and PPAR2. In particular, differential promoter usage and alternate splicing of the gene generates three mRNA isoforms. PPAR1 and PPAR3 mRNA both encode the PPAR1 protein product which is expressed in most tissues, whereas PPAR2 mRNA encodes the PPAR2 protein, which contains an additional 28 amino acids at the amino terminus and is specific to adipocytes (Gurnell 2003). PPARβ/δ was initially reported as PPARβ in Xenopus laevis and NUC1 in humans (Schmidt, Endo et al. 1992). Subsequently, a similar transcript was cloned from mice and termed PPARδ (Amri, Bonino et al. 1995). Though now

to block cell-mediated inflammation with different strategies such as humanized antibodies against ICAM-1, inhibitors of interleukin-1 beta or a interleukin-1 receptor antagonist. However, there have been no successful clinical trials of these anti-inflammatory agents so

The complexity of events in cerebral ischemia and the disappointing results from human clinical stroke trials using a single agent suggest that perhaps to treat the stroke a new pleiotropic approach is required. In the pharmacological perspective, the evaluation of drugs with multiple effects on the ischemic cascade may be more effective in reducing infarct size and improving outcome in respect to single target strategy, because the ischemic cascade is diverse and it is likely that many different mechanisms of ischemia induced cell death occur simultaneously. Therefore, the development of neuroprotective drugs with multiple effects on the ischemic cascade is potentially more appealing than drugs acting on only one component of the cascade, if the safety profile is reasonable and the preclinical assessment package fulfils recent recommendations. Most recent discoveries portray Peroxisome Proliferator-Activated Receptors (PPARs) as promising pharmacological targets for the treatment of acute ischemic stroke, thanks to their ability to simultaneously interfere with several mechanisms that underlie the pathophysiology of brain ischemia, thus leading to an interesting protective strategy to counteract the multiple deleterious effects of ischemic

Peroxisome Proliferator-Activated Receptors (PPARs) are members of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcription factors. There are three PPAR subtypes: α, β/δ and γ, named also NR1C1, NR1C2 and NR1C3, respectively, according to the unified nomenclature of nuclear receptors (Nuclear Receptors Nomenclature Committee, 1999). The three isoforms are the products of distinct genes: the human PPAR gene was mapped on chromosome 22 in the general region 22q12–q13.1, the PPAR gene is located on chromosome 3 at position 3p25, whereas PPARβ/δ has been assigned to chromosome 6, at position 6p21.1–p21.2 (Sher, Yi et al. 1993; Greene, Blumberg et al. 1995; Yoshikawa, Brkanac et al. 1996). PPARs were originally identified by Isseman and Green (Issemann and Green 1990) after screening the rat liver cDNA library with a cDNA sequence located in the highly conserved C domain of NHRs. The name PPAR is derived from the fact that activation of PPAR, the first member of the PPAR family to be cloned, results in peroxisome proliferation in rodent hepatocytes (Desvergne and Wahli 1999). Activation of neither PPARβ/δ nor PPAR, however, elicits this response and, interestingly, the phenomenon of peroxisome proliferation does not occur in humans. The molecular basis for this difference between species is not yet clear. With respect to the PPAR isotype, alternative splicing and promoter use results in the formation of two further isoforms: PPAR1 and PPAR2. In particular, differential promoter usage and alternate splicing of the gene generates three mRNA isoforms. PPAR1 and PPAR3 mRNA both encode the PPAR1 protein product which is expressed in most tissues, whereas PPAR2 mRNA encodes the PPAR2 protein, which contains an additional 28 amino acids at the amino terminus and is specific to adipocytes (Gurnell 2003). PPARβ/δ was initially reported as PPARβ in Xenopus laevis and NUC1 in humans (Schmidt, Endo et al. 1992). Subsequently, a similar transcript was cloned from mice and termed PPARδ (Amri, Bonino et al. 1995). Though now

far.

injury.

**2. PPAR** 

recognised as homologues for each other, it was not originally certain whether PPARβ from Xenopus was identical to murine PPARδ, hence the terminology PPARβ/δ.

All members of this superfamily share the typical domain organization of nuclear receptors (Figure 1). The N-terminal A/B domain contains a ligand-independent transactivation function. In the α and isotypes, the activity of this domain can be regulated by Mitogen-Activated Protein Kinase (MAPK) phosphorylation (Hu, Kim et al. 1996). The C domain is the DNA binding domain with its typical two zinc-finger-like motifs, as previously described for the steroid receptors, and the D domain is the co-factor docking domain (Schwabe, Neuhaus et al. 1990). The E/F domain is the ligand binding domain, it contains a ligand-dependent trans-activation function (AF)-2 (Fajas, Auboeuf et al. 1997), and is able to interact with transcriptional coactivators such as steroid receptor coactivator (SRC)-1 (Onate, Tsai et al. 1995) and CREB-binding protein (CBP) (Amri, Bonino et al. 1995).


Fig. 1. Schematic representation of the domain organization of human PPAR isoforms. The A/B domain contains the Activation Function 1 (AF-1) which has a ligand-independent transcriptional activity. The C domain corresponds to the DNA Binding Domain (DBD). The D domain is the co-factor docking domain. The E/F domain contains the Ligand Binding Domain (LBD) and carries the Activation Function 2 (AF-2), which has a liganddependent transcriptional activity. The human chromosome regions in which disting genes encoding for PPAR isoforms are mapped, the percentage of amino acid sequence identity (in comparison with PPAR) and the amino acid number of different isoforms are reported in the Table.

The highest PPARα expression has been found in the liver and in tissues with high fatty acid catabolism, such as the kidney, heart, skeletal muscle, and brown fat (Lefebvre, Chinetti et al. 2006). PPARα mainly regulates energy homeostasis, activating fatty acid catabolism and stimulating gluconeogenesis (Kersten, Seydoux et al. 1999). This increased fatty acid oxidation in response to PPARα activation with a selective agonist, WY14643, results in lower circulating triglyceride levels and reduction of lipid storage in liver, muscle, and adipose tissue (Chou, Haluzik et al. 2002), which is associated with improved insulin sensitivity (Kim, Haluzik et al. 2003). Consequently, fibrates (fenofibrate, bezafibrate,

PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke 515

humans. In contrast to the well-documented therapeutic effect, there is also evidence of liver toxicity induced by activation of PPARα, mainly hepatocarcinogenesis. The most serious safety risk associated with fibrates, although rare, is myopathy and rhabdomyolysis. Studies suggest that the mechanism of myotoxicity through fibrates is not entirely clear, because complex and multifactorial mechanisms are involved, including genetic predisposition, pharmacokinetics, drug interactions, and dose. It is of interest to note that increased expression of lipoprotein lipase, which is a known PPARα target gene, in skeletal muscle

The most widely used PPAR agonists belong to the thiazolidinedione (TZD) or glitazone class of anti-diabetic drugs used in the treatment of type-2 diabetes. Troglitazone, the first TZD approved for this use, was withdrawn from the market in March 2000 following the emergence of a serious hepatotoxicity in some patients. Since troglitazone induces CYP3A4, it has been hypothesized that potentially toxic quinones derived from CYP3A4-dependent metabolism could cause liver damage (Yamamoto, Yamazaki et al. 2002). Rosiglitazone and pioglitazone are the only available thiazolidinediones in North America, but meta-analyses of randomised controlled trials have suggested an increased risk of ischaemic cardiovascular events with rosiglitazone (Nissen and Wolski ; Singh, Loke et al. 2007). In contrast, metaanalysis of trials of pioglitazone indicates the possibility of an ischaemic cardiovascular benefit (Lincoff, Wolski et al. 2007). Robust evidence also shows that both drugs increase the risk of congestive heart failure and fractures, but whether any meaningful difference exists in the magnitude of risk between the two thiazolidinediones is not known (Singh, Loke et al. 2007; Loke, Singh et al. 2009). The European Medicines Agency has recommended the suspension of marketing authorisation for rosiglitazone, whereas the US Food and Drug Administration has

allowed the continued marketing of rosiglitazone with additional restrictions.

protein and pyruvate dehydrogenase kinase-4)(Shearer, Steger et al. 2008).

**2.2 Molecular mechanisms of PPAR activation** 

**2.2.1 Mechanism of transcriptional transactivation** 

independent transrepression (Figure 2).

On the contrary, there are no PPARβ/δ drugs in clinical use yet. However several selective PPARβ/δ ligands have been recently designed, including GW0742, GW2433, GW9578, L-783483, L-165041, or GW501516 (Berger, Leibowitz et al. 1999; Lim and Dey 2000; Martens, Visseren et al. 2002). As yet only one selective PPARβ/δ antagonist has been described GSK0660. In skeletal muscle myoblast cells in culture, GSK0660 inhibited GW0742 induction of established PPARβ/δ target genes (carnitine palmitoyltransferase 1A, angiopoietin-like 4

There are at least three primary mechanisms by which PPARs can regulate biological functions: transcriptional transactivation, transcriptional transrepression and ligand-

PPARs function as heterodimers with their obligatory partner the Retinoid X Receptor (RXR). Like other NHRs, the PPAR/RXR heterodimer most likely recruits co-factor complexes - either co-activators or co-repressors - that modulate its transcriptional activity (Shi, Hon et al. 2002). The PPAR/RXR heterodimer then binds to sequence specific PPAR Response Elements (PPREs), located in the 5'-flanking region of target genes, thereby acting as a transcriptional regulator (Palmer, Hsu et al. 1995). The PPRE consists of two direct repeats of the consensus sequence AGGTCA separated by a single nucleotide, which constitutes a DR-1 motif. PPAR binds 5' of RXR on the DR-1 motif and the 5'-flanking

leads to severe myopathy in mice.

gemfibrozil), which are synthetic agonists for PPARα, are in wide clinical use for the treatment of dyslipidaemias.

PPARγ is expressed in white and brown adipose tissue, gut, and immune cells (Feige, Gelman et al. 2006). It is involved in adipocyte differentiation and lipid storage in white adipose tissue (Rosen, Sarraf et al. 1999). Furthermore, PPARγ is involved in glucose metabolism via an improvement of insulin sensitivity (Hevener, He et al. 2003). Therefore, synthetic PPARγ agonists (thiazolidinediones) are in clinical use as insulin sensitizers to treat patients with type-2 diabetes.

PPARβ/δ remained an enigma for almost a decade after its cloning in 1992. It has been reported to be ubiquitously expressed in almost every tissue and, in the past, this widespread tissue expression has suggested a possible "general housekeeping" role for PPARβ/δ (Kliewer, Forman et al. 1994). More recently, the use of transgenic mouse models and the availability of high-affinity synthetic ligands has led researchers to a better understanding of its physiological role. Specifically, increasing evidence has shown a particular role for PPARβ/δ in insulin sensitivity regulation, lipid metabolism and the inflammation response. However, in contrast to PPARα and γ, PPARβ/δ agonists are not yet in clinical use.

### **2.1 Endogenous and synthetic PPAR ligands**

Although many fatty acids are capable of activating all three PPAR isoforms, some fatty acids are also specific for a particular PPAR isoform. X-ray crystallography studies of PPARβ/δ revealed an exceptionally large ligand-binding pocket of approximately 1,300 Ǻ3, similar to that of PPARγ but much larger than the pockets of other nuclear receptors (Xu, Lambert et al. 1999). The increased dimension is believed to accommodate the binding of various fatty acids or other amphipathic acids to PPARβ/δ via hydrogen bonds and hydrophobic interactions. The long-chain polyunsaturated fatty acids and their oxidized derivatives, especially eicosanoids such as 8-S-hydroxyeicosatetraenoic acid (8-S-HETE), leukotriene B4 (LTB4) and arachidonate monooxygenase metabolite epoxyeicosatrienoic acids have been shown to potently activate PPAR with high affinity (Theocharisa, Margeli et al. 2003; Feige, Gelman et al. 2006). PPAR can be activated by several prostanoids, such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and 12- and 15-hydroxy-eicosatetraenoic acid (12- and 15-HETE), which are derivatives of arachidonic acid synthesized through the lipoxygenase pathway, as well as modified oxidised lipids, 9- and 13 hydroxyoctadecadienoic acids (9- and 13-HODE) (Willson, Brown et al. 2000; Theocharisa, Margeli et al. 2003). PPARβ/δ agonists include linoleic acid, oleic acid, arachidonic acid and eicosapentaenoic acid (EPA), which have been shown to co-crystallize within the ligand binding domain of this nuclear receptor (Xu, Lambert et al. 1999). A number of eicosanoids, including prostaglandin (PG)A1 and PGD2, and carbaprostacyclin, a semi-synthetic prostaglandin, have micromolar affinities for PPARβ/δ (Forman, Chen et al. 1997). Recently, cows milk, ice cream, butter, and yoghurt were described as activators of PPARβ/δ in reporter assays, but a specific common compound was not identified (Suhara, Koide et al. 2009).

With respect to the synthetic ligands, fibrates (e.g. fenofibrate, clofibrate), which are hypolipidaemic drugs, are well-known ligands for PPAR (Willson, Brown et al. 2000). Fibrates are capable of activating PPARα at pharmacological doses leading to increased expression of lipid metabolizing enzymes that effectively lower serum lipid levels in humans. In contrast to the well-documented therapeutic effect, there is also evidence of liver toxicity induced by activation of PPARα, mainly hepatocarcinogenesis. The most serious safety risk associated with fibrates, although rare, is myopathy and rhabdomyolysis. Studies suggest that the mechanism of myotoxicity through fibrates is not entirely clear, because complex and multifactorial mechanisms are involved, including genetic predisposition, pharmacokinetics, drug interactions, and dose. It is of interest to note that increased expression of lipoprotein lipase, which is a known PPARα target gene, in skeletal muscle leads to severe myopathy in mice.

The most widely used PPAR agonists belong to the thiazolidinedione (TZD) or glitazone class of anti-diabetic drugs used in the treatment of type-2 diabetes. Troglitazone, the first TZD approved for this use, was withdrawn from the market in March 2000 following the emergence of a serious hepatotoxicity in some patients. Since troglitazone induces CYP3A4, it has been hypothesized that potentially toxic quinones derived from CYP3A4-dependent metabolism could cause liver damage (Yamamoto, Yamazaki et al. 2002). Rosiglitazone and pioglitazone are the only available thiazolidinediones in North America, but meta-analyses of randomised controlled trials have suggested an increased risk of ischaemic cardiovascular events with rosiglitazone (Nissen and Wolski ; Singh, Loke et al. 2007). In contrast, metaanalysis of trials of pioglitazone indicates the possibility of an ischaemic cardiovascular benefit (Lincoff, Wolski et al. 2007). Robust evidence also shows that both drugs increase the risk of congestive heart failure and fractures, but whether any meaningful difference exists in the magnitude of risk between the two thiazolidinediones is not known (Singh, Loke et al. 2007; Loke, Singh et al. 2009). The European Medicines Agency has recommended the suspension of marketing authorisation for rosiglitazone, whereas the US Food and Drug Administration has allowed the continued marketing of rosiglitazone with additional restrictions.

On the contrary, there are no PPARβ/δ drugs in clinical use yet. However several selective PPARβ/δ ligands have been recently designed, including GW0742, GW2433, GW9578, L-783483, L-165041, or GW501516 (Berger, Leibowitz et al. 1999; Lim and Dey 2000; Martens, Visseren et al. 2002). As yet only one selective PPARβ/δ antagonist has been described GSK0660. In skeletal muscle myoblast cells in culture, GSK0660 inhibited GW0742 induction of established PPARβ/δ target genes (carnitine palmitoyltransferase 1A, angiopoietin-like 4 protein and pyruvate dehydrogenase kinase-4)(Shearer, Steger et al. 2008).

### **2.2 Molecular mechanisms of PPAR activation**

514 Advances in the Preclinical Study of Ischemic Stroke

gemfibrozil), which are synthetic agonists for PPARα, are in wide clinical use for the

PPARγ is expressed in white and brown adipose tissue, gut, and immune cells (Feige, Gelman et al. 2006). It is involved in adipocyte differentiation and lipid storage in white adipose tissue (Rosen, Sarraf et al. 1999). Furthermore, PPARγ is involved in glucose metabolism via an improvement of insulin sensitivity (Hevener, He et al. 2003). Therefore, synthetic PPARγ agonists (thiazolidinediones) are in clinical use as insulin sensitizers to

PPARβ/δ remained an enigma for almost a decade after its cloning in 1992. It has been reported to be ubiquitously expressed in almost every tissue and, in the past, this widespread tissue expression has suggested a possible "general housekeeping" role for PPARβ/δ (Kliewer, Forman et al. 1994). More recently, the use of transgenic mouse models and the availability of high-affinity synthetic ligands has led researchers to a better understanding of its physiological role. Specifically, increasing evidence has shown a particular role for PPARβ/δ in insulin sensitivity regulation, lipid metabolism and the inflammation response. However, in contrast to PPARα and γ, PPARβ/δ agonists are not

Although many fatty acids are capable of activating all three PPAR isoforms, some fatty acids are also specific for a particular PPAR isoform. X-ray crystallography studies of PPARβ/δ revealed an exceptionally large ligand-binding pocket of approximately 1,300 Ǻ3, similar to that of PPARγ but much larger than the pockets of other nuclear receptors (Xu, Lambert et al. 1999). The increased dimension is believed to accommodate the binding of various fatty acids or other amphipathic acids to PPARβ/δ via hydrogen bonds and hydrophobic interactions. The long-chain polyunsaturated fatty acids and their oxidized derivatives, especially eicosanoids such as 8-S-hydroxyeicosatetraenoic acid (8-S-HETE), leukotriene B4 (LTB4) and arachidonate monooxygenase metabolite epoxyeicosatrienoic acids have been shown to potently activate PPAR with high affinity (Theocharisa, Margeli et al. 2003; Feige, Gelman et al. 2006). PPAR can be activated by several prostanoids, such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and 12- and 15-hydroxy-eicosatetraenoic acid (12- and 15-HETE), which are derivatives of arachidonic acid synthesized through the lipoxygenase pathway, as well as modified oxidised lipids, 9- and 13 hydroxyoctadecadienoic acids (9- and 13-HODE) (Willson, Brown et al. 2000; Theocharisa, Margeli et al. 2003). PPARβ/δ agonists include linoleic acid, oleic acid, arachidonic acid and eicosapentaenoic acid (EPA), which have been shown to co-crystallize within the ligand binding domain of this nuclear receptor (Xu, Lambert et al. 1999). A number of eicosanoids, including prostaglandin (PG)A1 and PGD2, and carbaprostacyclin, a semi-synthetic prostaglandin, have micromolar affinities for PPARβ/δ (Forman, Chen et al. 1997). Recently, cows milk, ice cream, butter, and yoghurt were described as activators of PPARβ/δ in reporter assays, but a specific common compound was not identified (Suhara, Koide et al.

With respect to the synthetic ligands, fibrates (e.g. fenofibrate, clofibrate), which are hypolipidaemic drugs, are well-known ligands for PPAR (Willson, Brown et al. 2000). Fibrates are capable of activating PPARα at pharmacological doses leading to increased expression of lipid metabolizing enzymes that effectively lower serum lipid levels in

treatment of dyslipidaemias.

treat patients with type-2 diabetes.

**2.1 Endogenous and synthetic PPAR ligands** 

yet in clinical use.

2009).

There are at least three primary mechanisms by which PPARs can regulate biological functions: transcriptional transactivation, transcriptional transrepression and ligandindependent transrepression (Figure 2).

### **2.2.1 Mechanism of transcriptional transactivation**

PPARs function as heterodimers with their obligatory partner the Retinoid X Receptor (RXR). Like other NHRs, the PPAR/RXR heterodimer most likely recruits co-factor complexes - either co-activators or co-repressors - that modulate its transcriptional activity (Shi, Hon et al. 2002). The PPAR/RXR heterodimer then binds to sequence specific PPAR Response Elements (PPREs), located in the 5'-flanking region of target genes, thereby acting as a transcriptional regulator (Palmer, Hsu et al. 1995). The PPRE consists of two direct repeats of the consensus sequence AGGTCA separated by a single nucleotide, which constitutes a DR-1 motif. PPAR binds 5' of RXR on the DR-1 motif and the 5'-flanking

PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke 517

target genes, transrepression does not involve binding to typical receptor specific response elements (Pascual and Glass 2006). Several lines of evidence suggest that PPARs may exert anti-inflammatory effects by negatively regulating the expression of pro-inflammatory genes. To date, several mechanisms have been suggested to account for this activity, but

Firstly, competition for limited amounts of essential, shared transcriptional co-activators may play a role in transrepression. The activated PPAR/RXR heterodimer reduces the availability of co-activators required for gene induction by other transcriptional factors.

Secondly, PPAR/RXR complexes may cause a functional inhibition by directly binding to transcription factors, preventing them from inducing gene transcription or inducing the expression of inhibitory proteins, such as the protein inhibitor of kappa B (IκB)α, which sequesters the NF-κB subunits in the cytoplasm and consequently reduces their DNA

Thirdly, PPAR/RXR heterodimers may also inhibit phosphorylation and activation of several members of the MAPK family. In general very little is known about the molecular

Recent studies have suggested another mechanism based on co-repressor-dependent transrepression by PPARs. Evidence has been presented in which PPARβ/δ controls the inflammatory status of macrophages based on its association with the transcriptional repressor BCL-6 (Lee, Chawla et al. 2003). Free BCL-6 suppresses the expression of multiple proinflammatory cytokines and chemokines. PPARβ/δ, but not PPARα and PPAR, exhibits BCL-6 binding ability (Barish, Atkins et al. 2008; Takata, Liu et al. 2008). In the absence of a ligand, PPARβ/δ sequesters BCL-6 from inflammatory response genes. In contrast, in the presence of a ligand, PPARβ/δ releases the repressor, which now distributes to NF-κBdependent promoters and exerts anti-inflammatory effects by repressing transcription from

PPARs may repress the transcription of direct target genes in the absence of ligands (ligandindependent repression). PPARs bind to response elements in the absence of any ligand and recruit co-repressor complexes that mediate active repression. The co-repressors are capable of fully repressing PPAR-mediated transactivation induced either by ligands or by cAMPregulated signalling pathways. This suggests co-repressors as general antagonists of the various stimuli inducing PPAR-mediated transactivation. Co-repressors can display different ligand selectivity: the nuclear receptor co-repressor NCoR interacted strongly with the ligand-binding domain of PPARβ/δ, whereas interactions with the ligand-binding domains of PPAR and PPAR were significantly weaker (Krogsdam, Nielsen et al. 2002). Very recently, a team of Harvard Medical School researchers has shown that PPAR is phosphorylated at Ser273 by cyclin dependent kinase 5 (CDK5) during obesity which results in deregulation of a subset of genes; including a number of key metabolic regulators, such as adipsin, the first fat cell-selective gene whose expression is altered in obesity and adiponectin, a central regulator of insulin sensitivity *in vivo* (Choi, Banks et al.). Ser273 phosphorylation did not alter the chromatin occupancy of PPAR, suggesting that other mechanisms, such as differential recruitment of co-regulators, may cause these differences in target gene expression. PPAR ligands inhibited Ser273 phosphorylation and reversed

Thus, without distinct co-factors, transcription factors cannot cause gene expression.

despite intensive investigation, unifying principles remain to be elucidated.

mechanisms by which PPARs and their ligands modulate kinase activities.

binding activity (Delerive, Martin-Nizard et al. 1999).

**2.2.3 Mechanism of ligand-independent transrepression** 

these genes.

Fig. 2. Molecular mechanisms of PPAR activation. After ligand binding, PPAR undergoes conformational changes, which lead to recruitment of Retinoid X Receptor (RXR) and coactivators. The resultant heterodimer binds to specific DNA response elements called PPAR response elements, causing target gene transcription (Transactivation). A second mechanism (Transrepression) involves interfering with other transcription-factor pathways by negatively regulating the expression of pro-inflammatory genes. Lastly, PPAR may repress the transcription of direct target genes in the absence of ligands (ligand-independent Transrepression) recruiting corepressor complexes that mediate active repression.

sequence conveys the selectivity of binding between different PPAR isotypes (Juge-Aubry, Pernin et al. 1997). In the absence of a ligand, to prevent PPAR/RXR binding to DNA, highaffinity complexes are formed between the inactive PPAR/RXR heterodimers and corepressor molecules, such as nuclear receptor co-repressor or silencing mediator for retinoic receptors. In response to ligand binding, PPAR undergoes a conformational change, leading to release of auxiliary proteins and co-repressors and recruitment of co-activators that contain histone acetylase activity. Acetylation of histones by co-activators bound to the ligand-PPAR complex leads to nucleosome remodelling, allowing for recruitment of RNA polymerase II causing target gene transcription. The search for PPAR target genes with identified PPREs has led to the identification of several genes involved in lipid metabolism, oxidative stress and inflammatory response, as widely documented in the literature.

### **2.2.2 Mechanism of transcriptional transrepression**

PPARs can also negatively regulate gene expression in a ligand-dependent manner by inhibiting the activities of other transcription factors, such as Activated Protein-1 (AP-1), Nuclear Factor-κB (NF-κB) and Nuclear Factor of Activated T cells (NFAT) (liganddependent transrepression). In contrast to transcriptional activation, which usually involves the binding of PPARs to specific response elements in the promoter or enhancer regions of

Fig. 2. Molecular mechanisms of PPAR activation. After ligand binding, PPAR undergoes conformational changes, which lead to recruitment of Retinoid X Receptor (RXR) and coactivators. The resultant heterodimer binds to specific DNA response elements called PPAR response elements, causing target gene transcription (Transactivation). A second mechanism (Transrepression) involves interfering with other transcription-factor pathways by negatively regulating the expression of pro-inflammatory genes. Lastly, PPAR may repress the transcription of direct target genes in the absence of ligands (ligand-independent

Transrepression) recruiting corepressor complexes that mediate active repression.

oxidative stress and inflammatory response, as widely documented in the literature.

PPARs can also negatively regulate gene expression in a ligand-dependent manner by inhibiting the activities of other transcription factors, such as Activated Protein-1 (AP-1), Nuclear Factor-κB (NF-κB) and Nuclear Factor of Activated T cells (NFAT) (liganddependent transrepression). In contrast to transcriptional activation, which usually involves the binding of PPARs to specific response elements in the promoter or enhancer regions of

**2.2.2 Mechanism of transcriptional transrepression** 

sequence conveys the selectivity of binding between different PPAR isotypes (Juge-Aubry, Pernin et al. 1997). In the absence of a ligand, to prevent PPAR/RXR binding to DNA, highaffinity complexes are formed between the inactive PPAR/RXR heterodimers and corepressor molecules, such as nuclear receptor co-repressor or silencing mediator for retinoic receptors. In response to ligand binding, PPAR undergoes a conformational change, leading to release of auxiliary proteins and co-repressors and recruitment of co-activators that contain histone acetylase activity. Acetylation of histones by co-activators bound to the ligand-PPAR complex leads to nucleosome remodelling, allowing for recruitment of RNA polymerase II causing target gene transcription. The search for PPAR target genes with identified PPREs has led to the identification of several genes involved in lipid metabolism, target genes, transrepression does not involve binding to typical receptor specific response elements (Pascual and Glass 2006). Several lines of evidence suggest that PPARs may exert anti-inflammatory effects by negatively regulating the expression of pro-inflammatory genes. To date, several mechanisms have been suggested to account for this activity, but despite intensive investigation, unifying principles remain to be elucidated.

Firstly, competition for limited amounts of essential, shared transcriptional co-activators may play a role in transrepression. The activated PPAR/RXR heterodimer reduces the availability of co-activators required for gene induction by other transcriptional factors. Thus, without distinct co-factors, transcription factors cannot cause gene expression.

Secondly, PPAR/RXR complexes may cause a functional inhibition by directly binding to transcription factors, preventing them from inducing gene transcription or inducing the expression of inhibitory proteins, such as the protein inhibitor of kappa B (IκB)α, which sequesters the NF-κB subunits in the cytoplasm and consequently reduces their DNA binding activity (Delerive, Martin-Nizard et al. 1999).

Thirdly, PPAR/RXR heterodimers may also inhibit phosphorylation and activation of several members of the MAPK family. In general very little is known about the molecular mechanisms by which PPARs and their ligands modulate kinase activities.

Recent studies have suggested another mechanism based on co-repressor-dependent transrepression by PPARs. Evidence has been presented in which PPARβ/δ controls the inflammatory status of macrophages based on its association with the transcriptional repressor BCL-6 (Lee, Chawla et al. 2003). Free BCL-6 suppresses the expression of multiple proinflammatory cytokines and chemokines. PPARβ/δ, but not PPARα and PPAR, exhibits BCL-6 binding ability (Barish, Atkins et al. 2008; Takata, Liu et al. 2008). In the absence of a ligand, PPARβ/δ sequesters BCL-6 from inflammatory response genes. In contrast, in the presence of a ligand, PPARβ/δ releases the repressor, which now distributes to NF-κBdependent promoters and exerts anti-inflammatory effects by repressing transcription from these genes.

### **2.2.3 Mechanism of ligand-independent transrepression**

PPARs may repress the transcription of direct target genes in the absence of ligands (ligandindependent repression). PPARs bind to response elements in the absence of any ligand and recruit co-repressor complexes that mediate active repression. The co-repressors are capable of fully repressing PPAR-mediated transactivation induced either by ligands or by cAMPregulated signalling pathways. This suggests co-repressors as general antagonists of the various stimuli inducing PPAR-mediated transactivation. Co-repressors can display different ligand selectivity: the nuclear receptor co-repressor NCoR interacted strongly with the ligand-binding domain of PPARβ/δ, whereas interactions with the ligand-binding domains of PPAR and PPAR were significantly weaker (Krogsdam, Nielsen et al. 2002).

Very recently, a team of Harvard Medical School researchers has shown that PPAR is phosphorylated at Ser273 by cyclin dependent kinase 5 (CDK5) during obesity which results in deregulation of a subset of genes; including a number of key metabolic regulators, such as adipsin, the first fat cell-selective gene whose expression is altered in obesity and adiponectin, a central regulator of insulin sensitivity *in vivo* (Choi, Banks et al.). Ser273 phosphorylation did not alter the chromatin occupancy of PPAR, suggesting that other mechanisms, such as differential recruitment of co-regulators, may cause these differences in target gene expression. PPAR ligands inhibited Ser273 phosphorylation and reversed

PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke 519

has been first established in animal models of acute myocardial infarction (Yue Tl, Chen et al. 2001). More recently, good evidence supporting the beneficial role of PPAR in stroke has been provided by several *in vivo* experimental models of cerebral IRI, evaluating the effects of both prophylactic and therapeutic administration of PPAR agonists. It has been demonstrated that a 14-day preventive treatment with fenofibrate reduced susceptibility to stroke in apolipoprotein E-deficient mice as well as decreased cerebral infarct volume in wild-type littermates (Deplanque, Gele et al. 2003). The authors demonstrated that fenofibrate administration was associated with a decrease in cerebral oxidative stress depending on the increase in activity of several anti-oxidant enzymes and with a reduced expression of adhesion molecules. In another study, it was confirmed that two different PPARα agonists, fenofibrate and WY14643, provided similar brain protection when administered 3 or 7 days, respectively, before the induction of cerebral ischemia (Inoue, Jiang et al. 2003). More recently, we have found that PPARα agonists may also reduce cerebral I/R injury when administered just before ischemia or during reperfusion (Collino, Aragno et al. 2006). We showed that the potential neuroprotective effects of PPARα agonists is manifested by modulation of protein S100B levels in the rat CNS. S100B is a calciumbinding protein, mainly expressed in the brain and recent preclinical and clinical studies indicate that increased S100B levels is a reliable indicator of infarct size in acute ischemic stroke (Buyukuysal 2005; Foerch, Singer et al. 2005). Pre-treatment of rats with the selective PPAR agonist, WY14643, prior to cerebral ischemia causes a marked reduction of S100B levels in the rat hippocampus. This protective effect is reversed by administration of the PPAR antagonist, MK886, thus confirming the involvement of PPAR activation in neuroprotection. Similarly, fenofibrate pretreatment for 14 days significantly reduced the cerebral infarct volume in an experimental model of Middle Cerebral Artery Occlusion (MCAO), although its withdrawal 3 days before induction of cerebral ischemia decreased the neuroprotective effect (Ouk, Laprais et al. 2009). Also prophylactic administration of gemfibrozil resulted in reduction of infarct size 24 h after MCAO and increased cortical blood flow in the ischemic hemisphere (Guo, Wang et al. 2009). However, the principal focus of studies of PPAR agonists has been on agonists of the PPARγ isoform. Emerging studies have reported the protective effects of PPAR agonist administration in animal models of cerebral IRI (Sundararajan, Gamboa et al. 2005; Collino, Aragno et al. 2006; Allahtavakoli, Shabanzadeh et al. 2007) and in models of permanent ischemia (Sayan-Ozacmak, Ozacmak et al.; Zhang, Xu et al.). The effect of delayed post ischemia administration of a PPARγ agonist, rosiglitazone, has been recently evaluated, demonstrating that post-treatment with rosiglitazone, 24 h after stroke induction, may reduce ischemic injury, improve neurological outcome, and prevent neutrophilia, thus supporting an extended therapeutic window for the treatment of ischemic stroke (Allahtavakoli, Moloudi et al. 2009). Recent experimental data confirmed that PPAR agonists are protective at clinically relevant doses, independent of any effects on systemic blood pressure or cerebral blood flow and, most notably, the timing of reperfusion relative to drug administration, may significantly influence the ability of PPAR agonists to reduce infarction volume and improve neurologic function following ischemic injury (Gamboa, Blankenship et al.). The relevance of PPARγ as an endogenous protective factor was also shown by the fact that treatment with a PPARγ antagonist increased infarct size (Victor, Wanderi et al. 2006). Moreover, it was demonstrated that in primary cortical neurons of PPARγ KO mice exposed to ischemia there was a reduced expression of numerous key gene products (including superoxide dismutase-1, catalase, and glutathione S-transferase) along

associated changes in gene expression. Critically, the extent to which PPAR ligands inhibit CDK5-mediated phosphorylation of PPAR is not correlated with the extent to which they exert PPAR agonism, suggesting that these compounds have two distinct and separable activities. Whether or not similar mechanisms of receptor phosphorylation lead to changes in gene expression also in the other two PPAR isoforms -α and β/δ is a very important question, so far not yet addressed.

### **3. PPAR in the brain**

All three PPAR isotypes are co-expressed in the nervous system during late rat embryogenesis. Their expression peaks in the central nervous system at mid-gestation. Whereas PPARβ/δ remains highly expressed in this tissue, the expression of PPARα and PPARγ decreases postnatally in the brain (Braissant, Foufelle et al. 1996). While PPARβ/δ has been found in neurons of numerous brain areas of adult rodents, PPARα and PPARγ have been localized to more restricted areas of the brain (Moreno, Farioli-Vecchioli et al. 2004). The localization of PPARs has also been investigated in purified cultures of neural cells. PPARβ/δ is expressed in immature oligodendrocytes where its activation promotes differentiation, myelin maturation and turnover. The PPARγ isotype is the dominant isoform in microglia. Astrocytes possess all three PPAR isotypes, although to different degrees depending on the brain area and animal age (Cristiano, Bernardo et al. 2001). The role of PPARs in the CNS is mainly related to lipid metabolism; however, these receptors have been implicated in neural cell differentiation and death as well as in inflammation and neurodegeneration. The expression of PPARγ in the brain has been extensively studied in relation to inflammation and neurodegeneration. PPARα has been suggested to be involved in acetylcholine metabolism, excitatory amino acid neurotransmission and oxidative stress defence. PPARβ/δ seems to play a critical role in regulating myelinogenesis and differentiation of cells within the CNS (Peters, Lee et al. 2000).

### **4. PPARs and cerebral ischemia**

### **4.1 Experimental data on the effects of PPAR ligands in ischemic stroke**

Although the relevance of animal models to the development of therapies for acute stroke has been often questioned, evidence demonstrates that animal models of stroke do have clinical relevance and are useful in the development of drugs that attenuate the ischemic damage. The characteristics of brain injury depends on the severity and the duration of cerebral blood flow reduction but it can be significantly exacerbated by the following phase of reperfusion; for this reason several animal models of the so-called "cerebral ischemia/reperfusion injury (IRI)" have been developed, demonstrating that often reperfusion after a long ischemic period may cause a larger infarct than that associated with permanent vessel occlusion. In general, the role of neuroprotective agents is to interfere with one or more of the mechanisms involved in the "IRI cascade" and thereby limit the resultant tissue damage. It seem reasonable to assume that drugs that work on a specific biochemical mechanism must be given at the time that the mechanism is active, mainly during ischemia and/or reperfusion. Accordingly, in general, two different experimental paradigms can be identified: prophylactic administration, aimed to evaluate drug effects on stroke prevention, and therapeutic administration, when the drug is administered during reperfusion to test its potential beneficial effects on IRI after stroke had occurred. A role for PPARs in reducing IRI

associated changes in gene expression. Critically, the extent to which PPAR ligands inhibit CDK5-mediated phosphorylation of PPAR is not correlated with the extent to which they exert PPAR agonism, suggesting that these compounds have two distinct and separable activities. Whether or not similar mechanisms of receptor phosphorylation lead to changes in gene expression also in the other two PPAR isoforms -α and β/δ is a very important

All three PPAR isotypes are co-expressed in the nervous system during late rat embryogenesis. Their expression peaks in the central nervous system at mid-gestation. Whereas PPARβ/δ remains highly expressed in this tissue, the expression of PPARα and PPARγ decreases postnatally in the brain (Braissant, Foufelle et al. 1996). While PPARβ/δ has been found in neurons of numerous brain areas of adult rodents, PPARα and PPARγ have been localized to more restricted areas of the brain (Moreno, Farioli-Vecchioli et al. 2004). The localization of PPARs has also been investigated in purified cultures of neural cells. PPARβ/δ is expressed in immature oligodendrocytes where its activation promotes differentiation, myelin maturation and turnover. The PPARγ isotype is the dominant isoform in microglia. Astrocytes possess all three PPAR isotypes, although to different degrees depending on the brain area and animal age (Cristiano, Bernardo et al. 2001). The role of PPARs in the CNS is mainly related to lipid metabolism; however, these receptors have been implicated in neural cell differentiation and death as well as in inflammation and neurodegeneration. The expression of PPARγ in the brain has been extensively studied in relation to inflammation and neurodegeneration. PPARα has been suggested to be involved in acetylcholine metabolism, excitatory amino acid neurotransmission and oxidative stress defence. PPARβ/δ seems to play a critical role in regulating myelinogenesis and

differentiation of cells within the CNS (Peters, Lee et al. 2000).

**4.1 Experimental data on the effects of PPAR ligands in ischemic stroke** 

Although the relevance of animal models to the development of therapies for acute stroke has been often questioned, evidence demonstrates that animal models of stroke do have clinical relevance and are useful in the development of drugs that attenuate the ischemic damage. The characteristics of brain injury depends on the severity and the duration of cerebral blood flow reduction but it can be significantly exacerbated by the following phase of reperfusion; for this reason several animal models of the so-called "cerebral ischemia/reperfusion injury (IRI)" have been developed, demonstrating that often reperfusion after a long ischemic period may cause a larger infarct than that associated with permanent vessel occlusion. In general, the role of neuroprotective agents is to interfere with one or more of the mechanisms involved in the "IRI cascade" and thereby limit the resultant tissue damage. It seem reasonable to assume that drugs that work on a specific biochemical mechanism must be given at the time that the mechanism is active, mainly during ischemia and/or reperfusion. Accordingly, in general, two different experimental paradigms can be identified: prophylactic administration, aimed to evaluate drug effects on stroke prevention, and therapeutic administration, when the drug is administered during reperfusion to test its potential beneficial effects on IRI after stroke had occurred. A role for PPARs in reducing IRI

**4. PPARs and cerebral ischemia** 

question, so far not yet addressed.

**3. PPAR in the brain** 

has been first established in animal models of acute myocardial infarction (Yue Tl, Chen et al. 2001). More recently, good evidence supporting the beneficial role of PPAR in stroke has been provided by several *in vivo* experimental models of cerebral IRI, evaluating the effects of both prophylactic and therapeutic administration of PPAR agonists. It has been demonstrated that a 14-day preventive treatment with fenofibrate reduced susceptibility to stroke in apolipoprotein E-deficient mice as well as decreased cerebral infarct volume in wild-type littermates (Deplanque, Gele et al. 2003). The authors demonstrated that fenofibrate administration was associated with a decrease in cerebral oxidative stress depending on the increase in activity of several anti-oxidant enzymes and with a reduced expression of adhesion molecules. In another study, it was confirmed that two different PPARα agonists, fenofibrate and WY14643, provided similar brain protection when administered 3 or 7 days, respectively, before the induction of cerebral ischemia (Inoue, Jiang et al. 2003). More recently, we have found that PPARα agonists may also reduce cerebral I/R injury when administered just before ischemia or during reperfusion (Collino, Aragno et al. 2006). We showed that the potential neuroprotective effects of PPARα agonists is manifested by modulation of protein S100B levels in the rat CNS. S100B is a calciumbinding protein, mainly expressed in the brain and recent preclinical and clinical studies indicate that increased S100B levels is a reliable indicator of infarct size in acute ischemic stroke (Buyukuysal 2005; Foerch, Singer et al. 2005). Pre-treatment of rats with the selective PPAR agonist, WY14643, prior to cerebral ischemia causes a marked reduction of S100B levels in the rat hippocampus. This protective effect is reversed by administration of the PPAR antagonist, MK886, thus confirming the involvement of PPAR activation in neuroprotection. Similarly, fenofibrate pretreatment for 14 days significantly reduced the cerebral infarct volume in an experimental model of Middle Cerebral Artery Occlusion (MCAO), although its withdrawal 3 days before induction of cerebral ischemia decreased the neuroprotective effect (Ouk, Laprais et al. 2009). Also prophylactic administration of gemfibrozil resulted in reduction of infarct size 24 h after MCAO and increased cortical blood flow in the ischemic hemisphere (Guo, Wang et al. 2009). However, the principal focus of studies of PPAR agonists has been on agonists of the PPARγ isoform. Emerging studies have reported the protective effects of PPAR agonist administration in animal models of cerebral IRI (Sundararajan, Gamboa et al. 2005; Collino, Aragno et al. 2006; Allahtavakoli, Shabanzadeh et al. 2007) and in models of permanent ischemia (Sayan-Ozacmak, Ozacmak et al.; Zhang, Xu et al.). The effect of delayed post ischemia administration of a PPARγ agonist, rosiglitazone, has been recently evaluated, demonstrating that post-treatment with rosiglitazone, 24 h after stroke induction, may reduce ischemic injury, improve neurological outcome, and prevent neutrophilia, thus supporting an extended therapeutic window for the treatment of ischemic stroke (Allahtavakoli, Moloudi et al. 2009). Recent experimental data confirmed that PPAR agonists are protective at clinically relevant doses, independent of any effects on systemic blood pressure or cerebral blood flow and, most notably, the timing of reperfusion relative to drug administration, may significantly influence the ability of PPAR agonists to reduce infarction volume and improve neurologic function following ischemic injury (Gamboa, Blankenship et al.). The relevance of PPARγ as an endogenous protective factor was also shown by the fact that treatment with a PPARγ antagonist increased infarct size (Victor, Wanderi et al. 2006). Moreover, it was demonstrated that in primary cortical neurons of PPARγ KO mice exposed to ischemia there was a reduced expression of numerous key gene products (including superoxide dismutase-1, catalase, and glutathione S-transferase) along

PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke 521

plasma levels of 15d-PGJ2 (the natural ligand for PPAR) have been associated with good neurological outcome and smaller infarct volume in patients with an acute atherothrombotic stroke (Blanco, Moro et al. 2005). Moreover, a recent report suggests that the Pro12Ala polymorphism of PPARγ2 is associated with a reduced risk for ischemic stroke (Lee, Olson et al. 2006), further supporting the importance of PPARs in cerebral ischemia. Nevertheless, as TZDs are hampered by adverse effects related to increased weight gain, fluid overload, and congestive heart failure, the risks associated with chronic TZD administration needs to

Abnormal levels of serum lipids, including triglycerides, low density lipoprotein (LDL) and high density lipoprotein (HDL), are regarded as other important risk factors for cerebrovascular disease, including stroke. The association between hypercholesterolemia and stroke has become more apparent because of data from prospective cohort studies that show higher risks of ischemic stroke with increasing levels of total cholesterol in both men and women. Increased HDL cholesterol levels have a protective effect against the occurrence of ischemic stroke and elevated triglyceride levels have also been reported as a risk factor for stroke. Overall, elevated total cholesterol confers an approximately two-fold relative increase in stroke risk for men and women. As fibrates are used as lipid-lowering agents, it has been supposed that these PPARα agonists could also protect the brain against noxious biological reactions induced by cerebral IRI. A recent systematic meta-analysis of randomized clinical trials shows that fibrates do not significantly reduce the odds of stroke (Saha, Kizhakepunnur et al. 2007). However, data from large trials specifically investigating the role of fibrates in stroke event reduction are needed to conclusively elucidate their potential neuroprotective role. For instance, a large clinical trial, named Action to Control Cardiovascular Risk in Diabetes (ACCORD) is currently testing the ability of fenofibrate to decrease stroke incidence in high-risk patients with type-2 diabetes (ACCORD study group

**5. Molecular mechanisms of beneficial effects of PPARs against cerebral** 

Cerebral IRI is known to induce generation of ROS, as well as the expression of cytokines, adhesion molecules and enzymes involved in the inflammatory response, and is known to be regulated by oxygen- or redox-sensitive mechanisms. Recent studies have confirmed the pivotal role of both oxidative stress and inflammatory response in the pathogenesis of acute ischemic stroke. Through various mechanisms PPARs can regulate both inflammatory and oxidative pathways and PPAR agonist-induced neuroprotection seems to be specific for injuries in which inflammation or free radical generation are the main causes of cell damage. For instance, PPARα activation can induce expression and activation of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH). We have demonstrated that administration of a highly selective PPARα agonist, WY14643, 30 min prior to IRI, decreased ROS production and lipid peroxidation in rats subjected to IRI and, at the same time, offered protection against GSH depletion (Collino, Aragno et al. 2006). Similar results on oxidative stress modulation have been reported when another PPARα agonist, fenofibrate, was tested in a mouse model of middle cerebral artery occlusion (Deplanque, Gele et al. 2003). Interestingly, PPAR KO mice have been found to exhibit significant increases in oxidative stress and lipid peroxidation much earlier in their life than

be better elucidated.

2007).

**ischemia** 

with an increased damage. PPARγ mRNA is up-regulated in ischemic brain, especially in the peri-infarct area. Increased PPARγ mRNA was detected in the infarcted brain as early as 6 h following focal ischemia (Ou, Zhao et al. 2006), and PPARγ immunopositive neurons were detected between 4 h and 14 days, whereas in neurons and microglia only transiently at 12 h in the post-ischemic brain (Zhao, Patzer et al. 2005; Victor, Wanderi et al. 2006). The beneficial role of PPARβ/δ in stroke has been demonstrated by two different studies in which PPARβ/δ knockout mice subjected to cerebral IRI showed significantly larger infarct size than wild-type littermates (Pialat, Cho et al. 2007). This finding is confirmed by another study demonstrating that intracerebroventricular administration of high affinity PPARβ/δ agonists such as L-165041 and GW501516 significantly decreased the infarct volume at 24 h of reperfusion after cerebral ischemia in rats (Iwashita, Muramatsu et al. 2007).

### **4.2 Clinical evidence of beneficial effects of PPAR ligands in ischemic stroke**

Although various PPAR agonists applied before the onset of ischemia can effectively protect the brain in animal models of acute IRI, these treatments are seldom possible in the clinical setting of stroke because patients with stroke present after onset of the ischemic attack. Neuroprotective interventions applied after the onset of ischemia would thus seem to have greater clinical potential. Although some preclinical data provide evidence that administration of PPAR agonists during reperfusion decreases cerebral IRI, to date, there are no clinical data on the therapeutic efficacy of PPAR agonists administration after the onset of the ischemic event. Nevertheless, it must be noted that there may be subgroups of patients at high risk for stroke that could benefit from taking neuroprotective agents as prophylactic treatment. As already mentioned, pioglitazone and rosiglitazone (the TZD class of PPAR agonists) have proven to be beneficial in type-2 diabetes mellitus patients. Diabetics are at an increased risk of stroke incidence and stroke causes more damage in diabetics compared to normoglycemic individuals. For this reason, such patients might benefit from taking an antidiabetic medication with neuroprotective properties, which might lessen the incidence and/or the severity of acute ischemic stroke. However, it's important to assess whether the potential benefits of taking an oral neuroprotective drug chronically outweighs the risks, including potential side effects. The use of a PPAR agonist, specifically pioglitazone, as a preventive approach to ischemic brain injury has been recently addressed by two large clinical trials: the Prospective Pioglitazone Clinical Trial in Macrovascular Events (PROactive) and the Insulin Resistance Intervention after Stroke Trial (IRIS trial). The PROactive study has demonstrated that pioglitazone significantly reduces the combined risk of heart attacks, strokes and death by 16% in high risk patients with type-2 diabetes (Dormandy, Charbonnel et al. 2005). Enhanced functional recovery was also reported in a small group of stroke patients with type-2 diabetes treated with pioglitazone (Lee, Olson et al. 2006). However, it remain unclear whether the suggested beneficial effects of pioglitazone are mediated by insulin sensitization or by additional observed reductions in risk factors, such as hyperthension and dyslipidemia. This question and that related to the potential beneficial effects of pioglitazone in non-diabetic patients with stroke will be addressed by the IRIS trial, a randomized, double-blind, placebo-controlled trial on more than 3000 non-diabetic subjects who are insulin resistant and have had a recent transient ischemic attack or ischemic stroke. The IRIS study (ClinicalTrials.gov Identifier: NCT00091949) began on February 2005 and it is still recruiting patients. Interestingly, high

with an increased damage. PPARγ mRNA is up-regulated in ischemic brain, especially in the peri-infarct area. Increased PPARγ mRNA was detected in the infarcted brain as early as 6 h following focal ischemia (Ou, Zhao et al. 2006), and PPARγ immunopositive neurons were detected between 4 h and 14 days, whereas in neurons and microglia only transiently at 12 h in the post-ischemic brain (Zhao, Patzer et al. 2005; Victor, Wanderi et al. 2006). The beneficial role of PPARβ/δ in stroke has been demonstrated by two different studies in which PPARβ/δ knockout mice subjected to cerebral IRI showed significantly larger infarct size than wild-type littermates (Pialat, Cho et al. 2007). This finding is confirmed by another study demonstrating that intracerebroventricular administration of high affinity PPARβ/δ agonists such as L-165041 and GW501516 significantly decreased the infarct volume at 24 h

of reperfusion after cerebral ischemia in rats (Iwashita, Muramatsu et al. 2007).

**4.2 Clinical evidence of beneficial effects of PPAR ligands in ischemic stroke** 

Although various PPAR agonists applied before the onset of ischemia can effectively protect the brain in animal models of acute IRI, these treatments are seldom possible in the clinical setting of stroke because patients with stroke present after onset of the ischemic attack. Neuroprotective interventions applied after the onset of ischemia would thus seem to have greater clinical potential. Although some preclinical data provide evidence that administration of PPAR agonists during reperfusion decreases cerebral IRI, to date, there are no clinical data on the therapeutic efficacy of PPAR agonists administration after the onset of the ischemic event. Nevertheless, it must be noted that there may be subgroups of patients at high risk for stroke that could benefit from taking neuroprotective agents as prophylactic treatment. As already mentioned, pioglitazone and rosiglitazone (the TZD class of PPAR agonists) have proven to be beneficial in type-2 diabetes mellitus patients. Diabetics are at an increased risk of stroke incidence and stroke causes more damage in diabetics compared to normoglycemic individuals. For this reason, such patients might benefit from taking an antidiabetic medication with neuroprotective properties, which might lessen the incidence and/or the severity of acute ischemic stroke. However, it's important to assess whether the potential benefits of taking an oral neuroprotective drug chronically outweighs the risks, including potential side effects. The use of a PPAR agonist, specifically pioglitazone, as a preventive approach to ischemic brain injury has been recently addressed by two large clinical trials: the Prospective Pioglitazone Clinical Trial in Macrovascular Events (PROactive) and the Insulin Resistance Intervention after Stroke Trial (IRIS trial). The PROactive study has demonstrated that pioglitazone significantly reduces the combined risk of heart attacks, strokes and death by 16% in high risk patients with type-2 diabetes (Dormandy, Charbonnel et al. 2005). Enhanced functional recovery was also reported in a small group of stroke patients with type-2 diabetes treated with pioglitazone (Lee, Olson et al. 2006). However, it remain unclear whether the suggested beneficial effects of pioglitazone are mediated by insulin sensitization or by additional observed reductions in risk factors, such as hyperthension and dyslipidemia. This question and that related to the potential beneficial effects of pioglitazone in non-diabetic patients with stroke will be addressed by the IRIS trial, a randomized, double-blind, placebo-controlled trial on more than 3000 non-diabetic subjects who are insulin resistant and have had a recent transient ischemic attack or ischemic stroke. The IRIS study (ClinicalTrials.gov Identifier: NCT00091949) began on February 2005 and it is still recruiting patients. Interestingly, high plasma levels of 15d-PGJ2 (the natural ligand for PPAR) have been associated with good neurological outcome and smaller infarct volume in patients with an acute atherothrombotic stroke (Blanco, Moro et al. 2005). Moreover, a recent report suggests that the Pro12Ala polymorphism of PPARγ2 is associated with a reduced risk for ischemic stroke (Lee, Olson et al. 2006), further supporting the importance of PPARs in cerebral ischemia. Nevertheless, as TZDs are hampered by adverse effects related to increased weight gain, fluid overload, and congestive heart failure, the risks associated with chronic TZD administration needs to be better elucidated.

Abnormal levels of serum lipids, including triglycerides, low density lipoprotein (LDL) and high density lipoprotein (HDL), are regarded as other important risk factors for cerebrovascular disease, including stroke. The association between hypercholesterolemia and stroke has become more apparent because of data from prospective cohort studies that show higher risks of ischemic stroke with increasing levels of total cholesterol in both men and women. Increased HDL cholesterol levels have a protective effect against the occurrence of ischemic stroke and elevated triglyceride levels have also been reported as a risk factor for stroke. Overall, elevated total cholesterol confers an approximately two-fold relative increase in stroke risk for men and women. As fibrates are used as lipid-lowering agents, it has been supposed that these PPARα agonists could also protect the brain against noxious biological reactions induced by cerebral IRI. A recent systematic meta-analysis of randomized clinical trials shows that fibrates do not significantly reduce the odds of stroke (Saha, Kizhakepunnur et al. 2007). However, data from large trials specifically investigating the role of fibrates in stroke event reduction are needed to conclusively elucidate their potential neuroprotective role. For instance, a large clinical trial, named Action to Control Cardiovascular Risk in Diabetes (ACCORD) is currently testing the ability of fenofibrate to decrease stroke incidence in high-risk patients with type-2 diabetes (ACCORD study group 2007).

### **5. Molecular mechanisms of beneficial effects of PPARs against cerebral ischemia**

Cerebral IRI is known to induce generation of ROS, as well as the expression of cytokines, adhesion molecules and enzymes involved in the inflammatory response, and is known to be regulated by oxygen- or redox-sensitive mechanisms. Recent studies have confirmed the pivotal role of both oxidative stress and inflammatory response in the pathogenesis of acute ischemic stroke. Through various mechanisms PPARs can regulate both inflammatory and oxidative pathways and PPAR agonist-induced neuroprotection seems to be specific for injuries in which inflammation or free radical generation are the main causes of cell damage. For instance, PPARα activation can induce expression and activation of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH). We have demonstrated that administration of a highly selective PPARα agonist, WY14643, 30 min prior to IRI, decreased ROS production and lipid peroxidation in rats subjected to IRI and, at the same time, offered protection against GSH depletion (Collino, Aragno et al. 2006). Similar results on oxidative stress modulation have been reported when another PPARα agonist, fenofibrate, was tested in a mouse model of middle cerebral artery occlusion (Deplanque, Gele et al. 2003). Interestingly, PPAR KO mice have been found to exhibit significant increases in oxidative stress and lipid peroxidation much earlier in their life than

PPAR Agonism as New Pharmacological Approach to the Management of Acute Ischemic Stroke 523

molecule-1 (VCAM-1) and ICAM-1 in two independent studies (Deplanque, Gele et al. 2003; Collino, Aragno et al. 2006). In the brain, the decreased expression of these adhesion molecules might contribute to inhibit the infiltration of the brain ischemic area by neutrophils. Studies addressing the molecular mechanisms of these anti-inflammatory actions demonstrated that the involvement of PPARs in the control of IRI-induced inflammation is mediated mainly through their transrepression capabilities. PPARs can suppress the activities of many distinct families of transcription factors. The range of transcription factors affected and the mechanisms involved may be different for each PPAR isotype, although a common mechanism of PPARα and PPAR neuroprotection appears to involve inhibition of p38 MAPK activation and NF-κB nuclear translocation. A recent study confirms that PPAR activation prevents the post-ischemic cerebral expression of proinflammatory transcription factors, such as Egr1, C/EBP and NF-κB, possibly by decreasing DNA binding (Tureyen, Kapadia et al. 2007). The inhibitory protein IκBα, which is an indicator of NF-κB transcriptional activity, is remarkably increased in the brain of rats that underwent cerebral ischemia and completely blocked by rosiglitazone and 15d-PGJ2 administration, thus further confirming that both endogenous and synthetic PPAR ligands inhibit NF-κB signalling (Pereira, Hurtado et al. 2006). Similarly, p38 MAPK and NF-κB activation by cerebral IRI has been demonstrated to be inhibited by pre-treatment with the PPARα agonist WY14643 or the PPAR agonist pioglitazone. However, as MAPK and NFκB are functionally interconnected and do not act independently, we cannot rule out the possibility that PPARs affect NF-κB activation by interfering with the MAPK signalling

The generation of ROS is known to be associated with the induction of apoptosis and, in neurons, inhibition of cell death is an important factor to prevent during IRI. PPAR activation may decrease the IRI-induced activation of apoptotic pathways depending on the increase in activity and expression of numerous anti-oxidant enzymes. Moreover, by their anti-inflammatory action on microglia and astrocytes, PPAR agonists prevent the release of neurotoxic agents, which induce neuronal apoptosis. PPAR agonists may attenuate ischemia-induced reactive oxygen species and subsequently alleviate the post-ischemic degradation of Bcl-2, Bcl-xl, and Akt, by increasing SOD/catalase and decreasing nicotinamide adenine dinucleotide phosphate oxidase levels (Fong, Tsai et al.). Chu and colleagues (Chu, Lee et al. 2006) have demonstrated that rosiglitazone-fed rats had better neurological scores and reduced number of TUNEL-positive cells following transient focal ischemia. Interestingly, these authors also reported an increased vasculature in the rosiglitazone-treated group with increased number of endothelial cells positive for BrdU, suggesting there may be enhanced angiogenesis following PPAR activation. Administration of a selective PPAR agonist (L-796449) 10 min prior to permanent cerebral artery occlusion, resulted in decreased apoptosis, measured as reduction of caspase-3 activity (Pereira, Hurtado et al. 2005). Another study confirmed inhibition on caspase-3 activity by both exogenous and endogenous PPAR agonists, rosiglitazone and 15d-PGJ2, in the ischemic cortex (Lin, Cheung et al. 2006). The same authors observed that rosiglitazone and 15d-PGJ2 exhibit a concentration-dependent paradoxical effect on cytotoxicity, when tested in an *in vitro* model of hydrogen peroxide induced neuronal apoptosis. The drugs induced pro-apoptotic effects when used at concentrations higher that 5 µmol/L but protect neurons from necrosis and apoptosis at concentrations lower than 1 µmol/L. The reason for

cascade or vice versa.

wild-type littermates (Poynter and Daynes 1998). The PPAR-induced protective effect on oxidative stress could be related to a direct effect on antioxidant enzyme expression, as the catalase and SOD gene promoters contain the PPRE. In fact, rats that have been treated with a diet containing PPARα ligands, WY14643 or fenofibrate, have demonstrated an enhanced expression of antioxidant enzymes such as SOD and catalase (Toyama, Nakamura et al. 2004). Based on gene expression microarray experiments, Coleman and colleagues (Coleman, Prabhu et al. 2007) have demonstrated that PPARβ/δ activation increased mRNA for aldheyde dehydrogenase and glutathione-S-transferase, thus protecting the cell from oxidative damage. In normotensive and hypertensive animals treated with rosiglitazone, ischemic hemispheres showed increased catalase and Cu/Zn-SOD activity in the peri-infarct region (Tureyen, Kapadia et al. 2007) and the level of Cu/Zn-SOD was demonstrated to increase in the ischemic cortex of animals treated with pioglitazone for 4 days prior to focal cerebral ischemia (Shimazu, Inoue et al. 2005). As we have recently shown, treatment of rats with either pioglitazone or rosiglitazone before occlusion of the common carotid artery decreased the production of ROS and nitrite, decreased lipid peroxidation and reversed the depleted stores of glutathione in the hippocampus (Collino, Aragno et al. 2006). These findings are supported by data from an *in vitro* model demonstrating that pre-treatment with PPARγ agonists protected an immortalized mouse hippocampal cell line against oxidative stress induced by glutamate or hydrogen peroxide (Aoun, Watson et al. 2003). Moreover, PPARγ agonists attenuate the expression of iNOS in inflammatory cells, which is an important source of nitric oxide (NO). NO may react with ROS to produce peroxynitrites, with deleterious effects on neuronal survival. Thus, iNOS inhibition may represent a further mechanism for neuroprotection by PPAR agonists. Mitochondria are the major source of ROS, which are mainly generated at complexes I and III of the respiratory chain. There is now evidence indicating that rosiglitazone and pioglitazone exert direct and rapid effects on mitochondrial respiration, inhibiting complex I and complex III activity (Brunmair, Lest et al. 2004). As PPARγ agonists partially disrupt the mitochondrial respiratory chain, both electron transport and superoxide anion generation are affected. Moreover, a novel mitochondrial target protein for PPARγ agonists ("mitoNEET") has recently been identified (Colca, McDonald et al. 2004). MitoNEET was found associated with components of complex III, suggesting how binding of PPARγ agonists to mitoNEET could selectively block different mitochondrial targets. The ability of PPARγ agonists to influence mitochondrial function might contribute to their inhibitory effects on ROS generation that is evoked by IRI.

Another mechanism through which PPAR agonists may provide neuroprotection is by down-regulating the inflammatory response associated with IRI. Depending on the affected tissue and which PPAR isoforms are involved, PPAR agonists can differently modulate the intensity, duration and consequences of inflammatory events. For instance, ischemiainduced COX-2 overexpression is prevented by PPAR agonists but not by PPARα agonists (Sundararajan, Gamboa et al. 2005; Collino, Aragno et al. 2006; Collino, Aragno et al. 2006). Activation of PPARγ attenuates the expression of matrix metalloproteinase (MMP)-9 and various inflammatory cytokines in ischemic brain tissue (Pereira, Hurtado et al. 2005). PPAR is constitutively expressed in macrophages and microglial cells and the systemic treatment of rodents with rosiglitazone reduces the infiltration of these cells into peri-infarct brain regions. Both chronic and acute administration of PPAR agonists has been demonstrated to prevent cerebral IRI-induced expression of vascular cell adhesion

wild-type littermates (Poynter and Daynes 1998). The PPAR-induced protective effect on oxidative stress could be related to a direct effect on antioxidant enzyme expression, as the catalase and SOD gene promoters contain the PPRE. In fact, rats that have been treated with a diet containing PPARα ligands, WY14643 or fenofibrate, have demonstrated an enhanced expression of antioxidant enzymes such as SOD and catalase (Toyama, Nakamura et al. 2004). Based on gene expression microarray experiments, Coleman and colleagues (Coleman, Prabhu et al. 2007) have demonstrated that PPARβ/δ activation increased mRNA for aldheyde dehydrogenase and glutathione-S-transferase, thus protecting the cell from oxidative damage. In normotensive and hypertensive animals treated with rosiglitazone, ischemic hemispheres showed increased catalase and Cu/Zn-SOD activity in the peri-infarct region (Tureyen, Kapadia et al. 2007) and the level of Cu/Zn-SOD was demonstrated to increase in the ischemic cortex of animals treated with pioglitazone for 4 days prior to focal cerebral ischemia (Shimazu, Inoue et al. 2005). As we have recently shown, treatment of rats with either pioglitazone or rosiglitazone before occlusion of the common carotid artery decreased the production of ROS and nitrite, decreased lipid peroxidation and reversed the depleted stores of glutathione in the hippocampus (Collino, Aragno et al. 2006). These findings are supported by data from an *in vitro* model demonstrating that pre-treatment with PPARγ agonists protected an immortalized mouse hippocampal cell line against oxidative stress induced by glutamate or hydrogen peroxide (Aoun, Watson et al. 2003). Moreover, PPARγ agonists attenuate the expression of iNOS in inflammatory cells, which is an important source of nitric oxide (NO). NO may react with ROS to produce peroxynitrites, with deleterious effects on neuronal survival. Thus, iNOS inhibition may represent a further mechanism for neuroprotection by PPAR agonists. Mitochondria are the major source of ROS, which are mainly generated at complexes I and III of the respiratory chain. There is now evidence indicating that rosiglitazone and pioglitazone exert direct and rapid effects on mitochondrial respiration, inhibiting complex I and complex III activity (Brunmair, Lest et al. 2004). As PPARγ agonists partially disrupt the mitochondrial respiratory chain, both electron transport and superoxide anion generation are affected. Moreover, a novel mitochondrial target protein for PPARγ agonists ("mitoNEET") has recently been identified (Colca, McDonald et al. 2004). MitoNEET was found associated with components of complex III, suggesting how binding of PPARγ agonists to mitoNEET could selectively block different mitochondrial targets. The ability of PPARγ agonists to influence mitochondrial function might contribute to their inhibitory effects on ROS generation that is

Another mechanism through which PPAR agonists may provide neuroprotection is by down-regulating the inflammatory response associated with IRI. Depending on the affected tissue and which PPAR isoforms are involved, PPAR agonists can differently modulate the intensity, duration and consequences of inflammatory events. For instance, ischemiainduced COX-2 overexpression is prevented by PPAR agonists but not by PPARα agonists (Sundararajan, Gamboa et al. 2005; Collino, Aragno et al. 2006; Collino, Aragno et al. 2006). Activation of PPARγ attenuates the expression of matrix metalloproteinase (MMP)-9 and various inflammatory cytokines in ischemic brain tissue (Pereira, Hurtado et al. 2005). PPAR is constitutively expressed in macrophages and microglial cells and the systemic treatment of rodents with rosiglitazone reduces the infiltration of these cells into peri-infarct brain regions. Both chronic and acute administration of PPAR agonists has been demonstrated to prevent cerebral IRI-induced expression of vascular cell adhesion

evoked by IRI.

molecule-1 (VCAM-1) and ICAM-1 in two independent studies (Deplanque, Gele et al. 2003; Collino, Aragno et al. 2006). In the brain, the decreased expression of these adhesion molecules might contribute to inhibit the infiltration of the brain ischemic area by neutrophils. Studies addressing the molecular mechanisms of these anti-inflammatory actions demonstrated that the involvement of PPARs in the control of IRI-induced inflammation is mediated mainly through their transrepression capabilities. PPARs can suppress the activities of many distinct families of transcription factors. The range of transcription factors affected and the mechanisms involved may be different for each PPAR isotype, although a common mechanism of PPARα and PPAR neuroprotection appears to involve inhibition of p38 MAPK activation and NF-κB nuclear translocation. A recent study confirms that PPAR activation prevents the post-ischemic cerebral expression of proinflammatory transcription factors, such as Egr1, C/EBP and NF-κB, possibly by decreasing DNA binding (Tureyen, Kapadia et al. 2007). The inhibitory protein IκBα, which is an indicator of NF-κB transcriptional activity, is remarkably increased in the brain of rats that underwent cerebral ischemia and completely blocked by rosiglitazone and 15d-PGJ2 administration, thus further confirming that both endogenous and synthetic PPAR ligands inhibit NF-κB signalling (Pereira, Hurtado et al. 2006). Similarly, p38 MAPK and NF-κB activation by cerebral IRI has been demonstrated to be inhibited by pre-treatment with the PPARα agonist WY14643 or the PPAR agonist pioglitazone. However, as MAPK and NFκB are functionally interconnected and do not act independently, we cannot rule out the possibility that PPARs affect NF-κB activation by interfering with the MAPK signalling cascade or vice versa.

The generation of ROS is known to be associated with the induction of apoptosis and, in neurons, inhibition of cell death is an important factor to prevent during IRI. PPAR activation may decrease the IRI-induced activation of apoptotic pathways depending on the increase in activity and expression of numerous anti-oxidant enzymes. Moreover, by their anti-inflammatory action on microglia and astrocytes, PPAR agonists prevent the release of neurotoxic agents, which induce neuronal apoptosis. PPAR agonists may attenuate ischemia-induced reactive oxygen species and subsequently alleviate the post-ischemic degradation of Bcl-2, Bcl-xl, and Akt, by increasing SOD/catalase and decreasing nicotinamide adenine dinucleotide phosphate oxidase levels (Fong, Tsai et al.). Chu and colleagues (Chu, Lee et al. 2006) have demonstrated that rosiglitazone-fed rats had better neurological scores and reduced number of TUNEL-positive cells following transient focal ischemia. Interestingly, these authors also reported an increased vasculature in the rosiglitazone-treated group with increased number of endothelial cells positive for BrdU, suggesting there may be enhanced angiogenesis following PPAR activation. Administration of a selective PPAR agonist (L-796449) 10 min prior to permanent cerebral artery occlusion, resulted in decreased apoptosis, measured as reduction of caspase-3 activity (Pereira, Hurtado et al. 2005). Another study confirmed inhibition on caspase-3 activity by both exogenous and endogenous PPAR agonists, rosiglitazone and 15d-PGJ2, in the ischemic cortex (Lin, Cheung et al. 2006). The same authors observed that rosiglitazone and 15d-PGJ2 exhibit a concentration-dependent paradoxical effect on cytotoxicity, when tested in an *in vitro* model of hydrogen peroxide induced neuronal apoptosis. The drugs induced pro-apoptotic effects when used at concentrations higher that 5 µmol/L but protect neurons from necrosis and apoptosis at concentrations lower than 1 µmol/L. The reason for

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Recently published data suggest that an increased uptake of cerebral extracellular glutamate levels after ischemia may represent an additional mechanism for the neuroprotection exerted by PPAR activation (Romera, Hurtado et al. 2007). Both *in vivo* and *in vitro* experiments showed that rosiglitazone administration increased the expression of the GLT1/EAAT2 glutamate transporter in the brain, thus preventing the extracellular glutamate levels from rising to neurotoxic values.

### **6. Conclusion**

Although clinical data are limited, a wide array of evidence obtained in animal models now shows that PPAR activation may be a rational and effective strategy against ischemic brain damage. The beneficial effects of PPAR agonists in experimental models of stroke are mediated by different mechanisms, as expected based on their pleiotropic pharmacological profile. The neuroprotective actions appear to be mainly related to the reduction in oxidative damage as well as anti-inflammatory and anti-apoptotic effects. These results have been essentially obtained with PPARα and PPAR agonists, while the PPARβ/δ pathway remains largely unexplored, despite a significant interest in this target. Selective activation of different isoforms of PPARs may account for the difference in molecular pathways underlying neuroprotection and these different features still remain far from being completely understood. In conclusion, currently available management protocols for patients with stroke may benefit from the use of PPAR agonists that target detrimental processes associated with IRI. However, several critical issues still need to be resolved. For instance, well-structured clinical trials aimed at evaluating the effects of PPAR ligands on stroke recovery are needed before firm conclusions are drawn about their therapeutic efficacy. A more stringent approach regarding the concentration range of PPAR agonists, especially within the CNS, and the duration of exposure should be applied. Also acceptable water solubility with satisfactory blood-brain barrier penetrability is an important aspect of PPAR agonists that needs to be optimized.

### **7. References**


this paradoxical action is unclear and further studies are needed to better clarify the effects

Recently published data suggest that an increased uptake of cerebral extracellular glutamate levels after ischemia may represent an additional mechanism for the neuroprotection exerted by PPAR activation (Romera, Hurtado et al. 2007). Both *in vivo* and *in vitro* experiments showed that rosiglitazone administration increased the expression of the GLT1/EAAT2 glutamate transporter in the brain, thus preventing the extracellular

Although clinical data are limited, a wide array of evidence obtained in animal models now shows that PPAR activation may be a rational and effective strategy against ischemic brain damage. The beneficial effects of PPAR agonists in experimental models of stroke are mediated by different mechanisms, as expected based on their pleiotropic pharmacological profile. The neuroprotective actions appear to be mainly related to the reduction in oxidative damage as well as anti-inflammatory and anti-apoptotic effects. These results have been essentially obtained with PPARα and PPAR agonists, while the PPARβ/δ pathway remains largely unexplored, despite a significant interest in this target. Selective activation of different isoforms of PPARs may account for the difference in molecular pathways underlying neuroprotection and these different features still remain far from being completely understood. In conclusion, currently available management protocols for patients with stroke may benefit from the use of PPAR agonists that target detrimental processes associated with IRI. However, several critical issues still need to be resolved. For instance, well-structured clinical trials aimed at evaluating the effects of PPAR ligands on stroke recovery are needed before firm conclusions are drawn about their therapeutic efficacy. A more stringent approach regarding the concentration range of PPAR agonists, especially within the CNS, and the duration of exposure should be applied. Also acceptable water solubility with satisfactory blood-brain barrier penetrability is an important aspect of

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### *Edited by Maurizio Balestrino*

This book reports innovations in the preclinical study of stroke, including - novel tools and findings in animal models of stroke, - novel biochemical mechanisms through which ischemic damage may be both generated and limited, - novel pathways to neuroprotection. Although hypothermia has been so far the sole "neuroprotection" treatment that has survived the translation from preclinical to clinical studies, progress in both preclinical studies and in the design of clinical trials will hopefully provide more and better treatments for ischemic stroke. This book aims at providing the preclinical scientist with innovative knowledge and tools to investigate novel mechanisms of, and treatments for, ischemic brain damage.

Photo by Sutthaburawonk / iStock

Advances in the Preclinical Study of Ischemic Stroke

Advances in the Preclinical

Study of Ischemic Stroke

*Edited by Maurizio Balestrino*