The generation of reactive oxygen species (ROS), reactive nitrogen species and free radicals is increased as a consequence of ischemic damage and particularly by the restoration of blood supply. Xantine oxidase, cyclooxygenase, NADPH oxidase, the mitochondrial respiratory chain and the inflammatory response are the major sources of free radicals in cerebral ischemia (Margaill et al., 2005). Reactive species play an important role in both necrotic and apoptotic cell death in cerebral ischemia and reperfusion. ROS generate oxidative stress and triggers tissue inflammation, cause damage to the cellular membrane by lipid peroxidation, DNA damage and disruption of cellular processes. Particularly, superoxide radical has an important responsibility of ischemia damage, since a number of ROS are derived from superoxide.

Using an *in vivo* real-time quantitative superoxide analysis system with an electrochemical sensor previously developed, it has been demonstrated the increase in superoxide in the jugular veins of rats during ischemia/reperfusion in a forebrain ischemia model (Aki et al., 2009). The effect of pre-ischemic hypothermia (32ºC) in the generation of superoxide or as a post ischemia treatment (immediately after reperfusion) was determined in the same model. Both pre and post ischemic hypothermia successfully decreased the superoxide generated by ischemic rats. Hypothermia also decreased oxidative stress, early inflammation and endothelial injury markers in both treatments (Koda et al., 2010).

It has been observed that hypothermia maintains the glutation potential of the liver in an *in vivo* acute hypoxia model, it also avoids the increase in malondialdehide and prevents tissue damage induced by hypoxia (Alva et al., 2010).

The inhibition of superoxide generation using hypothermia was also evaluated in an insulin-induced hypoglycemia model. They showed that intracellular accumulation of zinc promotes the production of ROS through NADPH oxidase activation after hypoglycemia (Shin et al., 2010). This work also provides evidence that hypothermia could affect other mechanisms such as vesicular Zn2+ release and translocation, which affects part of the excitotoxic neuronal death. Hypothermia prevents massive Zn2+ release which ends in cell death, and hyperthermia aggravates it, showing that Zn2+ release is dependent on temperature (Suh et al., 2004, Shin et al., 2010). However, despite all the evidences of ROS reduction and hypothermia protection, the determination of ROS during the first 60 minutes of ischemia in normothermic and hypothermic conditions using a global cerebral ischemiareperfusion model, showed that the widely hypothermia protection effect observed does not correlate with the oxidative stress induced by ROS, as observed with electron spin resonance system (Kunimatsu et al., 2001). The use of different ischemia models, time and

Hypothermia as an Alternative for the Management of Cerebral Ischemia 23

The same group evaluated the effect of hypothermia in the activation of a kinase implicated in neuroprotection *in vitro,* the epsilon protein kinase C (PKC), demonstrating that PKC preservation is an important component in the protective effect of hypothermia. Using the permanent distal middle cerebral artery occlusion plus 1 h of transient bilateral common carotid artery occlusion in normothermic (37ºC) and hypothermic (30ºC) rats, the neuronal full length PKC expression and localization was evaluated with inmmunofluorescence by confocal microscopy and western blot. Normothermic ischemic rats showed a decrease of PKC in the ischemic core at 4 h after common carotid artery release, hypothermia blocked this decrease. Hypothermia also affects cellular distribution. In non ischemic rats, PKC is in the cytoplasm of neurons. When cellular PKC distribution was determined, normothermic ischemic rats showed an PKC decrease in cytosol as well as in membranal fractions of the ischemic core, blocked by hypothermia. In the penumbra, the membranal PKC was decreased after the ischemic damage in normothermic rats and hypothermia blocked this decrease. Even hypothermia blocks PKC cleavage, inhibition of caspase-3 assays showed that this caspase is not involved in this process, suggesting the action of other proteases (Shimohata et al., 2007a). In contrast to PKC protective effect, delta protein kinase C (PKC) is involved in the tissue damage by ischemia. PKC activation depends on catalytic cleavage, phosphorylation and translocation to membranes. The inhibition of PKC activity using the specific inhibitor V1-1, decreased infarct size after transient cerebral ischemia (Brigth et al., 2004). The translocation to the nucleus and mitochondria, the caspase-3 dependent proteolytic cleavage to generate the PKC catalytic fragment (which increased in the membrane fraction, mitochondria, and nuclei); and the release of cytochrome c as earlier as 10 min after reperfusion, are processes triggered by common carotid artery occlusion. Hypothermia blocked all these processes; in fact, the subcellular translocation of the activated PKC was attenuated in the penumbra but not in the ischemic core (Shimohata et al., 2007b). All these protective effects were corroborated with the use of a specific PKC activator, RACK, in the hypothermic rats.

Primary targets of ischemia-reperfusion injury are vascular endothelial cells through the stimulation of calcium overload, ROS generation and the triggering of inflammatory process, which in turn begin apoptotic programs. In order to determine mechanisms involved in hypothermia protection to ischemia and reperfusion damage, human umbilical endothelial cells were used. Using hypothermia (33ºC), a clear reduction in cell apoptosis induced by ischemia/reperfusion was observed. Characterization of this process showed that hypothermia reduces ischemia/reperfusion-induced apoptosis as observed by TUNEL, expression of activated caspase-3 and poly-ADP ribose polymerase (PARP). Hypothermia also reversed Fas/caspase 8 activation pathway and attenuated the Bax/Bcl-2 ratio compared with normothermic cells. Since JNK1/2 and p38 MAPK signaling pathways play an important role in oxidative stress-induced apoptosis, the effect of hypothermia in this pathway was studied, showing that hypothermia inhibits both extrinsic- and intrinsicdependent apoptotic pathways and activation of JNK1/2 activation via MKP-1 induction

The decrease in apoptosis contributes in a significant manner to hypothermia protection. Ischemia and reperfusion have shown to increase the number of active caspase-3

(Yang et al., 2009).

**3.2.2 Apoptotic proteins**

temperature are probably the explanation to contradictory results with respect to ROS generation.

#### **3.1.5 Inflammation**

Hypothermia attenuates inflammation by suppressing activating kinases of nuclear factorkappa B (NFkappaB). In a global cerebral ischemia model by bilateral carotid artery occlusion, microglial activation was observed and hypothermia decreased this activation as well as nuclear NFkappaB translocation and activation (Webster et al., 2009). NFkappaB is activated in cerebral ischemia, controlling the expression of inflammatory genes. In a study using middle cerebral artery occlusion by 2 h and hypothermia at 33 ºC, it was observed that the decrease in temperature decreased NFkappaB translocation and binding activity. Regulatory proteins such as IkappaB kinase were also affected decreasing its activity, suggesting that hypothermia exerts its protective effect by NFkappaB inhibition (Han et al., 2003).

#### **3.2 Hypothermia and apoptosis signaling pathways**

#### **3.2.1 Protein kinases activated by hypoxia**

In order to understand the protective mechanisms of hypothermia several efforts have been performed along years. It has been proposed that the mitochondria and the phosphatidylinositol 3-kinase (PI3-K)/Akt (protein kinase B) signaling pathway are determinant for neuronal survival controlling proapoptosis and antiapoptosis in ischemic neurons during stroke. Akt activity has been implicated in the endogenous neuroprotection observed by preconditioning (Miyawaki et al., 2008) and as part of the neuroprotective response to cerebral ischemia (Kamada et al., 2007). Several pharmacological efforts have been performed to target PI3-K/Akt pathway, since it is known that PI3-K/Akt downstream phosphorylated Bad and proline-rich Akt substrate survival signaling cascades are upregulated in surviving neurons in the ischemic brain (Chan et al., 2004).

One of the most relevant efforts is the demonstration that PI3/Akt pathways are involved in neuroprotection by hypothermia. After the distal middle cerebral artery occlusion of rats using intra-ischemic hypothermia (30ºC), Zhao et al (2005) observed a reduction in infarct size and the improvement of neurological outcome up to two months. Relevant information was obtained from this work besides observed tissue protection and functional response: 1) decrease of Akt activity observed in normothermic animals after stroke was attenuated by hypothermia; 2) hypothermia improved phosphorylation and attenuates dephosphorylation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphoinositide-dependent protein kinase 1 (PDK1); 3) consequent to the observed tissue protection and the involvement of the Akt pathway, the inhibition of PI3K (an upstream activator of Akt) increases infarct size of hypothermic ischemic rats; 4) phosphorylation of forkhead transcription factor (FKHR) was improved by hypothermia attenuating its apoptotic effects, since dephosphorylated FKHR acts as a transcription factor increasing Bcl-2 interacting mediator of cell death (Bim) and Fas ligand; 5) the nuclear translocation of the transcription factor P--catenin, observed in stroke normothermic rats was blocked by hypothermia in the penumbra, but not in the ischemic core, suggesting an important role of -catenin in stroke excitotoxicity (Zhao et al., 2005).

temperature are probably the explanation to contradictory results with respect to ROS

Hypothermia attenuates inflammation by suppressing activating kinases of nuclear factorkappa B (NFkappaB). In a global cerebral ischemia model by bilateral carotid artery occlusion, microglial activation was observed and hypothermia decreased this activation as well as nuclear NFkappaB translocation and activation (Webster et al., 2009). NFkappaB is activated in cerebral ischemia, controlling the expression of inflammatory genes. In a study using middle cerebral artery occlusion by 2 h and hypothermia at 33 ºC, it was observed that the decrease in temperature decreased NFkappaB translocation and binding activity. Regulatory proteins such as IkappaB kinase were also affected decreasing its activity, suggesting that hypothermia exerts its protective effect by NFkappaB inhibition (Han et al.,

In order to understand the protective mechanisms of hypothermia several efforts have been performed along years. It has been proposed that the mitochondria and the phosphatidylinositol 3-kinase (PI3-K)/Akt (protein kinase B) signaling pathway are determinant for neuronal survival controlling proapoptosis and antiapoptosis in ischemic neurons during stroke. Akt activity has been implicated in the endogenous neuroprotection observed by preconditioning (Miyawaki et al., 2008) and as part of the neuroprotective response to cerebral ischemia (Kamada et al., 2007). Several pharmacological efforts have been performed to target PI3-K/Akt pathway, since it is known that PI3-K/Akt downstream phosphorylated Bad and proline-rich Akt substrate survival signaling cascades are

One of the most relevant efforts is the demonstration that PI3/Akt pathways are involved in neuroprotection by hypothermia. After the distal middle cerebral artery occlusion of rats using intra-ischemic hypothermia (30ºC), Zhao et al (2005) observed a reduction in infarct size and the improvement of neurological outcome up to two months. Relevant information was obtained from this work besides observed tissue protection and functional response: 1) decrease of Akt activity observed in normothermic animals after stroke was attenuated by hypothermia; 2) hypothermia improved phosphorylation and attenuates dephosphorylation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphoinositide-dependent protein kinase 1 (PDK1); 3) consequent to the observed tissue protection and the involvement of the Akt pathway, the inhibition of PI3K (an upstream activator of Akt) increases infarct size of hypothermic ischemic rats; 4) phosphorylation of forkhead transcription factor (FKHR) was improved by hypothermia attenuating its apoptotic effects, since dephosphorylated FKHR acts as a transcription factor increasing Bcl-2 interacting mediator of cell death (Bim) and Fas ligand; 5) the nuclear translocation of the transcription factor P--catenin, observed in stroke normothermic rats was blocked by hypothermia in the penumbra, but not in the ischemic core, suggesting an important role of

upregulated in surviving neurons in the ischemic brain (Chan et al., 2004).

**3.2 Hypothermia and apoptosis signaling pathways**

**3.2.1 Protein kinases activated by hypoxia**

generation.

2003).

**3.1.5 Inflammation**

The same group evaluated the effect of hypothermia in the activation of a kinase implicated in neuroprotection *in vitro,* the epsilon protein kinase C (PKC), demonstrating that PKC preservation is an important component in the protective effect of hypothermia. Using the permanent distal middle cerebral artery occlusion plus 1 h of transient bilateral common carotid artery occlusion in normothermic (37ºC) and hypothermic (30ºC) rats, the neuronal full length PKC expression and localization was evaluated with inmmunofluorescence by confocal microscopy and western blot. Normothermic ischemic rats showed a decrease of PKC in the ischemic core at 4 h after common carotid artery release, hypothermia blocked this decrease. Hypothermia also affects cellular distribution. In non ischemic rats, PKC is in the cytoplasm of neurons. When cellular PKC distribution was determined, normothermic ischemic rats showed an PKC decrease in cytosol as well as in membranal fractions of the ischemic core, blocked by hypothermia. In the penumbra, the membranal PKC was decreased after the ischemic damage in normothermic rats and hypothermia blocked this decrease. Even hypothermia blocks PKC cleavage, inhibition of caspase-3 assays showed that this caspase is not involved in this process, suggesting the action of other proteases (Shimohata et al., 2007a).

In contrast to PKC protective effect, delta protein kinase C (PKC) is involved in the tissue damage by ischemia. PKC activation depends on catalytic cleavage, phosphorylation and translocation to membranes. The inhibition of PKC activity using the specific inhibitor V1-1, decreased infarct size after transient cerebral ischemia (Brigth et al., 2004). The translocation to the nucleus and mitochondria, the caspase-3 dependent proteolytic cleavage to generate the PKC catalytic fragment (which increased in the membrane fraction, mitochondria, and nuclei); and the release of cytochrome c as earlier as 10 min after reperfusion, are processes triggered by common carotid artery occlusion. Hypothermia blocked all these processes; in fact, the subcellular translocation of the activated PKC was attenuated in the penumbra but not in the ischemic core (Shimohata et al., 2007b). All these protective effects were corroborated with the use of a specific PKC activator, RACK, in the hypothermic rats.

Primary targets of ischemia-reperfusion injury are vascular endothelial cells through the stimulation of calcium overload, ROS generation and the triggering of inflammatory process, which in turn begin apoptotic programs. In order to determine mechanisms involved in hypothermia protection to ischemia and reperfusion damage, human umbilical endothelial cells were used. Using hypothermia (33ºC), a clear reduction in cell apoptosis induced by ischemia/reperfusion was observed. Characterization of this process showed that hypothermia reduces ischemia/reperfusion-induced apoptosis as observed by TUNEL, expression of activated caspase-3 and poly-ADP ribose polymerase (PARP). Hypothermia also reversed Fas/caspase 8 activation pathway and attenuated the Bax/Bcl-2 ratio compared with normothermic cells. Since JNK1/2 and p38 MAPK signaling pathways play an important role in oxidative stress-induced apoptosis, the effect of hypothermia in this pathway was studied, showing that hypothermia inhibits both extrinsic- and intrinsicdependent apoptotic pathways and activation of JNK1/2 activation via MKP-1 induction (Yang et al., 2009).

#### **3.2.2 Apoptotic proteins**

The decrease in apoptosis contributes in a significant manner to hypothermia protection. Ischemia and reperfusion have shown to increase the number of active caspase-3

Hypothermia as an Alternative for the Management of Cerebral Ischemia 25

The decrease in temperature and/or the decrease in oxygen concentration involve a series of events that modulate transcription and translation. Novel proteins have been discovered in the last decades. Here we show the role of cold inducible proteins and hypoxia inducible factor-1 as proteins that regulate the efficient transcription and translation of proteins in

Cold inducible proteins have the function to ensure the efficient translation of specific mRNAs at temperatures below the physiological standard. Hypothermia induces the synthesis of amino terminal consensus sequence RNA-binding domain proteins (CS-RBD). The "cold-inducible RNA-binding protein" (CIRP) is one of these proteins, and has been involved in protection as the result of hypothermia treatment in *in vitro* studies. CIRP regulates gene expression at translational level. It binds to the 5´-untranslated region (5´- UTR) or 3´-UTR of specific transcripts, affecting translation and transcript stability (Lleonart,

CIRP has been proposed as a therapeutic target in cerebral ischemia by Liu et al (2010). They determined mRNA expression in hippocampus and cortex of rat brains subjected to hypothermia (30º), cerebral ischemia (by four vessel occlusion model of forebrain ischemia) and hypothermia plus cerebral ischemia by real time quantitative PCR analysis. mRNA CIRP expression was followed at 2, 6 and 24 h showing an increase in cortex after cerebral ischemia with a previous hypothermia treatment. In order to clarify the relationship between CIRP and energy metabolism, they determined lactate and piruvate concentrations, showing that CIRP has a neuroprotective effect in hypothermia; however, it is not related to

The contribution of CIRP to the neuroprotection observed by hypothermia has also been studied using MEMB5 cells, a neural stem cell line from mouse forebrain (Saito et al., 2010). These cells proliferate in the presence of epidermal growth factor (EGF). EGF deprivation at 37 ºC results in apoptosis induction, as well as a decrease of the nestin neural stem cell marker and an increase of the astrocyte marker glial fibrillary acidic protein (GFAP). In contrast, MEMB5 cells at moderate hypothermia prevented apoptosis and decreased the observed GFAP expression of normothermic cells. This observation is important because it suggests that hypothermia prevents neural stem cells differentiation, and it has been hypothesized that the preservation of neural stem cells is one of the neuroprotective mechanisms of therapeutic hypothermia, since it could maintain the capability of cells to differentiate and proliferate after an ischemic event. CIRP mRNA and protein was increased in the MEB5 in hypothermic cells, a response according to previous observations showing that ischemia/reperfusion decreases CIRP mRNA (Xue et al., 1999) whereas hypothermia

The relevance of CIRP expression was confirmed using CIRP iRNA, increasing apoptosis in hypothermic cells without EGF. This result suggests that the induced CIRP plays the role of a survival factor in neural stem cells. The prevention of apoptosis observed with induced CIRP at hypothermia has been suggested to be the result of the activation of extracellular signalregulated kinase ERK (Artero-Castro et al., 2009; Sakurai et al., 2006; Schmitt et al., 2007).

increases it in an *in vivo* cerebral ischemia model (Liu et al., 2010).

**3.3.1 Effect of hypothermia in proteins involved in gene expression**

ischemia and ischemia/hypothermia events. **<IP_ADDRESS> Cold-inducible RNA-binding proteins**

2010).

energy metabolism.

immunoreactive nuclei, and hypothermia clearly reduced this induction (Kunimatsu et al., 2001).

Recently, Li & Wang (2011) used mild hypothermia (33 ºC) in rats subjected to middle cerebral artery occlusion and determined neurological impairment and the expression of Second Mitochondrion-derived Activator of Caspases (SMAC) as an index of cellular apoptosis. Mild hypothermia significantly improved the neurological deficit scores while results from protein and transcript expression of SMAC showed a significantly decrease, suggesting that mild hypothermia could be protecting the functions of cells by attenuating apoptotic death (Li & Wang, 2011).

It has been previously shown that the pro-apoptotic protein SMAC/DIABLO expression is increased in cortex and hippocampus in transient cerebral ischemia as well as in ischemiareperfusion injury (Saito et al., 2003; Scarabelli & Stephanou, 2004; Siegelin et al., 2005). SMAC increases 3 h after cerebral ischemia with a peak at 24 h. Apparently, hypothermia could be down-regulating SMAC production attenuating caspases activation. Cell apoptosis via SMAC involve mitochondrial and death receptor pathways, inducing changes in mitochondrial membrane permeability and subsequently release membrane proteins, such as SMAC, into the cytoplasm. SMAC leads cells toward apoptosis through apoptosis-related protein. The prevention of loss of mitochondrial transmembrane potential, release of apoptotic proteins (citochrome c and apoptosis inducing factor [AIF]) and the activation of apoptotic proteins such as caspase 3, as well as the attenuation in the elevation of oxidative stress markers have been observed also in *in vitro* cells with hypothermia in a model of iron and ascorbic acid neurotoxicity (Hasegawa et al., 2009).

Rats subjected to global cerebral ischemia with the four-vessel occlusion model with hypothermia (31-32 ºC) and hyperthermia (41-42ºC) confirmed the protective effects of hypothermia in the decrease of mortality rate (at 72 and 168 h post reperfusion), and in the increase of surviving neurons in hippocampus under hypothermic conditions. Hypothermia clearly reduced p53 and increased bcl-2 proteins reducing neuronal death. In addition hyperthermia had the opposite effect in the expression of both proteins (Zhang et al., 2010).

#### **3.3 Regulation of gene and protein expression**

Hypothermia induces changes in inflammatory, apoptotic and metabolic genes as the result of gene expression regulation.

In general, it is considered that hypothermia downregulates gene expression. However, there are reports that show the upregulation of certain genes, particularly those involved in cell survival. The understanding of gene and protein expression as result of hypothermia in cerebral ischemia could lead to the possible therapeutic target genes or pathways regulated as a result of decreasing body temperature.

Recently, an analysis of gene and protein expression using DNA microarrays and proteomics approach in a 2 h middle cerebral artery occlusion model and mild hypothermia (35ºC) has shown that it is possible to determine target molecules. The authors proposed that suppression of neuroinflammatory cascades MIP-3-CCR could contribute to the neuroprotective effects of hypothermia and also identified Hsp 70 as a neuroprotective factor stimulated by hypothermia (Shintani et al., 2010; Terao et al., 2009,).

#### **3.3.1 Effect of hypothermia in proteins involved in gene expression**

The decrease in temperature and/or the decrease in oxygen concentration involve a series of events that modulate transcription and translation. Novel proteins have been discovered in the last decades. Here we show the role of cold inducible proteins and hypoxia inducible factor-1 as proteins that regulate the efficient transcription and translation of proteins in ischemia and ischemia/hypothermia events.

#### **<IP_ADDRESS> Cold-inducible RNA-binding proteins**

24 Advances in the Treatment of Ischemic Stroke

immunoreactive nuclei, and hypothermia clearly reduced this induction (Kunimatsu et al.,

Recently, Li & Wang (2011) used mild hypothermia (33 ºC) in rats subjected to middle cerebral artery occlusion and determined neurological impairment and the expression of Second Mitochondrion-derived Activator of Caspases (SMAC) as an index of cellular apoptosis. Mild hypothermia significantly improved the neurological deficit scores while results from protein and transcript expression of SMAC showed a significantly decrease, suggesting that mild hypothermia could be protecting the functions of cells by attenuating

It has been previously shown that the pro-apoptotic protein SMAC/DIABLO expression is increased in cortex and hippocampus in transient cerebral ischemia as well as in ischemiareperfusion injury (Saito et al., 2003; Scarabelli & Stephanou, 2004; Siegelin et al., 2005). SMAC increases 3 h after cerebral ischemia with a peak at 24 h. Apparently, hypothermia could be down-regulating SMAC production attenuating caspases activation. Cell apoptosis via SMAC involve mitochondrial and death receptor pathways, inducing changes in mitochondrial membrane permeability and subsequently release membrane proteins, such as SMAC, into the cytoplasm. SMAC leads cells toward apoptosis through apoptosis-related protein. The prevention of loss of mitochondrial transmembrane potential, release of apoptotic proteins (citochrome c and apoptosis inducing factor [AIF]) and the activation of apoptotic proteins such as caspase 3, as well as the attenuation in the elevation of oxidative stress markers have been observed also in *in vitro* cells with hypothermia in a model of iron

Rats subjected to global cerebral ischemia with the four-vessel occlusion model with hypothermia (31-32 ºC) and hyperthermia (41-42ºC) confirmed the protective effects of hypothermia in the decrease of mortality rate (at 72 and 168 h post reperfusion), and in the increase of surviving neurons in hippocampus under hypothermic conditions. Hypothermia clearly reduced p53 and increased bcl-2 proteins reducing neuronal death. In addition hyperthermia had the opposite effect in the expression of both proteins (Zhang et al., 2010).

Hypothermia induces changes in inflammatory, apoptotic and metabolic genes as the result

In general, it is considered that hypothermia downregulates gene expression. However, there are reports that show the upregulation of certain genes, particularly those involved in cell survival. The understanding of gene and protein expression as result of hypothermia in cerebral ischemia could lead to the possible therapeutic target genes or pathways regulated

Recently, an analysis of gene and protein expression using DNA microarrays and proteomics approach in a 2 h middle cerebral artery occlusion model and mild hypothermia (35ºC) has shown that it is possible to determine target molecules. The authors proposed that suppression of neuroinflammatory cascades MIP-3-CCR could contribute to the neuroprotective effects of hypothermia and also identified Hsp 70 as a neuroprotective

factor stimulated by hypothermia (Shintani et al., 2010; Terao et al., 2009,).

2001).

apoptotic death (Li & Wang, 2011).

and ascorbic acid neurotoxicity (Hasegawa et al., 2009).

**3.3 Regulation of gene and protein expression**

as a result of decreasing body temperature.

of gene expression regulation.

Cold inducible proteins have the function to ensure the efficient translation of specific mRNAs at temperatures below the physiological standard. Hypothermia induces the synthesis of amino terminal consensus sequence RNA-binding domain proteins (CS-RBD). The "cold-inducible RNA-binding protein" (CIRP) is one of these proteins, and has been involved in protection as the result of hypothermia treatment in *in vitro* studies. CIRP regulates gene expression at translational level. It binds to the 5´-untranslated region (5´- UTR) or 3´-UTR of specific transcripts, affecting translation and transcript stability (Lleonart, 2010).

CIRP has been proposed as a therapeutic target in cerebral ischemia by Liu et al (2010). They determined mRNA expression in hippocampus and cortex of rat brains subjected to hypothermia (30º), cerebral ischemia (by four vessel occlusion model of forebrain ischemia) and hypothermia plus cerebral ischemia by real time quantitative PCR analysis. mRNA CIRP expression was followed at 2, 6 and 24 h showing an increase in cortex after cerebral ischemia with a previous hypothermia treatment. In order to clarify the relationship between CIRP and energy metabolism, they determined lactate and piruvate concentrations, showing that CIRP has a neuroprotective effect in hypothermia; however, it is not related to energy metabolism.

The contribution of CIRP to the neuroprotection observed by hypothermia has also been studied using MEMB5 cells, a neural stem cell line from mouse forebrain (Saito et al., 2010). These cells proliferate in the presence of epidermal growth factor (EGF). EGF deprivation at 37 ºC results in apoptosis induction, as well as a decrease of the nestin neural stem cell marker and an increase of the astrocyte marker glial fibrillary acidic protein (GFAP). In contrast, MEMB5 cells at moderate hypothermia prevented apoptosis and decreased the observed GFAP expression of normothermic cells. This observation is important because it suggests that hypothermia prevents neural stem cells differentiation, and it has been hypothesized that the preservation of neural stem cells is one of the neuroprotective mechanisms of therapeutic hypothermia, since it could maintain the capability of cells to differentiate and proliferate after an ischemic event. CIRP mRNA and protein was increased in the MEB5 in hypothermic cells, a response according to previous observations showing that ischemia/reperfusion decreases CIRP mRNA (Xue et al., 1999) whereas hypothermia increases it in an *in vivo* cerebral ischemia model (Liu et al., 2010).

The relevance of CIRP expression was confirmed using CIRP iRNA, increasing apoptosis in hypothermic cells without EGF. This result suggests that the induced CIRP plays the role of a survival factor in neural stem cells. The prevention of apoptosis observed with induced CIRP at hypothermia has been suggested to be the result of the activation of extracellular signalregulated kinase ERK (Artero-Castro et al., 2009; Sakurai et al., 2006; Schmitt et al., 2007).

Hypothermia as an Alternative for the Management of Cerebral Ischemia 27

besides hypothermia in the search of a neuroprotective compound capable of blocking the metabolic insult. Combination therapy is based in the extension of the therapeutic window of hypothermia using pharmacotherapy and establishes an interesting approach in

The combined administration of the antioxidant tirilazad, magnesium and hypothermia has shown that the hypothermia protection increases with the use of pharmacotherapy, extending the therapeutic window of hypothermia treatment. Zausinger et al (2003) used hypothermia (2 h, 33ºC) at 0, 1, 3 and 5 h after transient focal ischemia induction combined

intervals. Infarct volume was reduced by 74%, 49% and 45% when hypothermia was applied at 0, 1 and 3 h. No improvements were observed at 5 h. The same combination therapy was performed in a permanent ischemia with middle cerebral artery occlusion. Two h at 33 ºC hypothermia and two times of drug administration (30 min before and 1 h after middle cerebral artery occlusion) showed a 52 % infarct size reduction after 6 h occlusion. Interestingly, a separate group with 7 days of permanent middle cerebral artery occlusion was followed daily with neurological tests and body weight. Even high mortality ocurred in this group, neurological recovery was observed in survivor rats, as well as a decrease in

Moderate hypothermia (30ºC) has shown higher protection than mild hypothermia (33ºC), however moderate hypothermia is associated with severe side effects. Nonetheless, the combined effect observed with mild hypothermia, magnesium and tirilazad showed a comparable protection with that observed using moderate hypothermia, suggesting that

Combination therapy with magnesium has generated controversy, because magnesium treatment alone has shown to be ineffective in normothermic rats subjected to ischemia (Zhu et al., 2004, a and b). The quantification of infarct volumes at magnesium 360 or 720 umol/kg in rats subjected to middle cerebral artery occlusion showed that the 360 umol/kg dose reduces the striatal infarct volume by 32 %. The authors observed that mild spontaneous hypothermia was responsible of the observed neuroprotective effect of

The therapeutic time window of combined magnesium and mild hypothermia treatment was determined after permanent middle cerebral artery occlusion. The administration of a magnesium sulfate infusion (360 μmol/kg, then 120 μmol/kg/h) and mild hypothermia (35ºC) after 2, 4 or 6 h ischemia showed that combination therapy considerably reduced infarct volumes at 2 and 4 h but not at 6 h, supporting the use of combined therapy even at

Although the controversy in the use of magnesium and hypothermia in cerebral ischemia exists, a lot of experimental evidence demonstrates its efficacy in laboratory studies and it is necessary to perform more clinical trials to translate this combinatorial therapy to clinical

L-1.kg-1) in 1 h

with two administrations of tirilazad (3 mg/kg) and magnesium (1 mmol.

combination therapy could be a promising approach in clinical applications.

hypothermia research.

infarct size (Schöller et al., 2004).

**4.2 Hypothermia and magnesium**

magnesium (Campbell et al 2008a).

delayed hours after ischemia onset (Campbell et al 2008b).

**4.1 Hypothermia, tirilazad and magnesium**

#### **<IP_ADDRESS> Hypoxia inducible factor**

Hypoxia inducible factor is a transcription factor which binds to hypoxic response elementsdriven promoters of genes that mediate adaptive reactions to reduction in oxygen availability. Hypoxia inducible factor is regulated by oxygen accessibility and has been considered as a therapeutic target in cerebral ischemia (Aguilera et al., 2009). Recently it has been reported that persisting low temperature affects its stabilization and protein accumulation. Since the normal regulatory degradation processes of HIF are not affected by hypothermia, it has been hypothesized that probably hypothermia elevates intracellular oxygen tension by decreasing oxygen consumption, suppressing in turn HIF-1 alpha subunit induction. These results were obtained with different cell lines (T98G cells from human glioblastoma multiform, HeLa cells (derived from human cervical carcinoma) and Hep3B cells (derived from human hepatoma) as well as mice subjected to hypoxia and hypothermia (18ºC in a mouse incubator). The translation of HIF-1 alpha protein showed to be dependent on time exposure to hypothermia. The down-regulation of HIF protein expression observed with hypothermia has relevant implications in ischemia and hypothermia studies, since this transcription factor is a master regulator of the hypoxic response to oxygen decrease (Tanaka et al., 2010).

#### **3.3.2 Gene and protein expression in CA1 neurons as result of hypothermia**

Hippocampal CA1 layer is a region that presents a typical apoptosis cell death after ischemic damage. As a matter of fact , accumulating evidence has indicated that the postischemic DNA fragmentation in the hippocampal CA1 area in experimental ischemic models is a key phenomenon for the delayed neuronal death and is considered as apoptosis. Hypothermia has shown to protect CA1 neurons attenuating the down-regulation of GluR2 mRNA in a model of forebrain ischemia using two days of mild hypothermia induced after 1 h cerebral ischemia, suggesting that the observed attenuation and CA1 neurons protection responds to cooling (Colbourne et al., 2003). Another interesting protein is the -galactosidase-binding lectin Galectin-3, which has been observed expressed in experimental models of stroke (Walther et al., 2000; Yan et al., 2009) and increased in microgial cells in the hippocampal CA1 layer after a transient ischemic insult. Galectin-3 is a protein involved in apoptotic regulation, inflammation and cell differentiation and used as a marker of activated microglia. After 5 min of bilateral common carotid arteries of gerbils, galectin-3 expression was observed in microglial cells in CA1 region. Hypothermia (31ºC) prevents galectin-3 expression suggesting that hypothermia protection occurs through the inhibition of microglial activation and probably by preventing neuronal death (Satoh et al., 2011). Even when galectin-3 has been considered apoptotic, its role as an inflammatory mediator in neonatal hypoxia ischemia injury through the modulation of the inflammatory response has been reported (Doverhag et al., 2010).

#### **4. Combined therapies**

The preservation of tissue and reduction of brain damage observed during hypothermia and the easiness to achieve and maintain low temperatures, constitute an attractive alternative against stroke. However, because of the complexity of the pathophysiological mechanisms involved in the ischemic cascade, it is common to observe the use of one or two drugs besides hypothermia in the search of a neuroprotective compound capable of blocking the metabolic insult. Combination therapy is based in the extension of the therapeutic window of hypothermia using pharmacotherapy and establishes an interesting approach in hypothermia research.

#### **4.1 Hypothermia, tirilazad and magnesium**

26 Advances in the Treatment of Ischemic Stroke

Hypoxia inducible factor is a transcription factor which binds to hypoxic response elementsdriven promoters of genes that mediate adaptive reactions to reduction in oxygen availability. Hypoxia inducible factor is regulated by oxygen accessibility and has been considered as a therapeutic target in cerebral ischemia (Aguilera et al., 2009). Recently it has been reported that persisting low temperature affects its stabilization and protein accumulation. Since the normal regulatory degradation processes of HIF are not affected by hypothermia, it has been hypothesized that probably hypothermia elevates intracellular oxygen tension by decreasing oxygen consumption, suppressing in turn HIF-1 alpha subunit induction. These results were obtained with different cell lines (T98G cells from human glioblastoma multiform, HeLa cells (derived from human cervical carcinoma) and Hep3B cells (derived from human hepatoma) as well as mice subjected to hypoxia and hypothermia (18ºC in a mouse incubator). The translation of HIF-1 alpha protein showed to be dependent on time exposure to hypothermia. The down-regulation of HIF protein expression observed with hypothermia has relevant implications in ischemia and hypothermia studies, since this transcription factor is a master regulator of the hypoxic

**3.3.2 Gene and protein expression in CA1 neurons as result of hypothermia**

Hippocampal CA1 layer is a region that presents a typical apoptosis cell death after ischemic damage. As a matter of fact , accumulating evidence has indicated that the postischemic DNA fragmentation in the hippocampal CA1 area in experimental ischemic models is a key phenomenon for the delayed neuronal death and is considered as apoptosis. Hypothermia has shown to protect CA1 neurons attenuating the down-regulation of GluR2 mRNA in a model of forebrain ischemia using two days of mild hypothermia induced after 1 h cerebral ischemia, suggesting that the observed attenuation and CA1 neurons protection responds to cooling (Colbourne et al., 2003). Another interesting protein is the -galactosidase-binding lectin Galectin-3, which has been observed expressed in experimental models of stroke (Walther et al., 2000; Yan et al., 2009) and increased in microgial cells in the hippocampal CA1 layer after a transient ischemic insult. Galectin-3 is a protein involved in apoptotic regulation, inflammation and cell differentiation and used as a marker of activated microglia. After 5 min of bilateral common carotid arteries of gerbils, galectin-3 expression was observed in microglial cells in CA1 region. Hypothermia (31ºC) prevents galectin-3 expression suggesting that hypothermia protection occurs through the inhibition of microglial activation and probably by preventing neuronal death (Satoh et al., 2011). Even when galectin-3 has been considered apoptotic, its role as an inflammatory mediator in neonatal hypoxia ischemia injury through the modulation of the inflammatory response has

The preservation of tissue and reduction of brain damage observed during hypothermia and the easiness to achieve and maintain low temperatures, constitute an attractive alternative against stroke. However, because of the complexity of the pathophysiological mechanisms involved in the ischemic cascade, it is common to observe the use of one or two drugs

**<IP_ADDRESS> Hypoxia inducible factor**

response to oxygen decrease (Tanaka et al., 2010).

been reported (Doverhag et al., 2010).

**4. Combined therapies**

The combined administration of the antioxidant tirilazad, magnesium and hypothermia has shown that the hypothermia protection increases with the use of pharmacotherapy, extending the therapeutic window of hypothermia treatment. Zausinger et al (2003) used hypothermia (2 h, 33ºC) at 0, 1, 3 and 5 h after transient focal ischemia induction combined with two administrations of tirilazad (3 mg/kg) and magnesium (1 mmol. L-1.kg-1) in 1 h intervals. Infarct volume was reduced by 74%, 49% and 45% when hypothermia was applied at 0, 1 and 3 h. No improvements were observed at 5 h. The same combination therapy was performed in a permanent ischemia with middle cerebral artery occlusion. Two h at 33 ºC hypothermia and two times of drug administration (30 min before and 1 h after middle cerebral artery occlusion) showed a 52 % infarct size reduction after 6 h occlusion. Interestingly, a separate group with 7 days of permanent middle cerebral artery occlusion was followed daily with neurological tests and body weight. Even high mortality ocurred in this group, neurological recovery was observed in survivor rats, as well as a decrease in infarct size (Schöller et al., 2004).

Moderate hypothermia (30ºC) has shown higher protection than mild hypothermia (33ºC), however moderate hypothermia is associated with severe side effects. Nonetheless, the combined effect observed with mild hypothermia, magnesium and tirilazad showed a comparable protection with that observed using moderate hypothermia, suggesting that combination therapy could be a promising approach in clinical applications.

#### **4.2 Hypothermia and magnesium**

Combination therapy with magnesium has generated controversy, because magnesium treatment alone has shown to be ineffective in normothermic rats subjected to ischemia (Zhu et al., 2004, a and b). The quantification of infarct volumes at magnesium 360 or 720 umol/kg in rats subjected to middle cerebral artery occlusion showed that the 360 umol/kg dose reduces the striatal infarct volume by 32 %. The authors observed that mild spontaneous hypothermia was responsible of the observed neuroprotective effect of magnesium (Campbell et al 2008a).

The therapeutic time window of combined magnesium and mild hypothermia treatment was determined after permanent middle cerebral artery occlusion. The administration of a magnesium sulfate infusion (360 μmol/kg, then 120 μmol/kg/h) and mild hypothermia (35ºC) after 2, 4 or 6 h ischemia showed that combination therapy considerably reduced infarct volumes at 2 and 4 h but not at 6 h, supporting the use of combined therapy even at delayed hours after ischemia onset (Campbell et al 2008b).

Although the controversy in the use of magnesium and hypothermia in cerebral ischemia exists, a lot of experimental evidence demonstrates its efficacy in laboratory studies and it is necessary to perform more clinical trials to translate this combinatorial therapy to clinical

Hypothermia as an Alternative for the Management of Cerebral Ischemia 29

Hypothermia has shown positive results in cardiac arrest and neonatal hypoxic ischemia and in patients with acute brain injury. However, there are no large clinical stroke studies that could assist in the comprehension of the hypothermia role in stroke. In laboratory studies, hypothermia is a consistent protective agent. The present chapter shows the contribution of hypothermia research in the last years to understand its participation during the different stages of the ischemic damage cascade. Blood brain barrier integrity, metabolic rate decrease, redox state changes, inflammatory, apoptotic, signalling and gene regulation are targets where hypothermia protection participates. Hypothermia is affected by diverse factors such as timing, duration and deepness. All these factors contribute to disparity in obtained results. However, it is clear that this research is necessary in order to determine the exact targets, the time when hypothermia begins and duration of hypothermia. Additionally, since brain ischemia is a multifactorial problem and hypothermia has demonstrated to decrease damage in several stages of the process, combination therapy is an alternative to improve treatments by the extension of the therapeutic time window of

Nares-López F. E. received a scholarship from BECANET, number 053103.

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Intraischemic hypothermia decreases the release of glutamate in the cores of

hypothermia protection.

**6. Acknowledgments**

ISSN 1574-3624

8993

**7. References**

use. Actually, combined mild hypothermia (35ºC) and magnesium is recognized as a neuroprotective treatment able to minimize ischemic damage, even in delayed treatments (Meloni et al., 2009).

#### **4.3 Hypothermia and citicoline**

Hypothermia has been combined with other compounds. One of them is citicoline, an endogenous compound that has shown to stabilize membrane function and reduce free radical generation during ischemia (Rao et al., 2000).

Mild hypothermia combined with citicoline resulted in an additive effect in attenuating apoptosis in focal cerebral ischemia/reperfusion injury. Hypothermia (341ºC) during middle cerebral artery occlusion (2 h) followed by 24 h reperfusion was used in combination with 4000 mg/kg *i.p.* citicoline. Combined therapy with citicoline and hypothermia resulted in reduced apoptotic cell death before the development of the apoptosome, thus preventing apoptosis and neuronal damage. Bcl-2, caspases 3 and 9 as well as Bax proteins were immunohistochemistry tested, showing that the use of citicoline with hypothermia is more effective than citicoline or hypothermia used alone. A decrease in cerebral injury was observed and apparently, the tissue protection is due to the suppression of apoptotic processes (Sahin et al., 2010).

#### **4.4 Hypothermia and ginkgolides**

Despite the neuroprotective role of hypothermia, the combined use of hypothermia with protective compounds not always has a synergistic effect. This is the case of hypothermia and ginkgolides in astrocytes subjected to ischemia and reperfusion in which hypothermia attenuates, rather than enhances, the protective effect of ginkgolides on astrocytes from ischemia and reperfusion-induced injury. Co-treatment with different doses of ginkgolides at 32 and 28 ºC hypothermia during 24, 48 and 72 h before 24 h ischemia followed by 24 h reperfusion showed that the use of ginkgolides without hypothermia treatment had an improvement in cell viabilities and in anti-apoptotic properties, and this protective effects were not observed in the co-treatment (Fang et al., 2009).

#### **4.5 Hypothermia and xenon**

Xenon has been considered a great promise used as a neuroprotectant in *in vivo* and *in vitro* studies. Combined therapy with hypothermia in a neonatal rat hypoxia-ischemia model has shown that the combination of 50% xenon and hypothermia of 32°C has a protective effect in the restoration of long-term functional outcomes and global histopathology, showing that the combined xenon/hypothermia has a greater protection (Hobbs et al., 2008).

#### **5. Conclusion**

Despite all the efforts to develop pharmacological treatments to contend with cerebral ischemic damage, nowadays, there are no efficient treatments to deal with this pathology. Non-pharmacological treatments emerge as reasonable approaches to compete against ischemic damage.

Hypothermia has shown positive results in cardiac arrest and neonatal hypoxic ischemia and in patients with acute brain injury. However, there are no large clinical stroke studies that could assist in the comprehension of the hypothermia role in stroke. In laboratory studies, hypothermia is a consistent protective agent. The present chapter shows the contribution of hypothermia research in the last years to understand its participation during the different stages of the ischemic damage cascade. Blood brain barrier integrity, metabolic rate decrease, redox state changes, inflammatory, apoptotic, signalling and gene regulation are targets where hypothermia protection participates. Hypothermia is affected by diverse factors such as timing, duration and deepness. All these factors contribute to disparity in obtained results. However, it is clear that this research is necessary in order to determine the exact targets, the time when hypothermia begins and duration of hypothermia. Additionally, since brain ischemia is a multifactorial problem and hypothermia has demonstrated to decrease damage in several stages of the process, combination therapy is an alternative to improve treatments by the extension of the therapeutic time window of hypothermia protection.

#### **6. Acknowledgments**

Nares-López F. E. received a scholarship from BECANET, number 053103.

#### **7. References**

28 Advances in the Treatment of Ischemic Stroke

use. Actually, combined mild hypothermia (35ºC) and magnesium is recognized as a neuroprotective treatment able to minimize ischemic damage, even in delayed treatments

Hypothermia has been combined with other compounds. One of them is citicoline, an endogenous compound that has shown to stabilize membrane function and reduce free

Mild hypothermia combined with citicoline resulted in an additive effect in attenuating apoptosis in focal cerebral ischemia/reperfusion injury. Hypothermia (341ºC) during middle cerebral artery occlusion (2 h) followed by 24 h reperfusion was used in combination with 4000 mg/kg *i.p.* citicoline. Combined therapy with citicoline and hypothermia resulted in reduced apoptotic cell death before the development of the apoptosome, thus preventing apoptosis and neuronal damage. Bcl-2, caspases 3 and 9 as well as Bax proteins were immunohistochemistry tested, showing that the use of citicoline with hypothermia is more effective than citicoline or hypothermia used alone. A decrease in cerebral injury was observed and apparently, the tissue protection is due to the suppression of apoptotic

Despite the neuroprotective role of hypothermia, the combined use of hypothermia with protective compounds not always has a synergistic effect. This is the case of hypothermia and ginkgolides in astrocytes subjected to ischemia and reperfusion in which hypothermia attenuates, rather than enhances, the protective effect of ginkgolides on astrocytes from ischemia and reperfusion-induced injury. Co-treatment with different doses of ginkgolides at 32 and 28 ºC hypothermia during 24, 48 and 72 h before 24 h ischemia followed by 24 h reperfusion showed that the use of ginkgolides without hypothermia treatment had an improvement in cell viabilities and in anti-apoptotic properties, and this protective effects

Xenon has been considered a great promise used as a neuroprotectant in *in vivo* and *in vitro* studies. Combined therapy with hypothermia in a neonatal rat hypoxia-ischemia model has shown that the combination of 50% xenon and hypothermia of 32°C has a protective effect in the restoration of long-term functional outcomes and global histopathology, showing that

Despite all the efforts to develop pharmacological treatments to contend with cerebral ischemic damage, nowadays, there are no efficient treatments to deal with this pathology. Non-pharmacological treatments emerge as reasonable approaches to compete against

the combined xenon/hypothermia has a greater protection (Hobbs et al., 2008).

(Meloni et al., 2009).

**4.3 Hypothermia and citicoline**

processes (Sahin et al., 2010).

**4.5 Hypothermia and xenon**

**5. Conclusion**

ischemic damage.

**4.4 Hypothermia and ginkgolides**

radical generation during ischemia (Rao et al., 2000).

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**3**

*Japan*

**Timing of Hypothermia (During or After Global Cerebral Ischemia) Differentially Affects Acute**

Hypothermia (HT) is one of the most effective neuroprotective therapies for brain injury caused by cardiac arrest in humans (Benard et al., 2002), although there is as yet no evidence of such an effect of HT on cerebrovascular diseases from a large-scale clinical trial. We recently reported the experimental studies on the importance of timing of HT using global cerebral ischemia in mice (Doshi et al., 2009, 2011). we summarized our findings in this

Transient forebrain ischemia (global cerebral ischemia), which is induced by occlusion of the bilateral common carotid arteries (BCCA) in mice, causes delayed neuronal death in the hippocampus and is known as a model of brain injury following transient cardiac arrest (Kawase et al., 1999). C57BL/6J mice are widely used as a background strain for genetic alterations and have been valuable for investigating the molecular mechanism of delayed neuronal death following transient forebrain ischemia (Yang et al., 1997; Tajiri et al., 2004). We recently demonstrated that acute brain edema, one of the most important disorders following cerebral ischemia, occurred in the forebrain in this C57BL/6J mouse model (Doshi et al., 2009). Forebrain ischemia was induced by BCCA occlusion with clips for 15 min under 1 % halothane anesthesia in air using a face mask. Rectal temperature was monitored using a digital thermometer and maintained at 37±0.5℃ with a heating blanket (normothermia, NT). The control mice underwent a sham operation without BCCA occlusion under halothane

Delayed neuronal death in the hippocampus 7 days after reperfusion was determined by both hematoxylin-eosin (HE) staining and TdT-mediated dUTP-biotin nick end labeling

chapter because our results may be relevant to clinical studies.

**2.2 Evaluation of delayed neuronal death and acute brain edema**

**2.1 Global cerebral ischemia using C57BL/6J mice**

**1. Introduction**

**2. Experimental methods**

anesthesia for 15 min.

**Brain Edema and Delayed Neuronal Death**

Masaru Doshi1 and Yutaka Hirashima2

*1Faculty of Pharmaceutical Sciences, Teikyo University 2Department of Neurosurgery, Imizu City Hospital*

Zausinger, S., Schöller, K., Plesnila, N., & Schmid-Elsaesser, R. (2003). Combination drug therapy and mild hypothermia after transient focal cerebral ischemia in rats. *Stroke*, Vol. 34, No. 9, (September 2003), pp. 2246-51, ISSN 0039-2499

### **Timing of Hypothermia (During or After Global Cerebral Ischemia) Differentially Affects Acute Brain Edema and Delayed Neuronal Death**

Masaru Doshi1 and Yutaka Hirashima2

*1Faculty of Pharmaceutical Sciences, Teikyo University 2Department of Neurosurgery, Imizu City Hospital Japan*

#### **1. Introduction**

36 Advances in the Treatment of Ischemic Stroke

Zausinger, S., Schöller, K., Plesnila, N., & Schmid-Elsaesser, R. (2003). Combination drug

Vol. 34, No. 9, (September 2003), pp. 2246-51, ISSN 0039-2499

therapy and mild hypothermia after transient focal cerebral ischemia in rats. *Stroke*,

Hypothermia (HT) is one of the most effective neuroprotective therapies for brain injury caused by cardiac arrest in humans (Benard et al., 2002), although there is as yet no evidence of such an effect of HT on cerebrovascular diseases from a large-scale clinical trial. We recently reported the experimental studies on the importance of timing of HT using global cerebral ischemia in mice (Doshi et al., 2009, 2011). we summarized our findings in this chapter because our results may be relevant to clinical studies.

#### **2. Experimental methods**

#### **2.1 Global cerebral ischemia using C57BL/6J mice**

Transient forebrain ischemia (global cerebral ischemia), which is induced by occlusion of the bilateral common carotid arteries (BCCA) in mice, causes delayed neuronal death in the hippocampus and is known as a model of brain injury following transient cardiac arrest (Kawase et al., 1999). C57BL/6J mice are widely used as a background strain for genetic alterations and have been valuable for investigating the molecular mechanism of delayed neuronal death following transient forebrain ischemia (Yang et al., 1997; Tajiri et al., 2004). We recently demonstrated that acute brain edema, one of the most important disorders following cerebral ischemia, occurred in the forebrain in this C57BL/6J mouse model (Doshi et al., 2009).

Forebrain ischemia was induced by BCCA occlusion with clips for 15 min under 1 % halothane anesthesia in air using a face mask. Rectal temperature was monitored using a digital thermometer and maintained at 37±0.5℃ with a heating blanket (normothermia, NT). The control mice underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min.

#### **2.2 Evaluation of delayed neuronal death and acute brain edema**

Delayed neuronal death in the hippocampus 7 days after reperfusion was determined by both hematoxylin-eosin (HE) staining and TdT-mediated dUTP-biotin nick end labeling

Timing of Hypothermia (During or After Global Cerebral Ischemia)

solved in NT mice.

NT (ischemia 15 min) 3 / 4 HT (ischemia 15 min) 0 / 5\* HT (ischemia 45 min) 0 / 5\* \*p<0.05 vs NT (Fisher's exact test)

hippocampus of C57BL/6J mice.

unclear.

Differentially Affects Acute Brain Edema and Delayed Neuronal Death 39

delayed neuronal death in the hippocampus under our experimental conditions. As shown in Table 1, there were no histological changes in the hippocampus 7 d after reperfusion in all HT mice, whereas delayed neuronal death occurred in three of the four NT mice. In this model, controlled NT during cerebral ischemia is important for the induction of neuronal death following cerebral ischemia (Ohtaki et al., 2006), supported by data revealing that HT during ischemia protects against neuronal death in the BCCA occlusion C57BL/6J mouse model (Yang et al., 1997). Our data in this study were consistent with previous data. However, the neuroprotective effect of HT after ischemia-reperfusion still remains to be

Mice The number of delayed neuronal death-positive mice / total mice

Table 1. Effect of HT during ischemia on occurrence of delayed neuronal death in

**4. Effect of timing of hypothermia on occurrence of acute brain edema**

Brain edema, defined as an increase in brain water content, is one of the major reperfusion pathologies following cerebral ischemia along with delayed neuronal death and infarction. The increase in brain water content results in serious pathologic situations such as elevation of intracranial pressure and reduction of cerebral blood flow, and subsequently causes cerebral herniation and death (Kempski, 2001). Even now, the treatment options for brain edema are limited to the use of hyperosmotic agents, such as glycerol or mannitol, and surgical decompression. That is, despite the clinical significance of brain edema, the mechanisms of brain water transport and edema formation in ischemic injuries remain

Several studies have shown that HT, which was monitored by measuring rectal temperature, protects against delayed neuronal death and infarction (Maier et al., 1998; Tsuchiya et al., 2002), but few studies have investigated the effects of HT on brain edema in mouse models of cerebral ischemia. Therefore, we investigated the effect of HT on the occurrence of acute brain edema following the global cerebral ischemia in C57BL/6J mice.

We first investigated the effect of HT during global cerebral ischemia on the occurrence of acute brain edema in the C57BL/6J mouse model. In both NT and HT mice, the water content 1 h after reperfusion was significantly higher than that of the control mice, but no significant differences were detected between the NT and HT mice (Fig. 2). This data indicated the ineffectiveness of HT during ischemia against acute brain edema, unlike its effect on neuronal death, suggesting that HT during ischemia may not necessarily be an effective therapy for all reperfusion pathologies following cerebral ischemia. However, we found that the rectal temperature of the HT mice recovers from HT during ischemia to NT mice levels within 1 hr after reperfusion. Therefore, we speculated that the ineffectiveness of

**4.1 Effect of hypothermia during ischemia on occurrence of acute brain edema**

(TUNEL) assay. To evaluate the acute brain edema, the water content in the forebrain 1 hour after reperfusion was determined by the weight differences between wet and dry samples. The percentage of water in the forebrain was calculated as follows: ((wet weight-dry weight) / wet weight)×100.

#### **2.3 Induction of hypothermia**

HT during ischemia was spontaneously induced by removing the heating blanket, and the mice were allowed to recover from anesthesia at room temperature (23-25℃) until 1 hr after reperfusion. In contrast, artificial HT after reperfusion (rHT) was induced by placing the mice on a refrigerant, which was maintained at 18-19℃ until 1 hr after reperfusion in a styrofoam box. In NT mice, rectal temperature was maintained at 37±0.5℃ during ischemia. In HT mice, rectal temperature significantly decreased by 28-30℃ during ischemia. On the other hand, the patterns of the change in rectal temperature after reperfusion in rHT mice were significantly lower than that in HT mice (Fig. 1).

Fig. 1. Changes in rectal temperature after reperfusion in both HT and rHT mice.

Forebrain ischemia was induced by BCCA occlusion for 15 min under 1 % halothane anesthesia in both HT and rHT mice, as described in the Materials and Methods. The control mice (Control) underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min. The rectal temperature was monitored 0, 30, and 60 min after reperfusion. Data are expressed as mean ± SE (control : n=3, I-R : n=7). Statistical analysis was performed by analysis of variance (ANOVA) of repeated measures for the comparison of the changes in rectal temperature between the HT and rHT groups.

#### **3. Effect of hypothermia during ischemia on occurrence of delayed neuronal death**

HT during ischemia has been shown to protect against delayed neuronal death in several animal models of cerebral ischemia including the global cerebral ischemia in C57BL/6J mice (Yang et al., 1997). We confirmed the protective effect of HT during ischemia against delayed neuronal death in the hippocampus under our experimental conditions. As shown in Table 1, there were no histological changes in the hippocampus 7 d after reperfusion in all HT mice, whereas delayed neuronal death occurred in three of the four NT mice. In this model, controlled NT during cerebral ischemia is important for the induction of neuronal death following cerebral ischemia (Ohtaki et al., 2006), supported by data revealing that HT during ischemia protects against neuronal death in the BCCA occlusion C57BL/6J mouse model (Yang et al., 1997). Our data in this study were consistent with previous data. However, the neuroprotective effect of HT after ischemia-reperfusion still remains to be solved in NT mice.

\*p<0.05 vs NT (Fisher's exact test)

38 Advances in the Treatment of Ischemic Stroke

(TUNEL) assay. To evaluate the acute brain edema, the water content in the forebrain 1 hour after reperfusion was determined by the weight differences between wet and dry samples. The percentage of water in the forebrain was calculated as follows: ((wet weight-dry weight)

HT during ischemia was spontaneously induced by removing the heating blanket, and the mice were allowed to recover from anesthesia at room temperature (23-25℃) until 1 hr after reperfusion. In contrast, artificial HT after reperfusion (rHT) was induced by placing the mice on a refrigerant, which was maintained at 18-19℃ until 1 hr after reperfusion in a styrofoam box. In NT mice, rectal temperature was maintained at 37±0.5℃ during ischemia. In HT mice, rectal temperature significantly decreased by 28-30℃ during ischemia. On the other hand, the patterns of the change in rectal temperature after reperfusion in rHT mice

Fig. 1. Changes in rectal temperature after reperfusion in both HT and rHT mice.

Time after reperfusion (min)

0 30 60

of the changes in rectal temperature between the HT and rHT groups.

Forebrain ischemia was induced by BCCA occlusion for 15 min under 1 % halothane anesthesia in both HT and rHT mice, as described in the Materials and Methods. The control mice (Control) underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min. The rectal temperature was monitored 0, 30, and 60 min after reperfusion. Data are expressed as mean ± SE (control : n=3, I-R : n=7). Statistical analysis was performed by analysis of variance (ANOVA) of repeated measures for the comparison

HT, Control

HT, I-R rHT, Control rHT, I-R

**3. Effect of hypothermia during ischemia on occurrence of delayed neuronal**

HT during ischemia has been shown to protect against delayed neuronal death in several animal models of cerebral ischemia including the global cerebral ischemia in C57BL/6J mice (Yang et al., 1997). We confirmed the protective effect of HT during ischemia against

/ wet weight)×100.

**death**

**2.3 Induction of hypothermia**

were significantly lower than that in HT mice (Fig. 1).

36

30

Rectal temperature (

℃)

28

32 34

26

22

24

Table 1. Effect of HT during ischemia on occurrence of delayed neuronal death in hippocampus of C57BL/6J mice.

#### **4. Effect of timing of hypothermia on occurrence of acute brain edema**

Brain edema, defined as an increase in brain water content, is one of the major reperfusion pathologies following cerebral ischemia along with delayed neuronal death and infarction. The increase in brain water content results in serious pathologic situations such as elevation of intracranial pressure and reduction of cerebral blood flow, and subsequently causes cerebral herniation and death (Kempski, 2001). Even now, the treatment options for brain edema are limited to the use of hyperosmotic agents, such as glycerol or mannitol, and surgical decompression. That is, despite the clinical significance of brain edema, the mechanisms of brain water transport and edema formation in ischemic injuries remain unclear.

Several studies have shown that HT, which was monitored by measuring rectal temperature, protects against delayed neuronal death and infarction (Maier et al., 1998; Tsuchiya et al., 2002), but few studies have investigated the effects of HT on brain edema in mouse models of cerebral ischemia. Therefore, we investigated the effect of HT on the occurrence of acute brain edema following the global cerebral ischemia in C57BL/6J mice.

#### **4.1 Effect of hypothermia during ischemia on occurrence of acute brain edema**

We first investigated the effect of HT during global cerebral ischemia on the occurrence of acute brain edema in the C57BL/6J mouse model. In both NT and HT mice, the water content 1 h after reperfusion was significantly higher than that of the control mice, but no significant differences were detected between the NT and HT mice (Fig. 2). This data indicated the ineffectiveness of HT during ischemia against acute brain edema, unlike its effect on neuronal death, suggesting that HT during ischemia may not necessarily be an effective therapy for all reperfusion pathologies following cerebral ischemia. However, we found that the rectal temperature of the HT mice recovers from HT during ischemia to NT mice levels within 1 hr after reperfusion. Therefore, we speculated that the ineffectiveness of

Timing of Hypothermia (During or After Global Cerebral Ischemia)

79.6

79.4

79.2

Water content (%)

79.0

78.8

C57BL/6J mouse model.

(\**p*<0.05 vs HT).

**5. Conclusion**

**6. References**

*Health Sci*, 57, 82-85.

Differentially Affects Acute Brain Edema and Delayed Neuronal Death 41

\*

Control

I-R

Fig. 3. Effect of HT after reperfusion on occurrence of brain edema in the BCCA occlusion

HT rHT

Statistical analysis was performed by two-way ANOVA and the unpaired Student's *t*-test

HT during ischemia protects against delayed neuronal death in the hippocampus. In addition, rHT suppresses aggravation of acute brain edema following global cerebral ischemia in mice. Therefore, these findings indicate that the timing of therapeutic HT differs depending on the pathology following global cerebral ischemia. Further experimental studies should be performed to establish a standard method for the clinical application of therapeutic HT because there are still several controversial issues in the development of

Benard, SA., Gray, TW., Buist, MD., Jones, BM., Silvester, W., Gutteridge, G., & Smith, K.

Doshi, M., Kuwatori, Y., Ishii, Y., Sasahara, M., & Hirashima, Y. (2009). Hypothermia during

Doshi, M., Higuchi, E., & Hirashima, Y. (2011). Hypothermia after reperfusion suppresses

transient forebrain ischemia in mice, *Biol Pharm Bull*, 32, 1957-1961.

(2002). Treatment of comatose survivors of out-of-hospital cardiac arrest with

ischemia protects against neuronal death but not acute brain edema following

aggravation of acute brain edema following transient forebrain ischemia in mice, *J*

cooling techniques and in the determination of optimal duration and temperature.

induced hypothermia, *N Engl J Med*, 346, 557-563.

Forebrain ischemia was induced by BCCA occlusion for 15 min under 1 % halothane anesthesia in both HT and rHT mice. The control mice (Control) underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min. The water content in the forebrain treated with BCCA occlusion for 15 min and 1 hr after reperfusion

(I-R) was measured. Data are expressed as mean ± SE (control : n=3, I-R : n=7).

HT during ischemia against acute brain edema is due to the immediate recovery of rectal temperature after reperfusion at room temperature in the C57BL/6J mouse model.

Fig. 2. Effect of HT during ischemia on occurrence of brain edema in C57BL/6J mice.

Forebrain ischemia was induced by BCCA occlusion for 15 min under 1 % halothane anesthesia in both normothermia (NT) and hypothermia (HT). The control mice (Control) underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min. The water content in the forebrain treated with BCCA occlusion for 15 min and 1 h after reperfusion (I-R) was measured. Data are expressed as the mean ± SE (control : n=3, I-R : n=5). Statistical analysis was performed by two-way ANOVA and an unpaired Student's *t*-test for comparison between control and I-R mice groups (\*p<0.01 vs control).

#### **4.2 Effect of hypothermia after reperfusion on occurrence of acute brain edema**

 We next investigated the effect of rHT on acute brain edema in the C57BL/6J mouse model. The water content 1 hr after reperfusion was significantly higher in both the HT and rHT mice than in the control mice (*p*<0.05). However, the water content 1 hr after reperfusion was significantly lower in the rHT mice than in the HT mice (Fig. 3). Already, we have shown an increase in brain water content during BCCA occlusion in a previous study (Doshi et al., 2009). Therefore, these results indicated that rHT suppresses the additional increase in brain water content after reperfusion, but not the increase in brain water content during BCCA occlusion.

Fig. 3. Effect of HT after reperfusion on occurrence of brain edema in the BCCA occlusion C57BL/6J mouse model.

Forebrain ischemia was induced by BCCA occlusion for 15 min under 1 % halothane anesthesia in both HT and rHT mice. The control mice (Control) underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min. The water content in the forebrain treated with BCCA occlusion for 15 min and 1 hr after reperfusion (I-R) was measured. Data are expressed as mean ± SE (control : n=3, I-R : n=7). Statistical analysis was performed by two-way ANOVA and the unpaired Student's *t*-test (\**p*<0.05 vs HT).

#### **5. Conclusion**

40 Advances in the Treatment of Ischemic Stroke

HT during ischemia against acute brain edema is due to the immediate recovery of rectal

\* \* Control

I-R

temperature after reperfusion at room temperature in the C57BL/6J mouse model.

Fig. 2. Effect of HT during ischemia on occurrence of brain edema in C57BL/6J mice.

NT HT

78.8

78.6

79.0

80.0 79.8 79.6 79.4 79.2

Water content (%)

*t*-test for comparison between control and I-R mice groups (\*p<0.01 vs control).

BCCA occlusion.

**4.2 Effect of hypothermia after reperfusion on occurrence of acute brain edema**

 We next investigated the effect of rHT on acute brain edema in the C57BL/6J mouse model. The water content 1 hr after reperfusion was significantly higher in both the HT and rHT mice than in the control mice (*p*<0.05). However, the water content 1 hr after reperfusion was significantly lower in the rHT mice than in the HT mice (Fig. 3). Already, we have shown an increase in brain water content during BCCA occlusion in a previous study (Doshi et al., 2009). Therefore, these results indicated that rHT suppresses the additional increase in brain water content after reperfusion, but not the increase in brain water content during

Forebrain ischemia was induced by BCCA occlusion for 15 min under 1 % halothane anesthesia in both normothermia (NT) and hypothermia (HT). The control mice (Control) underwent a sham operation without BCCA occlusion under halothane anesthesia for 15 min. The water content in the forebrain treated with BCCA occlusion for 15 min and 1 h after reperfusion (I-R) was measured. Data are expressed as the mean ± SE (control : n=3, I-R : n=5). Statistical analysis was performed by two-way ANOVA and an unpaired Student's HT during ischemia protects against delayed neuronal death in the hippocampus. In addition, rHT suppresses aggravation of acute brain edema following global cerebral ischemia in mice. Therefore, these findings indicate that the timing of therapeutic HT differs depending on the pathology following global cerebral ischemia. Further experimental studies should be performed to establish a standard method for the clinical application of therapeutic HT because there are still several controversial issues in the development of cooling techniques and in the determination of optimal duration and temperature.

#### **6. References**

**4**

*Japan*

**Molecular Mechanisms Underlying**

**Hypothermia in Cerebral Ischemia**

Cerebral ischemia is a serious dynamic event in the brain involving heterogeneous cell types. Neuroprotective agents represent a potential approach for the treatment of acute stroke. Presently, recombinant tissue plasminogen activator (rtPA) is the only drug that is approved for the management of acute ischaemic stroke, except for the antioxidant Edaravone in Japan (Yoshida et al., 2006). Although stroke patients can receive rtPA therapy within the initial 3 h therapeutic window, there is an increased risk of intracranial haemorrhage, disruption of the blood brain barrier, seizures, or the progression of neuronal damage (Laloux, 2001). Thus, there is a continued need to explore novel neuroprotective strategies for the management of ischemic stroke. A large number of therapeutic agents have been tested, including N-methyl-D-aspartate receptor antagonists, calcium channel blockers, and antioxidants, for the management of stroke, but none has provided significant

Therapeutic hypothermia lowers a patient's body temperature in order to reduce the risk of the ischemic injury to the brain following a period of insufficient blood flow (Lampe and Becker, 2011; Yenari and Hemmen, 2010). The normal human adult body temperature is between 34.4–37.8°C and is maintained at a constant level through homeostasis or thermoregulation. Therapeutic hypothermia is defined as the artificial maintenance of the body temperature at <35°C and is subdivided into 4 different categories: mild (32–35°C), moderate (28–32°C), severe (20–28°C), and profound (<20°C). Nowadays, mild-to-moderate hypothermia (31–33°C) is usually applied for neuroprotection. The practical usage of hypothermia for clinical purposes was begun by the ancient Egyptians, Greeks, and Romans (Polderman, 2004). Ancient people observed the clinical usefulness of hypothermia for accidents and applied it to various diseases/symptoms. Modern clinical interest in hypothermia began in the 1930s with the description of the successful rescue of a drowned person with hypothermia after a prolonged period of asphyxia. After the first scientific report in 1945, which described the clinical application of hypothermia to patients with a severe head injury, hypothermia was subsequently applied to intracerebral aneurysm

neuroprotection in clinical trials (Green et al., 2003; Kidwell et al., 2001).

surgery and cerebral protection during complete circulatory arrest.

**1. Introduction**

**the Neuroprotective Effect of**

Yasushi Shintani and Yasuko Terao1

*Takeda Pharmaceutical Company Ltd.,*

*Biology Research Laboratories 1CNS Drug Discovery Unit,*

### **Molecular Mechanisms Underlying the Neuroprotective Effect of Hypothermia in Cerebral Ischemia**

Yasushi Shintani and Yasuko Terao1 *Biology Research Laboratories 1CNS Drug Discovery Unit, Takeda Pharmaceutical Company Ltd., Japan*

#### **1. Introduction**

42 Advances in the Treatment of Ischemic Stroke

Kawase, M., Murakami, K., Fujimura, M., Morita-Fujimura, Y., Gasche, Y., Kondo, T., Scott,

Maier, CM., Ahern, K., Cheng, ML., Lee, JE., Yenari, MA., & Steinberg, GK. (1998). Optimal

Ohtaki, H., Nakamachi, T., Dohi, K., Yofu, S., Hodoyama, K., Matsunaga, M., Aruga, T., &

Tajiri, S., Oyadomari, S., Yano, S., Morioka, M., Gotoh, T., Hamada, J-I., Ushio, Y., & Mori,

reticulum stress pathway involving CHOP, *Cell Death Differ*, 11, 403-415. Tsuchiya, D., Hong, S., Suh, SW., Kayama, T., Panter, SS., & Weinstein, PR. (2002). Mild

transient global ischemia in mice, *J Cereb Blood Flow Metab*, 22, 1231-1238. Yang, G., Kitagawa, K., Matsushita, K., Mabuchi, T., Yagita, Y., Yanagihara, T., &

Kempski, O. (2001). Cerebral edema, *Seminars in Nephrology*, 21, 303-307.

1962-1968.

249-253.

ischemia, *Stroke*, 29, 2171-2180.

RW., & Chan, PH. (1999). Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency, *Stroke*, 30,

depth and duration of mild hypothermia in a focal model of transient cerebral

Shioda, S. (2006). Controlled normothermia during ischemia is important for the induction of neuronal cell death after global ischemia in mouse, *Acta Neurochir,* 96,

M. (2004). Ischemia-induced neuronal cell death is mediated by the endoplasmic

hypothermia reduces zinc translocation, neuronal cell death, and mortality after

Matsumoto, M. (1997). C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: selective neuronal death in the murine transient forebrain ischemia, *Brain Res*, 752, 209-218.

Cerebral ischemia is a serious dynamic event in the brain involving heterogeneous cell types. Neuroprotective agents represent a potential approach for the treatment of acute stroke. Presently, recombinant tissue plasminogen activator (rtPA) is the only drug that is approved for the management of acute ischaemic stroke, except for the antioxidant Edaravone in Japan (Yoshida et al., 2006). Although stroke patients can receive rtPA therapy within the initial 3 h therapeutic window, there is an increased risk of intracranial haemorrhage, disruption of the blood brain barrier, seizures, or the progression of neuronal damage (Laloux, 2001). Thus, there is a continued need to explore novel neuroprotective strategies for the management of ischemic stroke. A large number of therapeutic agents have been tested, including N-methyl-D-aspartate receptor antagonists, calcium channel blockers, and antioxidants, for the management of stroke, but none has provided significant neuroprotection in clinical trials (Green et al., 2003; Kidwell et al., 2001).

Therapeutic hypothermia lowers a patient's body temperature in order to reduce the risk of the ischemic injury to the brain following a period of insufficient blood flow (Lampe and Becker, 2011; Yenari and Hemmen, 2010). The normal human adult body temperature is between 34.4–37.8°C and is maintained at a constant level through homeostasis or thermoregulation. Therapeutic hypothermia is defined as the artificial maintenance of the body temperature at <35°C and is subdivided into 4 different categories: mild (32–35°C), moderate (28–32°C), severe (20–28°C), and profound (<20°C). Nowadays, mild-to-moderate hypothermia (31–33°C) is usually applied for neuroprotection. The practical usage of hypothermia for clinical purposes was begun by the ancient Egyptians, Greeks, and Romans (Polderman, 2004). Ancient people observed the clinical usefulness of hypothermia for accidents and applied it to various diseases/symptoms. Modern clinical interest in hypothermia began in the 1930s with the description of the successful rescue of a drowned person with hypothermia after a prolonged period of asphyxia. After the first scientific report in 1945, which described the clinical application of hypothermia to patients with a severe head injury, hypothermia was subsequently applied to intracerebral aneurysm surgery and cerebral protection during complete circulatory arrest.

Molecular Mechanisms Underlying the Neuroprotective Effect of Hypothermia in Cerebral Ischemia 45

ameliorated by hypothermia, including the reduction of oxygen radical production, with the subsequent reduction in peroxidase damage to lipids, proteins, and DNA, thereby supporting the behavioral recovery of patients. Hypothermia also decreases microglial activation, ischemic depolarization, cerebral metabolic demand for oxygen, and the release of glycerin and excitatory amino acids. We and others have demonstrated that inflammation potentiates cerebral ischemic injury and that hypothermia can reduce inflammation by suppressing the infiltration of neutrophils into ischemic regions (Ohta et al., 2007; Shintani et al., 2011; Terao et al., 2009; Zheng and Yonari, 2004). Furthermore, the inhibition of reactive oxygen species production by leukocytes and microglia in the ischemia brain (Kil et al., 1996), NF-κB activation (Han et al., 2003), neutrophil infiltration (Wang et al., 2002), and cytochrome c release (Yenari et al., 2002) has been also reported in hypothermia-treated

Recently, Lin et al., (2011) reported that whole-body hypothermia broadens the therapeutic window of intranasally administered recombinant human insulin-like growth factor 1 (IGF-1) in a neonatal rat cerebral hypoxia–ischemia model. They ligated the right common carotid artery of postnatal day 7 rat pups, followed by 8% oxygen inhalation for 2 h. After the hypoxia-ischemia treatment, the pups were divided into 2 groups and maintained under different temperatures, room temperature (24.5 ± 0.2°C) and a cool environment (21.5 ± 0.3°C), for 2 or 4 h before being returned to room temperature. IGF-1 was administered intranasally at 1 h intervals starting at 0, 2, or 4 h after hypothermia. Although the administration of hypothermia or IGF-1 alone at 2 h after hypoxia-ischemia treatment did not provide neuroprotection, the combined treatment of hypothermia and IGF-1 significantly protected the neonatal rat brain from hypoxia-ischemia injury. Hypothermia extended the therapeutic window of IGF-1 to 6 h after hypoxia-ischemia. It was observed that the combination therapy decreased the infiltration of polymorphonuclear leukocytes, the activation of microglia/macrophages, and the attenuation of nuclear factor kappa-B (NF-κB) activation. These findings broaden the potential application of hypothermia, i.e., not only for its neuroprotective effects but also for its synergistic effects by the combined use

of hypothermia with already existing therapeutic drugs.

**3.1 Proteins influenced by hypothermia**

**3. Molecular mechanisms of hypothermia-induced neuroprotection**

The detailed molecular mechanisms underlying the neuroprotection induced by hypothermia against ischemia have been approached by degrees. In most cases, molecules, whose expression is known to be affected by ischemia, have been identified and the effects of hypothermia on the molecule have been explored retrospectively. Table 1 lists the molecules whose expression is affected by hypothermia during ischemia and/or reperfusion. The majority of these proteins are apoptosis, inflammation-related, and signalling molecules, such as kinases and transcription factors. For example, apoptosisrelated molecules, such as B-cell lymphoma 2 (Bcl2), Bcl-associated X protein (Bax), caspases, calpains, cytochrome c, and Fas/Fas ligand (FasL), are up- or down-regulated by hypothermia in accordance with its neuroprotective effect, thereby preventing neuronal cell death. Inflammatory molecules, such as tumor necrosis factor alpha (TNF), interleukin (IL)-1, IL-6, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-3

ischemic rat brains.

In the past few decades, the neuroprotective effects of hypothermia have been well established in experimental animals (Kawai et al., 2000; Miyazawa et al., 2003; Yanamoto et al., 2001) and in patients with cardiac arrest (THCASG, 2002 and Bernard et al., 2002). The initiation of moderate hypothermia within a few hours of severe ischemia can reduce the subsequent neuronal death and profoundly improve behavioural recovery. Although hypothermia is the only clinical intervention that appears to be neuroprotective after the initial injury, its key mechanisms have not been clarified. In other words, the neuroprotective effects of hypothermia provide many insights into the pathology of stroke, and thus may reveal clues for novel drug targets.

#### **2. Neuroprotection against cerebral ischemia by hypothermia**

Ischemia and cerebral hemorrhage are the two main causes of strokes. Ischemia accounts for ~85% of all reported incidents of stroke, and occurs when a thrombus or embolus blocks cerebral blood flow, resulting in cerebral ischemia and consequently neuronal damage and cell death. Conversely, hemorrhage occurs following the rupture of any blood vessel in the brain, resulting in rapid cerebral damage, and accounts for the remaining 15% of stroke cases. In each situation, the interruption of blood flow to the brain results in the reduced supply of oxygen and nutrients to the neurons. At the molecular level, the pathophysiology of ischemia is complicated and involves multiple sequential steps: progressive neural injury beginning with the activation of glutamate receptors, followed by the production and release of proinflammatory cytokines, nitric oxide (NO), free oxygen radicals, and proteases. As a result, neurons in an ischemic brain suffer irreversible and fatal damage. From the electrophysiological viewpoint, neurons depolarize massively giving rise to anoxic depolarization (Bureš et al., 1974) or to peri-infarct depolarizations (Gyngell et al., 1995). They are both characterized by swelling of neurons, massive influx of Na+ and Ca2+ into neurons, massive release of K+ into the interstitial space, release of glutamate, acidification of the tissue (Somjen, 2001; Dreier, 2011; Balestrino, 1995).

The lack of blood supply results in two identifiable areas, namely the core and the penumbra. The core is a neuronal dead area that is not therapeutically accessible, whereas the penumbra is a still salvageable zone (Bandera et al., 2006). As a consequence of the reduced blood supply inside the core, adenosine triphosphate (ATP) levels are reduced, leading to the depression of cellular metabolism. Energy loss results in impaired ion homeostasis, which leads to rapid depolarization and a large influx of calcium and potassium ions. The increased levels of intracellular calcium induces the activation of excitotoxic glutamatergic transmission, NO synthase, caspase, xanthine oxidase, and the release of reactive oxygen species. Glutamate release activates phospholipases, phospholipid hydrolysis, and the release of arachidonic acid. The generation of free radicals and lipid peroxidation and the activation of immediate early genes, such as *c-fos, c-jun*, and the inflammatory cascade, lead to progressive ischemic damage, resulting in necrotic as well as apoptotic cell death. Conversely, the penumbra represents viable tissue surrounding the core and receives a trivial amount of blood from collateral arteries; therefore, the penumbra is the target for drug intervention and has the potential for recovery.

Precisely controlled mild hypothermia has been proven to have neuroprotective properties and reduces the risk of the detrimental effects that often occur during profound hypothermia. Large numbers of the phenomena observed in ischemic brains can be ameliorated by hypothermia, including the reduction of oxygen radical production, with the subsequent reduction in peroxidase damage to lipids, proteins, and DNA, thereby supporting the behavioral recovery of patients. Hypothermia also decreases microglial activation, ischemic depolarization, cerebral metabolic demand for oxygen, and the release of glycerin and excitatory amino acids. We and others have demonstrated that inflammation potentiates cerebral ischemic injury and that hypothermia can reduce inflammation by suppressing the infiltration of neutrophils into ischemic regions (Ohta et al., 2007; Shintani et al., 2011; Terao et al., 2009; Zheng and Yonari, 2004). Furthermore, the inhibition of reactive oxygen species production by leukocytes and microglia in the ischemia brain (Kil et al., 1996), NF-κB activation (Han et al., 2003), neutrophil infiltration (Wang et al., 2002), and cytochrome c release (Yenari et al., 2002) has been also reported in hypothermia-treated ischemic rat brains.

Recently, Lin et al., (2011) reported that whole-body hypothermia broadens the therapeutic window of intranasally administered recombinant human insulin-like growth factor 1 (IGF-1) in a neonatal rat cerebral hypoxia–ischemia model. They ligated the right common carotid artery of postnatal day 7 rat pups, followed by 8% oxygen inhalation for 2 h. After the hypoxia-ischemia treatment, the pups were divided into 2 groups and maintained under different temperatures, room temperature (24.5 ± 0.2°C) and a cool environment (21.5 ± 0.3°C), for 2 or 4 h before being returned to room temperature. IGF-1 was administered intranasally at 1 h intervals starting at 0, 2, or 4 h after hypothermia. Although the administration of hypothermia or IGF-1 alone at 2 h after hypoxia-ischemia treatment did not provide neuroprotection, the combined treatment of hypothermia and IGF-1 significantly protected the neonatal rat brain from hypoxia-ischemia injury. Hypothermia extended the therapeutic window of IGF-1 to 6 h after hypoxia-ischemia. It was observed that the combination therapy decreased the infiltration of polymorphonuclear leukocytes, the activation of microglia/macrophages, and the attenuation of nuclear factor kappa-B (NF-κB) activation. These findings broaden the potential application of hypothermia, i.e., not only for its neuroprotective effects but also for its synergistic effects by the combined use of hypothermia with already existing therapeutic drugs.

#### **3. Molecular mechanisms of hypothermia-induced neuroprotection**

#### **3.1 Proteins influenced by hypothermia**

44 Advances in the Treatment of Ischemic Stroke

In the past few decades, the neuroprotective effects of hypothermia have been well established in experimental animals (Kawai et al., 2000; Miyazawa et al., 2003; Yanamoto et al., 2001) and in patients with cardiac arrest (THCASG, 2002 and Bernard et al., 2002). The initiation of moderate hypothermia within a few hours of severe ischemia can reduce the subsequent neuronal death and profoundly improve behavioural recovery. Although hypothermia is the only clinical intervention that appears to be neuroprotective after the initial injury, its key mechanisms have not been clarified. In other words, the neuroprotective effects of hypothermia provide many insights into the pathology of stroke,

Ischemia and cerebral hemorrhage are the two main causes of strokes. Ischemia accounts for ~85% of all reported incidents of stroke, and occurs when a thrombus or embolus blocks cerebral blood flow, resulting in cerebral ischemia and consequently neuronal damage and cell death. Conversely, hemorrhage occurs following the rupture of any blood vessel in the brain, resulting in rapid cerebral damage, and accounts for the remaining 15% of stroke cases. In each situation, the interruption of blood flow to the brain results in the reduced supply of oxygen and nutrients to the neurons. At the molecular level, the pathophysiology of ischemia is complicated and involves multiple sequential steps: progressive neural injury beginning with the activation of glutamate receptors, followed by the production and release of proinflammatory cytokines, nitric oxide (NO), free oxygen radicals, and proteases. As a result, neurons in an ischemic brain suffer irreversible and fatal damage. From the electrophysiological viewpoint, neurons depolarize massively giving rise to anoxic depolarization (Bureš et al., 1974) or to peri-infarct depolarizations (Gyngell et al., 1995). They are both characterized by swelling of neurons, massive influx of Na+ and Ca2+ into neurons, massive release of K+ into the interstitial space, release of glutamate, acidification

The lack of blood supply results in two identifiable areas, namely the core and the penumbra. The core is a neuronal dead area that is not therapeutically accessible, whereas the penumbra is a still salvageable zone (Bandera et al., 2006). As a consequence of the reduced blood supply inside the core, adenosine triphosphate (ATP) levels are reduced, leading to the depression of cellular metabolism. Energy loss results in impaired ion homeostasis, which leads to rapid depolarization and a large influx of calcium and potassium ions. The increased levels of intracellular calcium induces the activation of excitotoxic glutamatergic transmission, NO synthase, caspase, xanthine oxidase, and the release of reactive oxygen species. Glutamate release activates phospholipases, phospholipid hydrolysis, and the release of arachidonic acid. The generation of free radicals and lipid peroxidation and the activation of immediate early genes, such as *c-fos, c-jun*, and the inflammatory cascade, lead to progressive ischemic damage, resulting in necrotic as well as apoptotic cell death. Conversely, the penumbra represents viable tissue surrounding the core and receives a trivial amount of blood from collateral arteries; therefore, the penumbra

Precisely controlled mild hypothermia has been proven to have neuroprotective properties and reduces the risk of the detrimental effects that often occur during profound hypothermia. Large numbers of the phenomena observed in ischemic brains can be

and thus may reveal clues for novel drug targets.

of the tissue (Somjen, 2001; Dreier, 2011; Balestrino, 1995).

is the target for drug intervention and has the potential for recovery.

**2. Neuroprotection against cerebral ischemia by hypothermia**

The detailed molecular mechanisms underlying the neuroprotection induced by hypothermia against ischemia have been approached by degrees. In most cases, molecules, whose expression is known to be affected by ischemia, have been identified and the effects of hypothermia on the molecule have been explored retrospectively. Table 1 lists the molecules whose expression is affected by hypothermia during ischemia and/or reperfusion. The majority of these proteins are apoptosis, inflammation-related, and signalling molecules, such as kinases and transcription factors. For example, apoptosisrelated molecules, such as B-cell lymphoma 2 (Bcl2), Bcl-associated X protein (Bax), caspases, calpains, cytochrome c, and Fas/Fas ligand (FasL), are up- or down-regulated by hypothermia in accordance with its neuroprotective effect, thereby preventing neuronal cell death. Inflammatory molecules, such as tumor necrosis factor alpha (TNF), interleukin (IL)-1, IL-6, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-3

Molecular Mechanisms Underlying the Neuroprotective Effect of Hypothermia in Cerebral Ischemia 47

etc.

Rat global cerebral ischemia (20 min); the mortality in rats was evaluated at 72 h and 168 h reperfusion.

In vitro cell culture; 5 h OGD at 37°C; human umbilical vein endothelial cells were used in this study.

In vitro cell culture; 5 h OGD at 37°C; human umbilical vein endothelial cells were used in this study.

2h MCAO; soluble FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased.

no change

1 h

etc.

hypotention

reperfusion

ischemia Increase Decrease 2h MCAO Liu et al., 2008

Increase Decrease 10 min MCAO and

Increase Decrease 3 h MCAO and 24 h

Eberspacher et al., 2005; Jieyong et al., 2006; Zhao et al., 2004; Yenari et al., 2002; Zhang et al., 2001

Zhang et al., 2010

Yang et al., 2009

Yang et al., 2009

Eberspacher et al, 2003, 2005

Liu et al., 2008; Phanithi et al., 2000

Tomimatsu et al., 2001

Fukuda et al., 2001; Pabello et al., 2005; Phanithi et al., 2000; Tomimatsu et al., 2001

Zhao

Liebetrau et al., 2004

et al., 2004, 2005

Molecule Hypothermia Expression Ischemia/observation Reference Ischemia Hypothermia

N.D. Increase

ischemia Increase Decrease Bcl‐2, p53, and Mdm‐2;

ischemia Increase Decrease Hypoxia-ischemia for

Bcl-2 30-34°C Down Increase 1 h hypoxia-ischemia

Bcl-2

31-32°C hypothermia for 60 min after MCAO

Bax 34°C during

Fas, FasL 33°C during

Akt 34°C after

Caspase 8 33°C during

33°C for 10 min before ischemia and maintained 3 h after reperfusion

32°C for 10 min before and during reperfusion

Caspase-3/9

Calpain

Bcl2 33°C culture Increase Decrease

Bax 33°C culture Decrease Increase

ischemia etc. Increase Decrease

Caspase-3 30-34°C Increase Decrease 1 h hypoxia-ischemia

et al., 2002

Ceulemans et al., 2011

et al., 2002

Meybohm et al., 2010

Choi et al., 2011; Inamasu et al., 2001; Koda et al., 2010

Han et al., 2003

Yenari et al., 2006

Webster et al., 2009

Rat endothelin (ET)-1 induced transient focal cerebral ischemia; hypothermia reduced astrogliosis at 1 and 3 d after stroke onset.

In vitro cell culture; 30 h oxygen– glucose deprivation

Increase Decrease Hypoxia-ischemia Fukui et al., 2006

Pigs were subjected to cardiac arrest following temporary coronary

occlusion.support.

2 h MCAO; NF-kB was translocated from cytoplasm to

Phosphorylation of IkB- was suppressed by hypothermia.

nucleus by hypothermia.

after MCAO Increase Decrease 2 h MCAO Han et al., 2003

2 h MCAO; extracellular signal-regulated kinase-1/2 (ERK-1/2) activation, and induction of leukocyte infiltration and inflammatory reaction by ischemia were inhibited by hypothermia.

(OGD)

MCAO Increase Decrease 2 h MCAO Terao et al., 2009

MCAO Increase Decrease 2 h MCAO Ohta et al., 2007

artery

Molecule Hypothermia Expression Ischemia/observation Reference Ischemia Hypothermia

IL-1 <sup>33</sup>°C culture N.D. Increase In vitro cell culture Yanagawa

IL-6 33°C culture N.D. Decrease In vitro cell culture Yanagawa

Increase Decrease

IL-1, TNF

IL-18

IL-1, IL-6, IL-10, TNF, ICAM-1

Intercellular adhesion molecule-1 (ICAM-1)

NF-B's inhibitory protein (IkB-)

IkB-, NOS,

2 h

hypothermia, started 20 min after ET-1 injection

32°C for 24 h after hypoxiaischemia

MIP-3 <sup>34</sup>°C after

MCP-1 34 °C after

NF-B 33°C for 2 h

33°C for 2 h

TNF 30-34°C Increase Decrease

IL-1TNF 33°C culture Increase Decrease

33°C for 24 h Increase Decrease

33°C etc. Increase Decrease

after MCAO No change No change

Molecular Mechanisms Underlying the Neuroprotective Effect of Hypothermia in Cerebral Ischemia 49

forebrain ischemia

Phosphorylation of b-catenin was increased and degraded by ischemia. Hypothermia did not inhibit the phosphorylation, but it blocked degradation in the ischemic penumbra.

Alternation of the assembly of the GluR6- PSD95-MLK3 signaling

module

ischemia N.D. Increase 2 h MCAO Lee et al., 2005

24 h Decrease Increase Permanent MCAO Xie et al. 2007

MCAO Decrease Increase 1 h MCAO Shimohata

MCAO Increase Decrease 1 h MCAO Shimohata

30 min ischemia followed by 60 min reperfusion;

hypothermia inhibited translocation of CaM kinase II and

Baumann et al., 2009

Zhang et al., 2008

Hu et al., 2008

et al., 2008

et al., 2007

Harada et al., 2002

et al., 2007

et al., 2007

Lee et al., 2005; Nagel et al., 2008

stroke in normothermia Zhang et al., 2008

Molecule Hypothermia Expression Ischemia/observation Reference Ischemia Hypothermia

<sup>32</sup>° C Decrease Increase Transient 20 min

Increase Decrease

Galanin 33°C Decrease Increase 60 min transient MCAO Theodorsson

COX-2 33-34°C Increase Decrease Global ischemic insult Xiang

N.D. N.D.

<sup>9</sup>33°C etc. Increase Decrease 90 min MCAO

GSK3 33°C N.D. Decrease Global cerebral ischemia Kelly et al., 2005

N.D. N.D. Dephosphorylated after

Agrin, SPARC (BM-40, osteonectin)

> Moderate hypothermia (30°C) blocked degradation of total GSK

3

32°C hypothermia for 10 min before ischemia and maintained for 3 h after ischemia

GSK 3

MLK3, MKK4/7, JNK3, c-Jun, FasL

MMP-2, MMP-

TIMP-2 33°C during

BDNF 33-34°C for

PKC 30°C during

PKC 30°C during

30 min of ischemia followed by 60 min of reperfusion

2 hr MCAO; cytochrome c release and AIF translocation from mitochondria to nuclei were stimulated by hypothermia.

50 min MCAO; cytochrome c release and AIF translocation from mitochondria to nuclei were stimulated by hypothermia.

1 h MCAO; Akt activity was inhibited by ischemia and stimulated by hypothermia.

1 h MCAO; c-Jun expression was not affected by hypothermia.

Ischemia-induced phosphorylation of AMPK was inhibited by

hypothermia.

Increase Increase 20 min MCAO Liu et al., 2010

Rat global cerebral ischemia (20 min); the mortality in rats was evaluated at 72 h and 168 h reperfusion.

2 h MCAO; DNA damage-dependent signaling events, including NAD depletion, p53 activation, and mitochondrial translocation of PUMA

and NOXA

culture Increase Decrease In vitro cell culture Tanaka et al., 2010

32°C N.T. Decrease Koda et al., 2010

Hsp70 Increase Increase 2 h MCAO Terao et al., 2009

Zhao et al., 2007

Zhu et al., 2006

Zhao et al., 2005

Akaji et al., 2003

Li et al., 2011

Zhang et al., 2010

Ji et al., 2007

Molecule Hypothermia Expression Ischemia/observation Reference Ischemia Hypothermia

N.D. N.D.

N.D. N.D.

Increase Decrease

N.D. N.D.

Cytochrome c,

Cytochrome c,

30 or 33°C for 2 h during and/or after MCAO

AIF 34 or 36°C Increase Decrease

30°C for 10 min before ischemia and maintained for 1 h after ischemia

Cold room (1°C) during MCAO

Moderate (30 ± 2°C) hypothermia for 2 h

p53 31-32°C N.D. Decrease

NOXA 33°C Increase Decrease

28–32°C

AIF

Akt

c-Fos, AP-1

cold-inducible RNA-binding protein (CIRP)

p53, PUMA,

hypoxiainducible factor-1 (HIF-1)

high-mobility group box 1 (HMGB1)

AMPK (phosphorylation)

Molecular Mechanisms Underlying the Neuroprotective Effect of Hypothermia in Cerebral Ischemia 51

during ischemia. And now it has become clear that the neuroinflammatory response could be detrimental and that even peripheral immune responses can be regulated by the brain

Another type of change evoked by hypothermia in ischemic brains is protein modification. For instance, Li et al. (2011) reported that hypothermia reduced the activation of 5′ adenosine monophosphate-activated protein kinase (AMPK), a ubiquitously distributed kinase, by the dephosphorylation of its regulatory residues in ischemic mice. They showed that hypothermic neuroprotection was ameliorated by compound C, an AMPK inhibitor, and that genetic deletion of one of the catalytic isoforms of AMPK completely reversed the effects of hypothermia on stroke outcome after acute and chronic survival. Their study provides evidence that hypothermia exerts its protective effects, in part, by inhibiting AMPK activation, at least in experimental focal stroke. AMPK is known to participate in an energy sensing cascade and to serve as a master regulator of metabolism in response to ATP depletion. Recently, additional roles of AMPK in a variety of other cellular processes have been revealed, in the cytoplasm and nucleus, as a controllor of cell polarity and a transcriptional regulator. A more interesting function of AMPK in signaling pathways is its role as a responder to cellular stress and damage, and the relevance of AMPK signaling in

various diseases is becoming a hot topic in the investigation of ischemic physiology.