Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: Rescue effect of mild hypothermia

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24 h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.     

Protection against Recurrent Stroke with Resveratrol: Endothelial Protection

Despite increased risk of a recurrent stroke following a minor stroke, information is minimal regarding the interaction between injurious mild cerebral ischemic episodes and the possible treatments which might be effective. The aim of the current study was to investigate recurrent ischemic stroke and whether resveratrol, a nutritive polyphenol with promising cardio- and neuro- protective properties, could ameliorate the associated brain damage. Experiments in adult rats demonstrated that a mild ischemic stroke followed by a second mild cerebral ischemia exacerbated brain damage, and, daily oral resveratrol treatment after the first ischemic insult reduced ischemic cell death with the recurrent insult (P<0.002). Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. The protection observed with resveratrol treatment could not be explained by systemic effects of resveratrol treatment including effects either on blood pressure or body temperature measured telemetrically. Investigation of resveratrol effects on the blood-brain barrier in vivo demonstrated that resveratrol treatment reduced blood-brain barrier disruption and edema following recurrent stroke without affecting regional cerebral blood flow. Investigation of the mechanism in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against an in vitro ‘ischemia’ resulting in improved viability against oxygen and glucose deprivation (39.6±6.6% and 81.3±9.5% in vehicle and resveratrol treated cells, respectively). An inhibition of nitric oxide synthesis did not prevent the improved cell viability following oxygen glucose deprivation but SIRT-1 inhibition with sirtinol partially blocked the protection (P<0.001) suggesting endothelial protection is to some extent SIRT-1 dependent. Collectively, the results support that oral resveratrol treatment provides a low risk strategy to protect the brain from enhanced damage produced by recurrent stroke which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.     

Troponin elevation in acute ischemic stroke (TRELAS) - protocol of a prospective observational trial

Background  

Levels of the cardiac muscle regulatory protein troponin T (cTnT) are frequently elevated in patients with acute ischemic stroke and elevated cTnT predicts poor outcome and mortality. The pathomechanism of troponin release may relate to co-morbid coronary artery disease and myocardial ischemia or, alternatively, to neurogenic cardiac damage due to autonomic activation after acute ischemic stroke. Therefore, there is uncertainty about how acute ischemic stroke patients with increased cTnT levels should be managed regarding diagnostic and therapeutic workup.     

Increased neural damage to global hemispheric hypoxic ischemia (GHHI) in febrile but not nonfebrile lipopolysaccharide Escherichia coli injected rats

Experiments were conducted to compare the impact of febrile versus nonfebrile lipopolysaccharide (LPS) induced bacterial infection at the time of global hemispheric hypoxic ischemia (GHHI) on the neural damage evoked by the GHHI insult. In the first study acute intraperitoneal (i.p.) sterile saline (SS) or LPS Escherichia coli (60 microg/kg) was given to groups of male, conscious Long Evans rats, and core (colonic, Tc) temperatures were monitored over 6 h postinjection. Peak febrile response occurred approximately 5 h after the LPS E. coli was injected. Upon sacrifice 7 days later, no hemispheric or regional brain damage occurred in the saline or LPS-injected groups of this first study. In the second study, GHHI was applied (ligation of right common carotid artery + 35 min of 12% O2) in groups of anesthetized, male Long Evans rats previously given an acute i.p. injection of sterile saline or 60 microg/kg LPS E. coli 5 h earlier. Temperatures (Tc) were monitored before, during, and 1.5 and 24 h following GHHI. The LPS-injected group was subdivided into a febrile (Tc > 38 degrees C before and (or) after GHHI) and nonfebrile (Tc < 38 degrees C before and after GHHI) subgroups. A significant correlation was found between the peak temperature rise from preinjection control values following drug administration of either saline or LPS E. coli and the resultant hemispheric damage caused by GHHI. Moreover, upon sacrifice 7 days later ipsilateral hemispheric and regional (i.e., hippocampal, thalamic) damage to GHHI of the febrile LPS E. coli group was significantly increased from respective hemispheric, hippocampal, and thalamic damage of the saline and nonfebrile, LPS groups given the same ischemic insult. Results suggest that the heightened Tc of a LPS infection at the time of global ischemia exacerbated the neural damage of GHHI, a finding similar to that reported with heightened core temperatures induced by external heating.     

Quantification of volume of tissue damage in stroke using T2- weighed MRI images

We have developed a quantitative technique for scoring of the severity of ischemic damage of the brain using quantitative data of the T2-weighted MRI images of brain in stroke. The principle of the method is the assumption that T2 signal increases proportionally to the severity of ischemic damage of cerebral tissue up to the level equal to intraventricular liquor signal in the case of postinfarction cystic degeneration. Depicting the mean T2-signal from the intraventricular liquor region as Iliq, the signal from ischemic brain area as Iinsult, and from the intact brain as Inorm, obviously, the volume quota of damaged tissue in the total volume of the stroke region is represented by the ratio (Iinsult - Inorm)/(Iliq - Inorm). The total volume of damaged tissue (VDT, cub.cm) in the stroke region is then the following sum taken over all slices i, where the stroke damage can be: VDT = sigma i d.Si.[(Iinsult - Inorm)/(Iliq - Inorm)]i, where d is the slice thickness, Si--area of the ischemic region in the slice i. The quota of damaged tissue in the physical volume of the stroke region is henceforth the following ratio: Q = [sigma i d.Si.[(Iinsult - Inorm)/(Iliq - Inorm)]i]/[sigma i d.Si]. The technique was applied in retrospective analysis of routine MRI studies in 15 patients referred because of acute ischemic stroke. The studies were performed using low-field MRI tomograph Magnetom-Open (Siemens Medical) with field strength 0.22 T. In patients studied during the first day after occurrence of ischemic insult with the minimal degree of acute neurologic deficit, who later have demonstrated clinically full recovery, the VDT was below 20 cm3, and Q was below 10%. In cases with VDT > 25 cm3 and Q > 20% the full regress was not observed in any patient. Henceforth, the quantification of cerebral damage in stroke using quantitative indices based on measurement of T2-parameters over ischemic and intact zones of the brain are of independent prognostic clinical value and improve clinical usefulness of the MRI in ischemic brain stroke.     

Perspectives on reperfusion-induced damage in rodent models of experimental focal ischemia and role of gamma-protein kinase C

Ischemic stroke represents the leading cause of death and disability among elderly people. Most stroke survivors are left with lifelong disability. With the exception of tissue-type plasminogen activator (t-PA), no effective therapy exists for the management of acute stroke. Understanding the role of various extrinsic and intrinsic pathogenic factors of ischemic damage represents a prime objective of ongoing stroke research. An important variable affecting stroke outcome is the presence or absence of reperfusion (recanalization of the occluded vessel) following an ischemic event. It appears that early reperfusion after a stroke is beneficial and capable of reversing the majority of ischemic dysfunctions. However, in some instances, late reperfusion may contrarily trigger deleterious processes and lead to more ischemic damage. Examples of ischemia/reperfusion damage using an experimental model of focal ischemia in rodents are provided, along with evidence that the brain-enriched gamma-isoform of protein kinase C may represent an important mediator of reperfusion-induced brain injury in mutant mice.     

Potential markers of oxidative stress in stroke

Free radical production is increased in ischemic and hemorrhagic stroke, leading to oxidative stress that contributes to brain damage. The measurement of oxidative stress in stroke would be extremely important for a better understanding of its pathophysiology and for identifying subgroups of patients that might receive targeted therapeutic intervention. Since direct measurement of free radicals and oxidized molecules in the brain is difficult in humans, several biological substances have been investigated as potential peripheral markers. Among lipid peroxidation products, malondialdehyde, despite its relevant methodological limitations, is correlated with the size of ischemic stroke and clinical outcome, while F2-isoprostanes appear to be promising, but they have not been adequately evaluated. 8-Hydroxy-2-deoxyguanosine has been extensively investigated as markers of oxidative DNA damage but no study has been done in stroke patients. Also enzymatic and nonenzymatic antioxidants have been proposed as indirect markers. Among them ascorbic acid, alpha-tocopherol, uric acid, and superoxide dismutase are related to brain damage and clinical outcome. After a critical evaluation of the literature, we conclude that, while an ideal biomarker is not yet available, the balance between antioxidants and by-products of oxidative stress in the organism might be the best approach for the evaluation of oxidative stress in stroke patients.     

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema, and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.     

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.     

In vivo imaging of autophagy in a mouse stroke model

Recent studies have suggested that autophagy is involved in a neural death pathway following cerebral ischemia. In vivo detection of autophagy could be important for evaluating ischemic neural cell damage for human stroke patients. Using novel green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (Tg) mice, in vivo imaging of autophagy was performed at 1, 3 and 6 d after 60 min transient middle cerebral artery occlusion (tMCAO). Ex vivo imaging of autophagy, testing of the autophagy inhibitor 3-methyladenine (3-MA), estern blot analysis, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and fluorescent analyses were performed on brain sections following tMCAO. In vivo fluorescent signals were detected above the ischemic hemisphere through the skull bone at 1, 3 and 6 d after tMCAO, with a peak at 1 d. Similar results were obtained with ex vivo fluorescence imaging. western blot analysis revealed maximum LC3-I and LC3-II expression at 1 d after tMCAO and fluorescence immunohistochemistry demonstrated that GFP-LC3-positive cells were primarily neuronal, not astroglial or microglial, cells. The number of GFP-LC3/TUNEL double-positive cells was greater in the periischemic area than in the core. These results provided evidence of in vivo autophagy detection, with a peak at 1 d, in a live animal model following cerebral ischemia. This novel technique could be valuable for monitoring autophagic processes in vivo in live stroke patients, as well as for clarifying the detailed role of autophagy in the ischemic brain, as well as in other neurological diseases.     

Acute ethanol administration and transient ischemia: a behavioral and neuropathological study

A pressing clinical question is how acute ethanol exposure might alter the outcome of a simultaneous transient ischemic attack (TIA), since ethanol is known to dysregulate key intermediary metabolites post-ischemia. Mongolian gerbils were administered ethanol (1 or 4 g/kg, s.c.) 1 hour before induction of transient ischemia, via bilateral carotid occlusions of 5 minutes duration. A control group was administered isotonic saline and rendered ischemic. All animals were maintained normothermic during the ischemic procedure. Subjects underwent behavioral assay of acquisition to the water maze 7 days after recovery from the surgery, and neuropathological examination 1-month after the ischemic brain insult. There were no behavioral or neuropathological between-group differences suggesting that mechanisms other than adverse ethanol-induced perturbations of ischemic processes predominate in mediating epidemiological findings of elevated stroke morbidity with high ethanol consumption.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.Swiss mice were fed with a standard diet or with a diet supplemented with 2.5% carbonyl iron to produce iron overload. Mice were submitted to permanent (by ligature and by in situ thromboembolic models) or transient focal ischemia (by ligature for 1 or 3 h).Treatment with iron diet produced an increase in the basal levels of ferritin in all the groups. However, serum ferritin did not change after ischemia. Animals submitted to permanent ischemia had the same infarct volume in the groups studied. However, in mice submitted to transient ischemia followed by early (1 h) but not late reperfusion (3 h), iron overload increased ischemic damage and haemorrhagic transformation.Iron worsens ischemic damage induced by transient ischemia and early reperfusion. In addition, ferritin is a good indicator of body iron levels but not an acute phase protein after ischemia.     

Rapid degradation of Bim by the ubiquitin-proteasome pathway mediates short-term ischemic tolerance in cultured neurons

A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke.     

Neuroprotection or increased brain damage mediated by temperature in stroke is time dependent

The control of temperature during the acute phase of stroke may be a new therapeutic target that can be applied in all stroke patients, however therapeutic window or timecourse of the temperature effect is not well established. Our aim is to study the association between changes in body temperature in the first 72 hours and outcome in patients with ischemic (IS) and hemorrhagic (ICH) stroke. We prospectively studied 2931 consecutive patients (2468 with IS and 463 with ICH). Temperature was obtained at admission, and at 24, 48 and 72 hours after admission. Temperature was categorized as low (<36°C), normal (36–37°C) and high (>37°C). As the main variable, we studied functional outcome at 3 months determined by modified Rankin Scale.Temperature in stroke patients is higher than in controls, and increases gradually in the first 72 hours after stroke. A positive correlation between temperature and stroke severity determined by NIHSS was found at 24 and 48 hours, but not at admission or 72 hours. In a logistic regression model, high temperature was associated with poor outcome at 24 hours (OR 2.05, 95% CI 1.59–2.64, p<0.0001) and 48 hours (OR 1.93, 95% CI 1.08–2.34, p = 0.007), but not at admission or 72 hours.Temperature increases in patients with stroke in the first 72 hours, with the harmful effect of high temperature occurring in the first 48 hours. The neuroprotective effect of low temperature occurs within the first 24 hours from stroke onset.     

Delayed treatment with a novel neurotrophic compound reduces behavioral deficits in rabbit ischemic stroke

Acute ischemic stroke is a major risk for morbidity and mortality in our aging population. Currently only one drug, the thrombolytic tissue plasminogen activator, is approved by the US Food and Drug Administration to treat stroke. Therefore, there is a need to develop new drugs that promote neuronal survival following stroke. We have synthesized a novel neuroprotective molecule called CNB-001 (a pyrazole derivative of curcumin) that has neurotrophic activity, enhances memory, and blocks cell death in multiple toxicity assays related to ischemic stroke. In this study, we tested the efficacy of CNB-001 in a rigorous rabbit ischemic stroke model and determined the molecular basis of its in vivo activity. CNB-001 has substantial beneficial properties in an in vitro ischemia assay and improves the behavioral outcome of rabbit ischemic stroke even when administered 1?h after the insult, a therapeutic window in this model comparable to tissue plasminogen activator. In addition, we elucidated the protein kinase pathways involved in neuroprotection. CNB-001 maintains the calcium-calmodulin-dependent kinase signaling pathways associated with neurotrophic growth factors that are critical for the maintenance of neuronal function. On the basis of its in vivo efficacy and novel mode of action, we conclude that CNB-001 has a great potential for the treatment of ischemic stroke as well as other CNS pathologies.     

Effect of paracetamol (acetaminophen) and ibuprofen on body temperature in acute ischemic stroke PISA, a phase II double-blind, randomized, placebo-controlled trial [ISRCTN98608690]

Background

Body temperature is a strong predictor of outcome in acute stroke. In a previous randomized trial we observed that treatment with high-dose acetaminophen (paracetamol) led to a reduction of body temperature in patients with acute ischemic stroke, even when they had no fever. The purpose of the present trial was to study whether this effect of acetaminophen could be reproduced, and whether ibuprofen would have a similar, or even stronger effect.

Methods

Seventy-five patients with acute ischemic stroke confined to the anterior circulation were randomized to treatment with either 1000 mg acetaminophen, 400 mg ibuprofen, or placebo, given 6 times daily during 5 days. Treatment was started within 24 hours from the onset of symptoms. Body temperatures were measured at 2-hour intervals during the first 24 hours, and at 6-hour intervals thereafter.

Results

No difference in body temperature at 24 hours was observed between the three treatment groups. However, treatment with high-dose acetaminophen resulted in a 0.3°C larger reduction in body temperature from baseline than placebo treatment (95% CI: 0.0 to 0.6 °C). Acetaminophen had no significant effect on body temperature during the subsequent four days compared to placebo, and ibuprofen had no statistically significant effect on body temperature during the entire study period.

Conclusions

Treatment with a daily dose of 6000 mg acetaminophen results in a small, but potentially worthwhile decrease in body temperature after acute ischemic stroke, even in normothermic and subfebrile patients. Further large randomized clinical trials are needed to study whether early reduction of body temperature leads to improved outcome.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

Copper chaperone for Cu,Zn-SOD supplement potentiates the Cu,Zn-SOD function of neuroprotective effects against ischemic neuronal damage in the gerbil hippocampus

In the present study, we investigated the chronological alterations in SOD1 and its copper chaperone (chaperone for superoxide dismutase, CCS) immunoreactivities and their neuroprotective effects against neuronal damage in the gerbil hippocampus after 5 min of transient forebrain ischemia. SOD1 and CCS immunoreactivities were significantly increased in the stratum pyramidale of the CA1 region at 24 and 12 h after ischemic insult, respectively. At 24 h after ischemic insult, the SOD1 and CCS immunoreactivities were colocalized in the CA1 pyramidal cells of the stratum pyramidale. Thereafter, their immunoreactivities were significantly decreased in the CA1 region. To elucidate the effects of CCS or CCS/SOD1, we constructed the expression vectors PEP-1-SOD and PEP-1-CCS. In the CCS-treated group and the CCS/SOD1-treated group, 43.9 and 78.9% pyramidal cells, respectively, compared to the sham-operated group, were stained with cresyl violet 5 or 7 days after ischemic insult. The distribution pattern of active astrocytes and microglia in the PEP-CCS/SOD1-treated group 5 days after ischemic insult was similar to that of the sham-operated group. In addition, the SOD activity in the PEP-CCS- or PEP-CCS/SOD1-treated group was maintained by 10 days after ischemic insult. The SOD activity was higher in the PEP-CCS/SOD1-treated group vs the CCS-treated group. These results suggest that the enhanced expression of SOD1 and CCS may be related to compensatory mechanisms against ischemic damage and that cotreatment with CCS and SOD1 has a greater neuroprotective effect than treatment with CCS or SOD1 in isolation.