Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia

Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damages in the brain. In the present study, ischemia-induced changes in GLP-1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP-1R agonist exendin-4 (EX-4) against ischemic damage was studied. GLP-1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP-1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP-1R protein level was increased again at 10 days after I/R, and GLP-1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX-4 treatment attenuated ischemia-induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose-dependent manner. EX-4 treatment also induced the elevation of GLP-1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP-1R is altered in the ischemic region after an ischemic insult and that EX-4 protects against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglial activation against transient cerebral ischemic damage.     

Antioxidant-like protein 1 is altered in non-pyramidal cells and expressed in astrocytes in the gerbil hippocampal CA1 region after transient forebrain ischemia

In the present study, we observed chronological changes of antioxidant-like protein 1 (AOP-1) in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia using immunohistochemistry and western blot. AOP-1 was significantly altered in the CA1 region after transient ischemia. In the sham-operated group, AOP-1 immunoreactivity was detected in pyramidal and non-pyramidal cells of the CA1 region. At 30 min after ischemic insult, AOP-1 immunoreactivity and protein level was decreased in the CA1 region. At 12 h after ischemic insult, AOP-1 immunoreactivity and protein level was highest in this region. At this time, after ischemia, AOP-1 immunoreactivity in non-pyramidal cells was high compared to the sham-operated group. Based on double immunofluorescence study, AOP-1-immunoreactive neurons were identified as GABAergic, which were stained with GAD or parvalbumin. Thereafter, AOP-1 immunoreactivity and protein levels were decreased time-dependently. From 4 days after ischemic insult, AOP 1 immunoreactivity was generally expressed in astrocytes. Five days after ischemic insult, AOP-1 immunoreactivity and protein level was increased again to 1.4 folds compared to that of the sham-operated group. In brief, AOP-1 immunoreactivity was increased in GABAergic non-pyramidal cells in the hippocampal CA1 region at early time after ischemic insult and was expressed in astrocytes at late time after ischemia. This result suggests that AOP-1 may be important role in homeostasis of GABAergic neurons because these neurons are resistant to ischemic damage.     

Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia

Several investigations have postulated evidence of the involvement of apoptosis in delayed neuronal death following brief periods of global cerebral ischemia. Apoptosis may be closely linked to mitochondrial dysfunction. Heat shock protein (HSP) 60 and HSP10 are mitochondrial matrix proteins induced by stress and form the chaperonin complex that is implicated in protein folding and assembly within the mitochondria. This study investigated the induction of these mitochondrial stress protein genes in the hippocampal CA1 region and less vulnerable regions following transient forebrain ischemia. In situ hybridization analysis revealed that the induction pattern of HSP60 mRNA was identical to that of HSP10 mRNA throughout the entire ischemic course. No changes occurred in the expression of both mRNAs after 2 min ischemia. Strong induction of both mRNAs occurred in the CA1 region after 10 min ischemia and persisted until 1 d after reperfusion. In contrast, induction of both mRNAs in the less vulnerable regions was terminated by 1 d after reperfusion. These results demonstrate that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in regions vulnerable to transient forebrain ischemia.     

Indole‐3‐propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus

Tryptophan‐derived indole compounds have been widely investigated as antioxidants and as free‐radical scavengers. Indole‐3‐propionic acid (IPA), one of these compounds, is a deamination product of tryptophan. In the present study, we used Mongolian gerbils to investigate IPA's neuroprotective effects against ischemic damage and its antioxidative effects in the hippocampal CA1 region (CA1) after 5 min of transient forebrain ischemia. The repeated oral administration of IPA (10 mg/kg) for 15 days before ischemic surgery protected neurons from ischemic damage. In this group, the percentage of cresyl violet–positive neurons in the CA1 was 56.8% compared with that in the sham group. In the vehicle‐treated group, glial fibrillary acidic protein (GFAP)‐, S‐100‐, and vimentin‐immunoreactive astrocytes and ionized calcium‐binding adapter molecule 1 (Iba‐1)– and isolectin B4 (IB4)–immunoreactive microglia were activated 4 days after ischemia/reperfusion, whereas in the IPA‐treated ischemic group, GFAP, S‐100, Iba‐1, and IB4, but not vimentin, immunoreactivity was distinctly lower than that in the vehicle‐treated ischemic groups. The administration of IPA significantly decreased the level of 4‐hydroxy‐2‐nonenal, a marker of lipid peroxidation, in ischemic hippocampal homogenates compared with that in the vehicle‐treated ischemic groups at various times after ischemia/reperfusion. In addition, immunostaining for 8‐hydroxy‐2′‐deoxyguanosine showed DNA damage in pyramidal neurons in the ischemic CA1 was significantly lower in the IPA‐treated ischemic groups than in the vehicle‐treated ischemic groups. These results suggest that IPA protects neurons from ischemia‐induced neuronal damage by reducing DNA damage and lipid peroxidation. © 2009 Wiley‐Liss, Inc.     

Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and αB crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in αB crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not αB crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.     

Ischemia-related changes in naive and mutant forms of ubiquitin and neuroprotective effects of ubiquitin in the hippocampus following experimental transient ischemic damage

Ubiquitin binds to short-lived proteins and denatured proteins produced by various forms of injury. The loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structure and function. The aim of the present study is to observe the chronological changes in ubiquitin naive form and its mutant form (ubiquitin⁺¹) in the hippocampal CA1 region (CA1) after transient cerebral ischemia in gerbils. Delayed neuronal death in the CA1 was confirmed 4 days after ischemic insult with NeuN immunohistochemistry. Ubiquitin immunoreactivity and protein level in the CA1 were lowest at 12 h after ischemia/reperfusion; thereafter, they were increased with time. Ubiquitin⁺¹ immunoreactivity and protein levels in the CA1 were slightly decreased at 3 h after ischemia/reperfusion, and they were significantly increased 1 day after ischemia/reperfusion. In addition, ubiquitin and ubiquitin⁺¹ immunoreaction was expressed in astrocytes after delayed neuronal death in the ischemic CA1. To elucidate the protective effect of ubiquitin on ischemic damage, the animals were treated with ubiquitin (1.5 mg/kg body weight) intravenously via the femoral vein. Ubiquitin treatment significantly reduced ischemia-induced locomotor hyperactivity, neuronal death and reactive gliosis such as astrocytes and microglia. In addition, 5 days after ubiquitin treatment in the ischemic group, ubiquitin immunoreactivity was similar to that in the ubiquitin-treated sham group, however, ubiquitin⁺¹ immunoreactivity was higher than that in the ubiquitin-treated sham group. These findings indicate that the depletion of ubiquitin and the accumulation of ubiquitin⁺¹ in CA1 pyramidal neurons after transient cerebral ischemia may inhibit ubiquitin proteolytic pathway and this leads to delayed neuronal death of CA1 pyramidal neurons directly or indirectly after transient cerebral ischemia.     

Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils

In the present study, we investigated chronological changes of galanin (GAL), well known as the potassium channel opener, immunoreactivity and GAL protein level in the hippocampus of the gerbil at the various times after 5 min transient forebrain ischemia. In the sham-operated group, weak GAL immunoreactivity was found in non-pyramidal cells. At 12 h after ischemia-reperfusion, the number of GAL-immunoreactive neurons and GAL immunoreactivity were significantly increased in the hippocampus compared to 3 h after ischemic insult, especially in the hippocampal CA1 region. Thereafter the number of GAL-immunoreactive neurons and GAL immunoreactivity decrease time-dependently in the hippocampus. Four days after transient ischemia, GAL immunoreactivity was low as compared with the sham-operated group. At this time point after ischemic insult, GAL immunoreactivity was shown in microglia in the CA1 region because delayed neuronal death happened in the CA1 pyramidal cells. The result of Western blot showed the pattern of GAL expression similar to that of immunohistochemical data. These results suggest that the early increase of GAL in the CA1 pyramidal cells may be associated with the reduction of the excitotoxic damage, that long-lasting enhanced expression of endogenous GAL at 12 h-2 days after ischemia may be associated with efflux of potassium ion into the extracellular space, and that GAL expression in microglia 4 days after ischemia may be associated with reduction of ischemic damage.     

Induction of the HSP110/105 family in the rat hippocampus in cerebral ischemia and ischemic tolerance.

Recently, the authors isolated a novel gene of the HSP110 family, ischemia responsive protein 94 kDa (irp94), and demonstrated the expression of this gene after transient forebrain ischemia. In the current study, the authors investigated the expression profiles of all HSP110 family members including hsp110/105 and osp94/apg-1, after transient forebrain ischemia using rat four-vessel occlusion model. Among three members of the HSP110 family, induction of hsp110/105 was the most prominent after ischemia. hsp110/105 mRNA expression was clearly enhanced from 4 to 24 hours after a 6-minute or longer ischemic period. First, hsp110/105 mRNA expression was induced in the dentate gyrus, and later in the pyramidal layer. HSP110/105 protein expression also was enhanced by a 6-minute or longer period of ischemia. Profiles of HSP110/105 expression after ischemia were similar to those of inducible HSP70. After transient forebrain ischemia for 10 minutes, HSP110/105 protein was induced in the dentate gyrus and the CA3 pyramidal layer, but not in the CA1 pyramidal neurons. However, 6 minutes of ischemia induced the HSP110/105 protein, as well as the HSP70 protein, in the CA1 region. CA1 pyramidal neurons expressing HSP110/105 acquired tolerance against subsequent severe ischemia. In conclusion, HSP110/105 showed the most prominent induction after ischemia among the three HSP110 gene family members. Colocalization of HSP110/105 and HSP70 in the CA1 neurons that acquired tolerance suggested that induced HSP110/105 might contribute to ischemic tolerance together with HSP70.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.     

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.     

Chronological alterations of neurofilament 150 immunoreactivity in the gerbil hippocampus and dentate gyrus after transient forebrain ischemia

In this study, we observed the chronological alterations of neurofilament 150 (NF-150) immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. NF-150 immunoreactivity in the sham-operated group was mainly detected in mossy fibers and in the hilar region of the dentate gyrus. NF-150 immunoreactivity and protein contents of NF-150 and RT 97 (polyphosphorylation epitopes of neurofilament) were significantly decreased at 15 min after ischemic insult. Between 30 min and 12 h after ischemic insult, NF-150 immunoreactivity and protein content were significantly increased as compared with the sham-operated group. Thereafter, NF-150 immunoreactivity and protein content started to decrease. At 12 h after ischemic insult, unlike dentate gyrus, NF-150 immunoreactivity increased in pyramidal cells of the CA1 region. Thereafter, NF-150 immunoreactivity in the CA1 region started to decrease, and 4 days after ischemic insult, NF-150 immunoreactivity nearly was similar to that of the sham-operated group. These biphasic patterns of NF-150 immunoreactivity in the hippocampus and dentate gyrus are reverse correlated with that of the intracellular calcium influx. For calcium detection in the CA1 region, we also conducted alizarin red staining. Alizarin red positive neurons were detected in some neurons at 15-30 min after ischemic insult. At 12 h after ischemia, alizarin red positive neurons were decreased. Thereafter, alizarin red positive neurons started to decrease, but alizarin positive neurons were significantly increased in dying neurons 4 days after ischemia. These results suggest that ischemia-related changes of NF-150 expression may be caused by the calcium following transient forebrain ischemia.     

The role of peroxisome proliferator-activated receptor γ, and effects of its agonist, rosiglitazone, on transient cerebral ischemic damage

Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in neurons and glia, and its synthetic agonist, rosiglitazone (RSG), regulates inflammatory process and has neuroprotective effects against neurological disorders. In the present study, we examined the role of PPARγ in the hippocampal CA1 region (CA1) after transient cerebral ischemia and the neuroprotective effects of RSG on ischemic damage. RSG attenuated neuronal damage in the ischemic CA1, not showing perfect neuroprotection: the RSG appeared to delay neuronal death after ischemia/reperfusion (I/R). PPARγ immunoreactivity and protein levels were increased after I/R, and most of PPARγ-immunoreactive cells colocalized with microglia, not astrocytes. In addition, RSG attenuated glial activation and increased IL-4 and IL-13 levels in the ischemic CA1. These results indicate that PPARγ increases and expresses in microglia after I/R, and that RSG delays neuronal damage by interfering with glial activations and increases anti-inflammatory cytokines in response to ischemic damage.     

Time course of changes in pyridoxal 5'-phosphate (vitamin B6 active form) and its neuroprotection in experimental ischemic damage

In the present study, we investigated ischemia-induced changes of pyridoxal 5'-phosphate synthesizing enzyme and degrading enzyme and neuroprotective effects and roles of pyridoxal 5'-phosphate against ischemic damage in the gerbil hippocampal CA1 region. Pyridoxal 5'-phosphate oxidase and pyridoxal phosphate phosphatase immunoreactivities were changed in neurons up to 2 days after ischemia, while 4 days after ischemia their immunoreactivities were expressed in astrocytes. Pyridoxal 5'-phosphate oxidase immunoreactivity and its protein level were highest 12 h after ischemia, while those in pyridoxal phosphate phosphatase were highest 2 days after ischemia. Total activities of these enzymes were changed after ischemia, but specific activities of the enzymes were not altered. Treatment with pyridoxal 5'-phosphate into brains (4 microg/5 microl, i.c.v.) at 30 min before transient ischemia protected about 80% of CA1 pyramidal cells 4 days after ischemia and induced elevation of glutamic acid decarboxylase 67 immunoreactivity in the CA1 region. However, pyridoxal 5'-phosphate treatment into ischemic brains decreased GABA transaminase immunoreactivity in the CA1 region after ischemia. These results indicate that pyridoxal 5'-phosphate may be associated with the inhibitory discharge of GABA in the hippocampal CA1 neurons, and the increased level of GABA may protect hippocampal CA1 pyramidal cells from ischemic damage.     

脑缺血后脑内HSP70表达的实验研究

热休克蛋白(HSP)是细胞对缺血等应激反应的敏感标记.通过建立大鼠全脑缺血模型,采用HSP70单克隆抗体LSAB免疫组化技术对脑缺血再灌注后的神经元进行了检测.发现在海马CA_3区,齿状回,尾壳核及杏仁核等处的神经元中有HSP70过量表达,并于再灌注后48h达到高峰.初步研究了HSP70在不同神经元中的分布情况,基本肯定不同神经元中HSP70的表达与神经元对缺血的耐受性有关.     

Na+/Ca2+ exchanger 1 alters in pyramidal cells and expresses in astrocytes of the gerbil hippocampal CA1 region after ischemia

Alterations of immunoreactivity and protein contents of Na(+)/Ca(2+) exchanger 1 (NCX1) were observed in the gerbil hippocampus proper after 5 min of transient forebrain ischemia. NCX1 immunoreactivity was significantly changed in the hippocampal CA1 region, but not in the CA2/3 region after ischemia/reperfusion. In the sham-operated group, NCX1 immunoreactivity was mainly detected in CA1 pyramidal cells. However, 30 min after ischemia/reperfusion, NCX1 immunoreactivity was significantly decreased and then increased at 1 day after ischemia. At this time, NCX1 immunoreactivity in CA1 pyramidal cells was similar to that of the sham-operated group. At 3 days after ischemia, NCX1 immunoreactivity was significantly reduced in the CA1 region compared to that of the sham-operated group and NCX1 immunoreactivity was significantly increased again 4 days after ischemia. Thereafter, NCX1 immunoreactivity was decreased time-dependently in ischemia groups. Between 15 min and 6 h post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum of the CA1 region. From 3 days post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum. Ischemia-induced changes in NCX1 protein contents in the hippocampus proper concurred with immunohistochemical data post-ischemia. Our results suggest that changes in NCX1 in CA1 pyramidal cells and astrocytes after ischemia are associated with intracellular Na(+) concentrations and that NCX1 may induce an intracellular calcium overload, which may be related to neuronal death.     

Transient ischemia-induced changes of interleukin-2 and its receptor beta immunoreactivity and levels in the gerbil hippocampal CA1 region

Interlukin-2 (IL-2) is an important cytokine in the brain: IL-2 and its receptors are involved with inflammatory processes. Chronological changes in IL-2 level in serum, and IL-2 and its receptor (IL-2 receptor beta, IL-2Rbeta) immunoreactivities and levels were examined in the hippocampal CA1 region after transient forebrain ischemia in gerbils. IL-2 level in serum significantly decreased 12 h after ischemia/reperfusion. IL-2 immunoreactivity was detected in the somata of pyramidal cells in sham-operated group. At 15 min after ischemia, IL-2 immunoreactivity was shown in non-pyramidal cells as well as pyramidal cells. One day after ischemia, IL-2 immunoreactivity was lowest, and IL-2 immunoreactivity is shown in non-pyramidal cells from 2 days after ischemia. Four days after ischemia, IL-2 immunoreactivity was shown in dying pyramidal cells. IL-2Rbeta immunoreactivity in the sham-operated and 15 min-3 min post-ischemic groups is detected in the cell membrane of pyramidal cells. From 3 h after ischemia, IL-2Rbeta immunoreactivity is found in cytoplasm and nuclei, but not in cell membrane. IL-2Rbeta immunoreactivity decreases from 6 h after ischemia and is shown mainly in non-pyramidal cells from 3 days after ischemia. The data of Western blot analyses for IL-2 and IL-2Rbeta was similar to the immunohistochemical data. IL-2 infusion into cerebrospinal fluid did not protect hippocampal neurons from ischemic damage. These results suggest that IL-2 and IL-2Rbeta show malfunction from 3 h after ischemia, and exogenous IL-2 does not protect ischemic neuronal damage.     

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.