Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia

Recently, we demonstrated that transient forebrain ischemia in rats leads to an early and strong induction of basic fibroblast growth factor (bFGF) synthesis in astrocytes in the injured brain regions. In this study, in order to clarify the targets of such raised endogenous bFGF levels, the messenger RNA (mRNA) expression of its receptors (flg and bek) at in the hippocampus following transient forebrain ischemia induced by four-vessel occlusion for 20 min was investigated using an in situ hybridization technique. Transient forebrain ischemia induced an increase in the number of flg mRNA-positive cells from an early stage (24 h after ischemia) in the hippocampal CA1 subfield where delayed neuronal death occurred later (48–72 h after ischemia). This increase became more marked with the progression of neuronal death and was still evident in the same area 30 days later. The time course of the appearance and distribution pattern of flg mRNA-positive cells in the CA1 subfield were quite similar to those of bFGF mRNA-positive cells. On the other hand, in situ hybridization for bek mRNA showed only slight and transient (observed 72 h and 5 days after ischemia) increases in the number of mRNA-positive cells in the CA1 subfield following ischemia. The use of in situ hybridization and glial fibrillary acidic protein immunohistochemistry in combination demonstrated that the cells in the CA1 subfield that exhibited ischemia-induced flg or bek mRNA expression were astrocytes. These data indicate that transient forebrain ischemia induces upregulation of fibroblast growth factor-receptor expression, accompanied by increased bFGF expression in astrocytes, and suggest that the increased astrocytic bFGF levels in injured brain regions act on the astrocytes via autocrine systems and are involved in the development and maintenance of astrocytosis.     

Expression of nerve growth factor in astrocytes of the hippocampal CA1 area following transient forebrain ischemia.

We have examined by immunoassay and immunohistochemistry, the expression of nerve growth factor in the rat hippocampus, one to 28 days after transient forebrain ischemia. In the CA1 area, the overall level of nerve growth factor expression remained constant over the first three days of reperfusion while it increased by about 45% of control levels after longer postischemic periods. In contrast, a slight decrease in nerve growth factor levels, which was most prominent at three days postlesion, was observed in the other hippocampal regions. Immunohistochemical analysis of the distribution of nerve growth factor showed that its expression was up-regulated in astrocytes but not in microglia of the postischemic CA1 region and that the intensity and temporal profile of the changes in nerve growth factor immunostaining in these cells, was consistent with that observed in the immunoassay. Interestingly, the regulation of the nerve growth factor expression in reactive astrocytes of the postischemic CA1 area closely parallels that of kainate receptor subunits GluR5-7, raising the possibility of a cause-effect relationship. These results indicate that after ischemia nerve growth factor expression is up-regulated in reactive astrocytes suggesting that these cells may contribute to rescuing damaged neurons by means of increasing nerve growth factor production.     

Mineralocorticoid and glucocorticoid receptor expressions in astrocytes and microglia in the gerbil hippocampal CA1 region after ischemic insult

In the present study, we observed expression and changes of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the gerbil hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia. In blood, corticosterone levels were increased biphasically at 30 min and 12 h after ischemia/reperfusion, and thereafter its levels were decreased. In the sham-operated group, MR and GR immunoreactivities were weakly detected in the CA1 region. By 3 days after ischemia, MR and GR were not significantly altered in the CA1 region: at 12 h after ischemia, GR was expressed in a few neurons in the CA1 region, whereas MR was not expressed in any neurons after ischemic insult. From 4 days after ischemia, MR and GR immunoreactivities were detected in astrocytes and microglia in the CA1 region, and at 7 days after ischemia, MR and GR immunoreactivities peaked in the hippocampal CA1 region. At this time, 55% of astrocytes and 30% of microglia showed MR immunoreactivity, and 20% of astrocytes and 40% of microglia showed GR immunoreactivity. Western blot analyses showed that the pattern of changes in MR and GR protein levels was similar to the immunohistochemical changes observed after transient forebrain ischemia. From 4 days after ischemia, MR and GR protein levels were increased time-dependently after ischemia. In conclusion, enhanced MR and GR expressions in astrocytes and microglia were detected in the hippocampal CA1 region 4-7 days after ischemia/reperfusion. At this time, GR immunoreactivity was abundant in microglia, whereas MR immunoreactivity was prominent in astrocytes. The specific distribution of corticosteroid receptors in the astrocytes and microglia may be associated with the differences of MR and GR functions against ischemic damage.     

Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage.

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.     

Post-ischemic synaptic plasticity in the rat hippocampus after long-term survival: histochemical and autoradiographic study.

The hippocampus provides a suitable area in the brain for the analysis of neuronal plasticity after application of a selective lesioning technique. Using histochemistry and autoradiography, we studied synaptic reorganization in the rat hippocampus with selective CA1 pyramidal cell lesioning caused by transient forebrain ischemia after long-term survival. An autoradiographic study was performed on second messenger systems ([3H]inositol 1,4,5-trisphosphate, [3H]forskolin and [3H]phorbol 12,13-dibutyrate binding). One-hundred days after ischemia, depletion of CA1 pyramidal cells and marked shrinkage of the CA1 subfield was noted in spite of unaltered thickness of the CA3 band and of the dentate molecular layers. Although neuronal density in the CA3 region of animals killed seven days after ischemia was not different from the normal group, 78% of animals showed neuronal loss of 30-50% in the stratum pyramidale of the CA3b 100 days after recirculation. Sixty-seven per cent of animals exhibited supragranular mossy fiber sprouting in the dentate gyrus. However, CA3 neuronal loss did not correlate with mossy fiber sprouting. Succinic dehydrogenase was depleted in the CA1 100 days after ischemia, and animals with CA3 damage showed a reduction of succinic dehydrogenase activity in the CA3. In contrast to the unaltered acetylcholinesterase in the animals killed seven days after ischemia, high density bands of acetylcholinesterase activity in the stratum pyramidale of the CA1 were found to be broadened 100 days after ischemia. In the CA1 subfield, subnormal activity of [3H]phorbol 12,13-dibutyrate and [3H]forskolin binding were observed in spite of the depleted [3H]inositol 1,4,5-triphosphate binding. [3H]Forskolin binding in the hilus had increased by 62% 100 days after ischemia, although binding in the stratum lucidum of the CA3 and in the stratum moleculare of the dentate gyrus was unaltered. However, no visible supragranular increase in [3H]forskolin binding was observed. These results indicate that long-term survival after CA1 pyramidal cell depletion caused by transient forebrain ischemia induced the modulation of neuronal activity and synaptic rearrangements in the whole hippocampal formation.     

Differential effects and changes of ceruloplasmin in the hippocampal CA1 region between adult and aged gerbils after transient cerebral ischemia

In this study, we examined the differential effects and changes of ceruloplasmin between adult and aged gerbil hippocampus after transient forebrain ischemia. Ceruloplasmin in the hippocampal CA1 region of adult and aged gerbils was significantly changed after ischemia/reperfusion. Whereas, it was not significantly changed in the CA2/3 region compared to the CA1 region after ischemia. Ceruloplasmin immunoreactivity and its protein level in aged gerbil CA1 region were higher than those in adult gerbil CA1 region. Ceruloplasmin in the CA1 region was highest in adult gerbils and aged gerbils at 24h and 12h after transient ischemia, respectively. At these time points, strong ceruloplasmin immunoreactivity was observed in CA1 pyramidal cells. Thereafter, ceruloplasmin was decreased with time after ischemia. Four days after ischemia/reperfusion, ceruloplasmin immunoreactivity in both adult and aged gerbils was expressed in astrocytes in the CA1 region. Ceruloplasmin treatment in adult ischemic gerbils showed strong protective effect against ischemic damage in CA1 pyramidal cells compared to that in aged ischemic gerbils. We conclude that ceruloplasmin early increases in the aged gerbil CA1 region compared to that of the adult gerbil CA1 region may be associated with the earlier induction of reactive oxygen species, and ceruloplasmin shows strong neuroprotective effects in adults compared to those in aged gerbils.     

Accumulation of 4-hydroxynonenal-modified proteins in hippocampal CA1 pyramidal neurons precedes delayed neuronal damage in the gerbil brain

It has been proposed that reactive oxygen species and lipid peroxidation have a role in the delayed neuronal death of pyramidal cells in the CA1 region. To explore the in situ localization and serial changes of 4-hydroxy-2-nonenal-modified proteins, which are major products of membrane peroxidation, we used immunohistochemistry of the gerbil hippocampus after transient forebrain ischemia with or without preconditioning ischemia. The normal gerbil hippocampus showed weak immunoreactivity for 4-hydroxy-2-nonenal-modified proteins in the cytoplasm of CA1 pyramidal cells. 4-hydroxy-2-nonenal immunoreactivity showed no marked changes after preconditioning ischemia. In the early period after ischemia and reperfusion, there was a transient increase of nuclear 4-hydroxy-2-nonenal immunoreactivity in CA1 pyramidal neurons. In contrast, cytoplasmic immunoreactivity transiently disappeared during same period and then increased markedly from 8h to seven days. One week after ischemia, 4-hydroxy-2-nonenal immunoreactivity was observed within reactive astrocytes in the CA1 region. Early nuclear accumulation of 4-hydroxy-2-nonenal in CA1 neurons may indicate a possible role in signal transduction between the nucleus and cytoplasm/mitochondria, while delayed accumulation of 4-hydroxy-2-nonenal-modified proteins in the cytoplasm may be related to mitochondrial damage.We conclude that 4-hydroxy-2-nonenal may be a key mediator of the oxidative stress-induced neuronal signaling pathway and may have an important role in modifying delayed neuronal death.     

Protein disulfide isomerase immunoreactivity and protein level changes in neurons and astrocytes in the gerbil hippocampal CA1 region following transient ischemia

We investigated the temporal and spatial alterations of protein disulfide isomerase (PDI) immunoreactivity and protein level in the hippocampus proper after 5 min transient forebrain ischemia in gerbils. PDI immunoreactivity was significantly altered in the hippocampal CA1 region. PDI immunoreactivity in the sham-operated animals was found in non-pyramidal cells. At 30 min after ischemia, PDI immunoreactivity was shown in the pyramidal cells of the stratum pyramidale (SP): the PDI immunoreactivity in the pyramidal cells was increased up to 12 h after ischemia. Thereafter PDI immunoreactivity was decreased, and the PDI immunoreactivity was shown in non-pyramidal cells 2 days after ischemia. Four to 5 days after ischemia, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred. At this time period, PDI immunoreactivity was expressed in some astrocytes as well as some neurons. The results of the Western blot analysis were consistent with the immunohistochemical data. These findings suggest that increase of PDI in pyramidal cells may play a critical role in resistance to ischemic damage at early time after ischemic insult, and that expression of this protein in astrocytes at late time after ischemic insult is partly implicated in the acquisition of tolerance against ischemic stress.     

Pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, reduces delayed neuronal death following transient forebrain ischemia in the adult rat hippocampus

Recent evidence indicates that statins have beneficial effects on the brain in the ischemic condition. However, there is a lack of studies related to the effect of statins on delayed neuronal death. We investigated the effect of prophylactic therapy with pravastatin on delayed neuronal death in the rat hippocampus. The rats were given a daily dose of 20 mg/kg of pravastatin orally for 14 days. Transient forebrain ischemia was induced by the four-vessel occlusion method. Three days after ischemia, surviving neurons of the hippocampal CA1 subfield were counted. Our results demonstrated that prophylactic statin treatment significantly reduced delayed neuronal death after transient forebrain ischemia. Our findings suggest that prophylactic statin treatment may be useful in preventing functional neurological disorders after transient cerebral ischemic insult.     

短暂性前脑缺血再灌流后海马本部与齿状回cGMP反应细胞的差异

观察脑缺血再灌流后海马本部与齿状回 c GMP反应细胞的差异。钳夹沙土鼠的双侧颈总动脉制造脑缺血模型 ,应用免疫荧光组织化学方法。结果表明 :海马本部 c GMP合成增加 ,c GMP主要分布于 CA1 区的放射层及腔隙分子层 ,双重免疫荧光反应证实多数 c GMP阳性细胞是星形胶质细胞。齿状回与海马本部不同 ,较对照组增加了一些中小圆形的 c GMP阳性细胞 ,分布在齿状回各层。本实验结果揭示 ,短暂性前脑缺血再灌流可增加海马本部 c GMP的合成 ,多数 c GMP阳性细胞是星形胶质细胞。意味着星形胶质细胞在脑缺血再灌流早期扮演着重要角色。     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.     

GABA(B) receptor expression and cellular localization in gerbil hippocampus after transient global ischemia

Using in situ hybridization, the expression of the GABA receptor subtype B subunit 1 (GABA(B) R1) and subunit 2 (GABA(B) R2) following transient global ischemia in the gerbil hippocampus was investigated. In sham-operated animals, mRNAs of both subunits were mainly detected in hippocampal pyramidal cells and interneurons with lower expression levels of the GABA(B) R2 in the CA1 field. Four days after transient cerebral ischemia, neuronal message decreased in conjunction with neuronal death and both receptor subunits disappeared from the pyramidal cell layer. However, GABA(B) R1 and GABA(B) R2 were still expressed in a few cells. In situ hybridization of the GABA synthesizing enzyme glutamic acid decarboxylase 67 (GAD67) remained unchanged after the ischemic insult. Double-labeling experiments revealed that in the postischemic hippocampus GABA(B) R1 and GABA(B) R2 were not present in GFAP-reactive astrocytes, but that the surviving parvalbumin-containing interneurons possessed GABA(B) R1 and GABA(B) R2 mRNA.     

LAMP-2A ablation in hippocampal CA1 astrocytes confers cerebroprotection and ameliorates neuronal injury after global brain ischemia

Reactive astrogliosis and neuronal death are major features of brain tissue damage after transient global cerebral ischemia/reperfusion (I/R). The CA1 subfield in the hippocampus is particularly susceptible to cell death after I/R. Recently, attention has focused on the relationship between the autophagy–lysosomal pathway and cerebral ischemia. Lysosomal-associated membrane protein type-2A (LAMP-2A) is a key protein in chaperone-mediated autophagy (CMA). However, LAMP-2A expression in astrocytes of the hippocampus and its influence on brain injury following I/R remain unknown. Here, we show that LAMP-2A is elevated in astrocytes of the CA1 hippocampal subfield after I/R and in primary cultured astrocytes after transient oxygen–glucose deprivation (OGD). Conditional LAMP-2A knockdown in CA1 astrocytes inhibited astrocyte activation and prevented neuronal death by inhibiting the mitochondrial pathway of apoptosis after I/R, suggesting that elevated astrocytic LAMP-2A contributes to regional ischemic vulnerability. Furthermore, astrocytic LAMP-2A ablation ameliorated the spatial learning and memory deficits caused by I/R. Conditional astrocytic LAMP-2A knockdown also prevented the loss of hippocampal synapses and dendritic spines, improved the synaptic ultrastructure, and inhibited the reduced expression of synaptic proteins after ischemia. Thus, our findings demonstrate that astrocytic LAMP-2A expression increases upon I/R and that LAMP-2A ablation specifically in hippocampal astrocytes contributes to cerebroprotection, suggesting a novel neuroprotective strategy for patients with global ischemia.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5'-Bromo-2'-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ).     

Effect of pitavastatin against expression of S100beta protein in the gerbil hippocampus after transient cerebral ischaemia

AIM AND METHODS: We investigated the immunohistochemical alterations of S100beta-, S100-, glial fibrillary acidic protein (GFAP)- and isolectin B4-positive cells in the hippocampus after 5 min of transient cerebral ischaemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor pitavastatin against neuronal damage in the hippocampal CA1 sector after ischaemia. RESULTS: Severe neuronal damage was observed in the hippocampal CA1 pyramidal neurons from 5 days after ischaemia. GFAP-positive cells increased gradually in the hippocampus from 5 days after ischaemia. Five and 14 days after ischaemia, significant increases in the number of GFAP-positive cells and isolectin B4-positive cells were observed in the hippocampal CA1 and CA3 sector. Mild increases in the number of S100 and S100beta-positive cells were observed in the hippocampal CA1 sector from 1 h to 2 days after ischaemia. Thereafter, S100beta-positive cells increased in the hippocampal CA1 sector after ischaemia, whereas S100-positive cells decreased in this region. In our double-labelled immunostainings, S100 and S100beta immunoreactivity was found in GFAP-positive astrocytes, but not in isolectin B4-positive microglia. Pharmacological study showed that HMG-CoA reductase inhibitor, pitavastatin, can protect against the hippocampal CA1 neuronal damage after ischaemia. This drug also prevented increases in the number of GFAP-positive astrocytes, isolectin B4-positive microglia, S100-positive astrocytes and S100beta-positive astrocytes after ischaemia. CONCLUSION: The present study demonstrates that pitavastatin can decrease the neuronal damage of hippocampal CA1 sector after ischaemia. This beneficial effect may be, at least in part, mediated by inhibiting the expression of astrocytic activation in the hippocampus at the acute phase after ischaemia. Thus the modulation of astrocytic activation may offer a novel therapeutic strategy of ischaemic brain damage.     

Glial cell line-derived neurotrophic factor protects against delayed neuronal death after transient forebrain ischemia in rats

Glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily, is a potent neurotrophic factor, which has a variety of biological activities that affect several types of neurons in both the central and peripheral nervous systems. In this study, we examined the effects of glial cell line-derived neurotrophic factor on delayed neuronal death in the hippocampal CA1 region of rats after transient forebrain ischemia. In the control rats pretreated with the vehicle, transient forebrain ischemia-induced delayed neuronal death in the hippocampal CA1 region was observed seven days after reperfusion. Pretreatment with glial cell line-derived neurotrophic factor (1.0 microg), which was directly microinjected into the right hippocampal CA1 region, gave significant protection against the delayed hippocampal neuronal death. On the contralateral side of the hippocampus, which was not injected with glial cell line-derived neurotrophic factor, delayed neuronal death similar to that seen in vehicle-treated control animals was observed. Intracerebroventricular glial cell line-derived neurotrophic factor (2.5 microg) injection also protected against delayed neuronal death. In addition, pretreatment with glial cell line-derived neurotrophic factor gave significant protection against apoptotic cell death induced by brain ischemia in the hippocampal CA1 region, as determined by in situ staining for DNA fragmentation. These findings suggest that glial cell line-derived neurotrophic factor plays an important role in delayed neuronal death induced by brain ischemia.