脑缺血再灌流大鼠脑胶质源性神经营养因子基因表达的变化

探讨脑缺血再灌流不同时程及不同程度缺血对海马及皮层胶质源性神经营养因子（ｇｌｉａｌｃｅｌｌｌｉｎｅ ｄｅｒｉｖｅｄ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ, ＧＤＮＦ）基因表达的影响,以及Ｎ甲基Ｄ天冬氨酸（Ｎｍ ｅｔｈｙｌＤｓａｐａｒｔａｔｅ, ＮＭＤＡ）受体拮抗剂,钙离子通道阻断剂是否能调节缺血病态下ＧＤＮＦｍ ＲＮＡ的表达。参照Ｓｍ ｉｔｈ 等方法建立大鼠前脑缺血再灌流动物模型。用ＤＩＧＯｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ３′ｅｎｄ ｌａｂｅｌｉｎｇ Ｋｉｔ,标记５１ ｍ ｅｒ的ＧＤＮＦ寡核苷酸探针在含有海马结构的冰冻组织切片上进行原位杂交检测ＧＤＮＦｍ ＲＮＡ的表达。１０ ｍ ｉｎ 缺血再灌流２ ｈ,齿状回ＧＤＮＦｍ ＲＮＡ表达上调。再灌流６ ｈ,ＣＡ１,ＣＡ３ 和皮层ＰＡＲ区ＧＤＮＦｍ ＲＮＡ表达亦见增多,２４ ｈ 达高峰。Ｋｅｔａｍ ｉｎｅ 可使ＧＤＮＦ的基因表达在海马结构及皮层ＰＡＲ区明显低于相应的缺血再灌流组,统计学差异显著（Ｐ＜ ００５）。脑缺血再灌流时ＧＤＮＦ基因表达增加,对缺血神经元可能起保护作用。Ｋｅｔａｍ ｉｎｅ可阻断缺血后ＧＤＮＦｍ ＲＮＡ 的表达增加,提示ＮＭＤＡ谷氨酸受体很可能参与介导了缺     

Bimodal upregulation of glial cell line-derived neurotrophic factor (GDNF) in the neonatal rat brain following ischemic/hypoxic injury

In order to delineate the spatial and temporal patterns of glial cell line-derived neurotrophic factor (GDNF) expression following ischemic/hypoxic injury in immature and neonatal brain, GDNF protein levels and immunocytochemistry were studied in rats subjected to a modified Levine procedure. Significant upregulation of GDNF protein occurred in a bimodal fashion in the damaged left cerebral cortex and hippocampus, while the levels in the right cerebral hemisphere of both control and ischemic groups remained relatively unchanged. Immunocytochemical studies indicated that the early rise in GDNF levels was most likely to be related to enhanced neuronal release of GDNF. The second rise was probably related to progressive astrogliosis that occurred in response to injury. In contrast to the lack of GDNF expression among astrocytes in normal mature brains, reactive astrocytes in the neonate appear to possess a ready capacity to express GDNF. Spatial and temporal changes in the pattern of GDNF expression following injury, as determined in this study may provide insight into the functions of GDNF in vivo and into possible therapeutic approaches toward prevention of damage or rescue of neurons following brain injury.     

脑缺血对大鼠皮层及海马铜蓝蛋白表达的影响

目的 探讨脑缺血对大鼠皮层及海马中铜蓝蛋白(Ceruloplasmin,Cp)表达的影响.方法 雄性Wistar大鼠60只,随机分为脑缺血1、3、7、28 d组和假手术对照组,每组各12只.实验组结扎双侧颈总动脉造成大鼠脑缺血,假手术对照组仅分离出双侧颈总动脉但不结扎.采用反转录聚合酶链反应(RT-PCR)检测皮层及海马组织中Cp mRNA的表达,免疫组织化学观察皮层及海马组织中Cp的表达.结果 大鼠皮层和海马均表达Cp mRNA.皮层和海马Cp mRNA的表达随缺血时间的延长逐渐降低,缺血1、3、7、28 d组表达均低于假手术组(P＜0.01).脑组织脉络丛细胞、室管膜细胞、皮层和海马的星形胶质细胞、血管内皮细胞均表达Cp;而皮层和海马的锥体细胞和颗粒细胞均不表达Cp.缺血1 d组皮层及海马Cp表达与对照组差异不显著(P＞0.05);缺血3 d组皮层和海马Cp表达低于假手术组(P＜0.05);缺血第7、28 d组Cp表达减少极为显著(P＜0.01).脑缺血大鼠皮层和海马中铁含量与Cp的表达呈负相关,相关系数分别为-0.831(P＜0.01)和-0.809(P＜0.01).结论 脑缺血可诱导大鼠皮层及海马中Cp表达降低.脑缺血后Cp表达减少可能参与了脑缺血引起的铁含量升高及神经元铁沉积的过程.     

Brain glutamine synthetase increases following cerebral ischemia in the rat.

Changes in astrocyte glutamine synthetase (GS) in postischemic rat brain were evaluated and correlated with regional neuronal vulnerability or resistance to ischemia. Rats subjected to 20 or 30 min of cerebral ischemia were allowed to survive for 3 or 24 h after ischemia; normal animals served as controls. Resultant neuronal necrosis was severe in the striatum by 24 h and in the CA1 region of the hippocampus at 72 h; neurons in paramedian cortex and CA3 region of the hippocampus were not permanently damaged. Glutamine synthetase (GS) immunocytochemistry was performed on vibratome sections of paraformaldehyde-fixed brains and enzyme activity was assayed in frozen samples of cerebral cortex, striatum and hippocampus. At 3 and 24 h after ischemia, GS immunoreactivity increased and was secondary to enlargement of GS-positive cell bodies and processes as well as to increased numbers of GS-positive astrocytes. Enzyme activity also increased in cortex, striatum and hippocampus at 3 and 24 h (P less than or equal to 0.03). This study shows that increase in astrocyte GS occurs rapidly after ischemia, and prior studies indicate that this increase occurs in parallel with proliferative changes in astrocyte organelles. The results also suggest that astrocyte metabolism of glutamate increases after ischemia. The increased capacity for glutamine synthetase may be important in normalizing extracellular glutamate following ischemia and protecting brain from the neurotoxic effects of this excitatory amino acid.     

Expression of the glial cell line-derived neurotrophic factor gene in rat brain after transient MCA occlusion

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.     

The expression of transforming growth factor-beta1 (TGF-beta1) in hippocampal neurons: a temporary upregulated protein level after transient forebrain ischemia in the rat

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.     

Ischemia-related changes of glial-derived neurotrophic factor and phosphatidylinositol 3-kinase in the hippocampus: their possible correlation in astrocytes

In the present study, we observed the changes of endogenous expression of glial-cell-line-derived neurotrophic factor (GDNF) and phosphatidylinositol 3-kinase (PI-3 kinase) in the gerbil hippocampus after transient forebrain ischemia and investigated the correlation between GDNF and PI-3 kinase in the ischemic hippocampus. In the sham-operated group, GDNF and PI-3 kinase immunoreactivity was not found in any cells in the hippocampal CA1 region. GDNF, not PI-3 kinase, immunoreactivity was expressed in non-pyramidal cells in the CA1 region at 6 h after ischemic insult. At 12-24 h after ischemia, GDNF and PI-3 kinase immunoreactivity in the CA1 region was similar to that of the sham-operated group. From 2 days after ischemic insult, GDNF- and PI-3-kinase-immunoreactive astrocytes were detected in the CA1 region, and GDNF and PI-3 kinase immunoreactivity in astrocytes was highest in the CA1 region 4 days after ischemic insult. Moreover, at this time point, GDNF and PI-3 kinase were co-localized in some astrocytes. Western blotting showed that ischemia-related changes of GDNF and PI-3 kinase protein levels were similar to the immunohistochemical changes after ischemia. These results suggest that GDNF and PI-3 kinase may be related to delayed neuronal death and that GDNF and PI-3 kinase may be involved in activation of astrocytes.     

Dexamethasone enhances upregulation of nerve growth factor mRNA expression in ischemic rat brain.

Nerve growth factor (NGF) is well-established as a trophic factor that plays a crucial role in neuroregeneration and plasticity after brain insults. Dexamethasone (DEX), a powerful glucocorticoid steroid, has long been used in the clinical management of neurological disorders. We examined the relationship between NGF and DEX after an ischemic insult to the brain. In situ hybridization was used to measure NGF mRNA expression in the rat hippocampus after 20 min of transient forebrain ischemia. Immunostaining for NGF protein was performed using the avidin-biotin peroxidase method. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was also used to study the astrocyte reaction in the hippocampal CA1 area. Ischemic brain from rats not treated with DEX had a 2 and 3 fold increase in NGF mRNA compared to sham-operated rats at 4 and 6 h after ischemia, respectively. The NGF mRNA expression returned to basal levels 12 h to 7 days post-ischemia. Treatment with DEX potentiated the ischemia-induced increase of NGF mRNA to 4 times that of sham-operated rats at 6 h following reperfusion and NGF protein expression was similarly elevated. Additionally, the number of GFAP positive astrocytes in the CA1 region in the ischemic rats was markedly increased. These data suggest that DEX may play a role in modulating NGF mRNA expression in the hippocampal neuronal response to brain ischemia.     

Differential sensitivity of murine astrocytes and neurons from different brain regions to injury

Different brain regions show differential vulnerability to ischemia in vivo. Despite this, little work has been done to compare vulnerability of brain cells isolated from different brain regions to injury. Relatively pure neuronal and astrocyte cultures were isolated from mouse cortex, hippocampus, and striatum. Astrocyte vulnerability to 6 h oxygen-glucose deprivation was greatest in striatum (81.8 +/- 4.6% cell death), intermediate in hippocampus (59.8 +/- 4.8%), and least in cortex (37.0 +/- 3.5%). In contrast neurons deprived of oxygen and glucose for 3 h showed greater injury to cortical neurons (71.1 +/- 5.2%) compared to striatal (39.0 +/- 3.1%) or hippocampal (39.0 +/- 5.3%) neurons. Astrocyte injury from glucose deprivation or H(2)O(2) exposure was significantly greater in cells from cortex than from striatum or hippocampus. Neuronal injury resulting from serum deprivation was greater in cortical neurons than in those from striatum or hippocampus, while excitotoxic neuronal injury was equivalent between regions. Antioxidant status and apoptosis-regulatory genes were measured to assess possible underlying differences. Glutathione was higher in astrocytes and neurons isolated from striatum than in those from hippocampus. Superoxide dismutase activity was significantly higher in striatal astrocytes, while glutathione peroxidase activity and superoxide did not differ by brain region. Bcl-x(L) was significantly higher in striatal astrocytes than in astrocytes from other brain regions and higher in striatal and hippocampal neurons than in cortical neurons. Both neurons and astrocytes isolated from different brain regions demonstrate distinct patterns of vulnerability when placed in primary culture. Antioxidant state and levels of expression of bcl-x(L) can in part account for the differential injury observed. This suggests that different protective strategies may have different efficacies depending on brain region.     

The dual response of protein kinase Fyn to neural trauma: early induction in neurons and delayed induction in reactive astrocytes

In the developing central nervous system, a src-related protein-tyrosine kinase fyn participates in the myelination process, neuronal growth, and cytoskeletal organization. In adults, fyn has been implicated in learning and memory formation. To test if fyn expression is modulated by neuronal activity, we performed quantitative in situ hybridization (ISH) using brain sections of the adult rats that had undergone either kainic acid (KA)-induced seizures or neuronal deafferentation (entorhinal cortex lesion, ECL). In the KA model, a few hours after seizure activities, fyn mRNA was elevated in the dentate gyrus (DG) (+45%), cerebral cortex layer III (+35%), and piriform cortex (+25%). Conversely, fyn mRNA consistently decreased in the hippocampal neurons after transection of the major axonal inputs from the entorhinal cortex. Although fyn expression in the brain has been allegedly limited to neurons and oligodendrocytes, we provide in this study the first evidence that fyn mRNA is highly expressed in the astrocytes involved in reactive gliosis. In the KA model, the occurrence of fyn-overexpressing astrocytes increased with the progress of neuronal damage in the CA1 and CA3 regions of the hippocampus. In contrast, fyn-overexpressing astrocytes were not observed in the granular cell layer of dentate gyrus (DG), where neurons were not damaged. Likewise, in the ECL model, the most drastic change in fyn mRNA expression took place at the reactive astrocytes near the stab wound sites, where fyn mRNA levels were doubled 4-10 d after the lesion. Collectively, our data suggest that (i) an early induction of fyn mRNA in neurons is linked to neuronal activity, and (ii) the delayed induction of fyn mRNA in reactive astrocytes near the damaged cells may play novel signaling roles during glial response.     

Expression of neuron specific phosphatase, striatal enriched phosphatase (STEP) in reactive astrocytes after transient forebrain ischemia

We studied the distribution and change of striatal enriched phosphatase (STEP) in the gerbil hippocampus after transient forebrain ischemia. STEP was expressed in the perikarya and in neuronal processes; it was not detected in non-neuronal cells of control animals. After 5-min forebrain ischemia, STEP immunoreactivity (STEP-IR) was preserved for 2 days; it disappeared 4 and more days after ischemia with completion of delayed neuronal death (DND) in the CA1 subfield. Furthermore, only in the CA1 after ischemia, STEP was expressed in reactive astrocytes for 4 to 28 days, showing different patterns of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. After non-or less-than lethal ischemia, STEP expression in reactive astrocytes corresponded with the degree of neuronal degeneration. Immunoblot analysis of the CA1 subfield revealed the expression of three isoforms, STEP45, -56 and -61; their expression patterns changed with time after ischemia. These data suggest that neuronal STEP is preserved until cell degeneration after ischemia and that STEP is expressed in reactive astrocytes only after lethal ischemia, with different expression patterns for its isoforms. Of STEP45, -56 and -61, STEP61 was the most strongly expressed in the reactive astrocytes; both STEP45 and -61 were expressed in neurons and the expression of STEP56 was weak. STEP may play an important role not only in neurons but also in reactive astrocytes after ischemia, depending on neuronal degeneration.     

Involvement of the calcium-independent receptor for alpha-latrotoxin in brain ischemia

Cerebral ischemia is caused by a reduced blood supply to neurons, and vulnerability to neurodegeneration varies considerably among neuronal types. In hippocampus, neurons in the CA1 region are more susceptible to ischemia-induced neuronal death than neurons in the CA3 region, and in response to transient forebrain ischemia a family of calcium-dependent receptors for alpha-latrotoxin is differentially expressed in the two regions. Here, we report that an ischemic insult up-regulated a family of calcium-independent receptors for alpha-latrotoxin (CIRL) mRNAs in CA1 neurons and down-regulated their mRNAs in CA3 neurons. Furthermore, antisense oligonucleotides complementary to CIRL-1 mRNA or CIRL-3 mRNA suppressed neuronal death associated with hypoxia in hippocampal and cortical cell cultures. The observed region-specific CIRL mRNA expression in hippocampus and an in vitro rescue experiment by antisense oligonucleotides against CIRL mRNAs suggest a functional importance of CIRL in neurodegeneration.     

Hyperglycemic but not normoglycemic global ischemia induces marked early intraneuronal expression of beta-amyloid precursor protein

Preischemic hyperglycemia is known to accentuate acute ischemic injury to neurons, microglia, and endothelia. In the present study, we used a monoclonal antibody to the N-terminal portion of beta-APP to examine how the immunoreactivity of this normal membrane glycoprotein is differentially influenced by transient cerebral ischemia when carried out under normoglycemic vs. hyperglycemic conditions. Anesthetized, physiologically regulated rats received 12.5 min of global forebrain ischemia by bilateral carotid artery occlusions plus systemic hypotension. Hyperglycemia was induced by intraperitoneal dextrose administration prior to ischemia. One or three days later, brains were examined by beta-APP immunohistochemistry. Ischemia under hyperglycemic conditions led to the robust, widespread intraneuronal expression of beta-APP immunoreactivity in neocortex, hippocampus, thalamus, and striatum of all 11 rats; this was most prominent at 24 h postischemia. Compared to rats with normoglycemic ischemia, numbers of beta-APP-immunopositive neurons in the parietal cortex of hyperglycemic rats were increased by 5.9 fold at 24 h, and by 10.6 fold at 3 days postischemia. beta-APP-immunopositive neurons in hyperglycemic rats often exhibited striking morphological alterations typical of ischemic necrosis; however, no beta-APP immunoreaction was observed in zones of frank infarction. Brains of normoglycemic rats (n=11), by contrast, showed only weak beta-APP immunostaining in occasional non-necrotic pyramidal neurons of parietal neocortex; no necrosis was present in thalamus. In sham-operated hyperglycemic rats, beta-APP immunostaining of thalamic neurons was somewhat increased at 24 h. Western analysis revealed that the hyperglycemia-induced intraneuronal overexpression of beta-APP was not associated with an overall increase in tissue levels. The results of this study demonstrate that transient forebrain ischemia under hyperglycemic conditions leads to the early intraneuronal expression of beta-APP within neuronal populations showing a heightened susceptibility to hyperglycemia-induced accentuation of ischemic injury. Our data suggest that beta-APP or its metabolites may be involved in the injury process.     

Calpain activity in the rat brain after transient forebrain ischemia.

Activity of the Ca(2+)-dependent protease calpain is increased in neurons after global and focal brain ischemia, and may contribute to postischemic injury cascades. Understanding the time course and location of calpain activity in the post-ischemic brain is essential to establishing causality and optimizing therapeutic interventions. This study examined the temporal and spatial characteristics of brain calpain activity after transient forebrain ischemia (TFI) in rats. Male Long Evans rats underwent 10 min of normothermic TFI induced by bilateral carotid occlusion with hypovolemic hypotension (MABP 30 mm Hg). Brain calpain activity was examined between 1 and 72 h after reperfusion. Western blot analysis of regional brain homogenates demonstrated a bimodal pattern of calpain-mediated alpha-spectrin degradation in the hippocampus, cortex, and striatum with an initial increase at 1 h followed by a more prominent secondary increase at 36 h after reperfusion. Immunohistochemical analysis revealed that calpain activity was primarily localized to dendritic fields of selectively vulnerable neurons at one hour after reperfusion. Between 24 and 48 h after reperfusion neuronal calpain activity progressed from the dorsal to ventral striatum, medial to lateral CA1 hippocampus, and centripetally expanded from watershed foci in the cerebral cortex. This progression was associated with fragmentation of dendritic processes, calpain activation in the neuronal soma and subsequent neuronal degeneration. These observations demonstrate a clear association between calpain activation and subsequent delayed neuronal death and suggest broad therapeutic window for interventions aimed at preventing delayed intracellular Ca(2+) overload and pathologic calpain activation.     

Microglia/macrophages proliferate in striatum and neocortex but not in hippocampus after brief global ischemia that produces ischemic tolerance in gerbil brain.

The current study determined whether short durations of ischemia that produce ischemia-induced tolerance stimulate glial proliferation in brain. Adult male gerbils were injected with BrdU (50 mg/kg) and dividing cells were detected using immunocytochemistry after sham operations, 2.5 or 5 minutes of global ischemia, or ischemia-induced tolerance. The 2.5-minute ischemia and the ischemia-induced tolerance did not kill hippocampal CA1 pyramidal neurons, whereas the 5-minute ischemia did kill the neurons. At 4 days after 2.5-minute global ischemia, when cell proliferation was maximal, BrdU-labeled cells increased in striatum and in neocortex, but not in hippocampus. The majority of the BrdU-labeled cells were double-labeled with isolectin B4, showing that these dividing cells were primarily microglia or macrophages, or both. Similarly, BrdU-labeled microglia/macrophages were found in striatum and neocortex but not in hippocampus of most animals 4 days after ischemia-induced tolerance (2.5 minutes of global ischemia followed 3 days later by 5 minutes of global ischemia). No detectable neuronal cell death existed in striatal and cortical regions where the microglia/macrophage proliferation occurred. Though 3 of 7 animals subjected to 2.5 minutes of ischemia showed decreased myelin-associated glycoprotein (MAG) immunostaining and increased numbers of adenomatous polyposis coli-stained oligodendrocytes in lateral striatum, this did not explain the microglia/macrophage proliferation. Data show that ischemia-induced tolerance in the gerbil is associated with proliferation of microglia/macrophages in striatum and cortex but not in hippocampus. Because there is no apparent neuronal death, it is postulated that the microglia/macrophage proliferation occurs in response to an unknown nonlethal injury to neurons or glia and may be beneficial.     

Enhanced expression of p53 in reactive astrocytes following transient focal ischemia

The present study used immunohistochemistry to investigate p53 expression in rat brain following transient occlusion of the middle cerebral artery. In the control group, no p53-immunoreactive cells were found in any region of the central nervous system. P53 expression in reactive astrocytes was not obvious in the forebrain one day or three days following ischemic insults. Seven days following ischemic injury, increased expression of p53 was clearly detectable in reactive astrocytes in affected cortical regions, such as forelimb area, hindlimb area, and parietal cortex. At seven days of recirculation, there was also a significant increase in the number of p53-immunoreactive neurons in the cerebral cortex, striatum, and hippocampal CA1-3 regions. Although the present study has not addressed multiple mechanisms contributing to cell death following ischemic injury, the first demonstration of a significant increase in p53 expression in glial cells may prove useful for future investigations of the pathophysiology of ischemia.     

Activation of the JAK/STAT pathway following transient focal cerebral ischemia: signaling through Jak1 and Stat3 in astrocytes

JAK/STAT is one of the pathways bearing signals from the cell membrane to the nucleus in response to extracellular growth factors and cytokines. In the present study, we examined the cellular distribution of Jak1 and Stat3, and activation of the JAK/STAT pathway following transient focal cerebral ischemia in the rat. Jak1 was mainly seen in white matter astrocytes and in certain neurons. Notably, large pyramidal neurons of cortical layer V showed the highest neuronal Jak1 expression within cerebral cortex and, in addition, expressed Stat3 indicating that the JAK/STAT pathway is involved in signaling in the corticofugal projection system. Shortly following ischemia, Jak1 immunoreactive astrocytes located in the ipsilateral neighbouring white matter and ischemic cortex and striatum showed nuclear translocation of Stat3. These features were maintained in large reactive astrocytes that surrounded the infarct from 3 to 7 days. At these later times, the abundant reactive microglia/macrophages were strongly immunoreactive to Stat3 and, to a lesser extent, Jak1. Two main protein complexes showing DNA binding activity at the sis-inducible element site were found under basal conditions, followed by changes in this pattern following ischemia concomitant with neuronal cell loss and activation of glia. This study showed basal cerebral activity of JAK/STAT signaling pathway, involving Jak1 and Stat3 proteins, and selective activation following ischemia. It is suggested that the kinase activity of Jak1 mediates nuclear translocation of Stat3 in astrocytes, and that this signaling pathway is involved in the astroglial response to focal cerebral ischemia.     

Time Course Study of GFRα-1 Expression in an Animal Model of Stroke

Previous studies have shown that intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) reduces ischemia-mediated cerebral infarction. The biological effects of GDNF are mediated by GDNF-family receptor α-1 (GFRα-1) and c-Ret. In this study, we examined the levels of expression of GFRα-1 and c-Ret in a rat model of stroke. Adult Sprague–Dawley rats were anesthetized with chloral hydrate. The right middle cerebral artery was ligated at its distal branch for 90 min. Animals were sacrificed at 0, 6, 12, and 24 h after reperfusion and levels of expression of GFRα-1 and c-Ret mRNA were determined by in situ hybridization histochemistry. We found that GFRα-1 mRNA was up-regulated in CA3, dentate gyrus (DG), cortex, and striatum. The peak of up-regulation in DG was 6 h after reperfusion. GFRα-1 mRNA levels in CA3 were gradually up-regulated over the 24-h reperfusion period. In cortex, GFRα-1 mRNA was up-regulated at all time points; however, the peak of up-regulation was observed at 0 and 24 h after reperfusion. In striatum, an initial up-regulation of GFRα-1 was found at 0 h after ischemia. In striatum, up-regulation of c-Ret mRNA was detected as early as 0 h after reperfusion. A gradual increase was found at 6, 12, and 24 h after reperfusion. In conclusion, our results indicate that there are both regional and temporal differences in up-regulation of GFRα-1 and c-Ret after ischemia. Since GDNF is neuroprotective, up-regulation of GFRα-1 and c-Ret could enhance the responsiveness to GDNF and reduce neuronal damage. The selective up-regulation of GFRα-1 and c-Ret in different brain areas suggests that there may be regional differences in GDNF-induced neuroprotection in stroke.