脑缺血再灌流后bFGF基因的表达及MK-801的影响

目的 研究脑缺血再灌流后ｂＦＧＦ基因表达的意义及其机制。方法 采用原位杂交和１免疫组化的方法,观察大鼠脑缺血再灌流后ｂＦＧＦ基因的表达及ＭＫ－８０１对它们的影响。结果 缺血２ｈ再灌流１ｈ可见ｂＦＧＦ表达增高（Ｐ〈０．０５）,ｂＦＧＦ ｍＲＮＡ表达于１２ｈ达高峰,ｂＦＧＦ蛋白于２４ｈ,达高峰;ＭＫ－８０１组与缺血组相比,于再灌流６ｈ～４８ｈ ｂＧＦＧ表达减弱（Ｐ〈０．０５）。结论 局灶性脑缺血再灌流     

大鼠全脑缺血再灌流后脑组织P53及P21蛋白的表达

目的　探讨大鼠全脑缺血再灌流后Ｐ５３、Ｐ２１蛋白的表达及其与迟发性神经元死亡（ＤＮＤ）的关系。方法　在４ 血管闭塞法全脑缺血模型上,采用ＨＥ及ＬＳＡＢ染色法,观察脑组织病理改变,检测脑组织Ｐ５３、Ｐ２１蛋白的表达,以及蛋白合成抑制剂放线菌酮对其的影响。结果　全脑缺血１５ ｍ ｉｎ 再灌流后,脑组织Ｐ５３、Ｐ２１蛋白表达增加,且两者分布接近。海马结构、丘脑、下丘脑等白质区（再灌流后６ ｈ）较皮层、海马的神经细胞核（２４ ｈ）先检测到Ｐ５３、Ｐ２１蛋白,７２ ｈ 表达达高峰。并且以缺血损伤最严重的海马ＣＡ１ 区Ｐ５３、Ｐ２１蛋白表达为强。另外,放线菌酮可抑制脑组织Ｐ５３、Ｐ２１蛋白的表达,并对ＤＮＤ具有一定的保护作用。结论　全脑缺血再灌流损伤后,脑组织Ｐ５３、Ｐ２１蛋白表达增加,放线菌酮可抑制其表达,并对ＤＮＤ起保护作用,提示Ｐ５３、Ｐ２１蛋白参与了全脑缺血后ＤＮＤ的凋亡机制,并对其起促进作用     

汉防己碱对缺血—再灌流大鼠脑NOS活性及超微结构的影响

用ＮＡＤＰＨｄ 组织化学和电镜技术研究了汉防己碱（Ｔｅｔ）对大鼠缺血再灌流脑一氧化氮合酶（ＮＯＳ）活性及超微结构变化的作用。结果发现,缺血１５ ｍ ｉｎ,再灌流２ ｈ 后,海马超微结构出现明显的病理学损害,主要包括核周出现髓鞘样变,核周间隙增大,胞核均质化,大量片状软化灶,毛细血管周围水肿。Ｔｅｔ １０ 和２０ｍ ｇ／ｋｇ 缺血前３０ ｍ ｉｎ 腹腔注射可明显改善或避免出现上述缺血组织学改变。缺血１５ ｍ ｉｎ,再灌流２４ ｈ 后,皮层和海马ＮＯＳ阳性细胞数目显著增多,Ｔｅｔ５,１０ 和１５ ｍ ｇ／ｋｇ 缺血前３０ ｍ ｉｎ 腹腔注射,浓度依赖地抑制缺血再灌流所致的ＮＯＳ阳性细胞数目增加。这些结果提示,Ｔｅｔ可抑制缺血再灌流脑ＮＯＳ活性,减少ＮＯ产生,这可能与Ｔｅｔ减轻脑缺血再灌流的损害有关。     

实验性局灶性脑缺血及再灌流后AP-1复合物的结合活性测定

本研究应用凝胶迁移法检测局灶脑缺血区中Ｆｏｓ与Ｊｕｎ基因的蛋白产物形成异源二聚体复合物—转录因子ＡＰ１（ａｃｔｉｖａｔｏｒｐｒｏｔｅｉｎ１）的ＤＮＡ结合活性。缺血３０ｍｉｎ或９０ｍｉｎ伴随再灌流４ｈ，结果表明ＡＰ１结合活性增加。提高的ＤＮＡ结合活性持续２４ｈ。以上结果说明提高转录因子ＡＰ１的表达水平在缺血病变的基因调节改变中起一定作用     

脑缺血选择性海马CA1区神经元损害的实验研究

采用Ｐｕｌｓｉｎｅｌｉ－Ｂｒｉｅｒｌｅｙ４血管阻塞脑缺血模型观察了大鼠全脑缺血２０ｍｉｎ再灌流８ｈ，ｃ－ｆｏｓ基因表达及再灌流７ｄ海马ＣＡ１区迟发性神经元损害。在缺血再灌流早期（８ｈ）海马ＣＡ１区极少ｃ－ｆｏｓ表达，而齿状回、海马ＣＡ３区、杏仁核大量ｃ－ｆｏｓ表达。缺血再灌流晚期（７ｄ）镀银染色显示海马ＣＡ１区神经元及其突触终末带呈黑色溃变相，而齿状回、海马ＣＡ３区、杏仁核呈金黄色正常相。相邻切片ＨＥ染色示缺血组海马ＣＡ１区核完整的锥体细胞数（５±２．６个／２００μｍ）与对照组（４０±２．９个／μｍ）比较差异有显著意义（Ｐ＜０．０１）。脑缺血诱导的ｃ－ｆｏｓ基因表达对于缺血易损海马ＣＡ１区迟发性神经元坏死可能起直接的调控作用。     

老龄大鼠全脑缺血再灌注部分脑区NGF、BDNF的表达

目的　探讨老龄大鼠全脑缺血再灌注损伤对颞叶、杏仁核和尾状核 Ｎ Ｇ Ｆ、 Ｂ Ｄ Ｎ Ｆ 的含量影响。方法　采用 Ｐｕｌｓｉｎｅｌｌｉ Ｂｒｉｅｒｌｅｙ ４ 血管阻塞法制作全脑缺血 １５ｍ ｉｎ、再灌注 １ｈ、６ｈ、２ｄ、４ｄ、９ｄ 模型，检测颞叶、杏仁核和尾状核 Ｎ Ｇ Ｆ、 Ｂ Ｄ Ｎ Ｆ 的表达。结果　全脑缺血再灌注损伤时，颞叶迅速出现中量 Ｎ Ｇ Ｆ 表达；杏仁核仅再灌注早期出现 Ｎ Ｇ Ｆ 表达量增加；尾状核 Ｎ Ｇ Ｆ 的表达很快消失。缺血再灌注损伤没有引起颞叶、杏仁核的 Ｂ Ｄ Ｎ Ｆ表达量改变；但引起尾状核 Ｂ Ｄ Ｎ Ｆ表达量增加。结论　颞叶具有较好的 Ｎ Ｇ Ｆ 保护机制，杏仁核对缺血再灌注损伤具有反应迅速、短期的 Ｎ Ｇ Ｆ 保护机制，而尾状核缺乏良好的 Ｎ Ｇ Ｆ 保护机制。颞叶和杏仁核具有良好的 Ｂ Ｄ Ｎ Ｆ 保护机制，尾状核具有反应迅速、作用持久的 Ｂ Ｄ Ｎ Ｆ保护机制。     

谷氨酸载体在鼠脑缺血神经元死亡中的作用

目的研究谷氨酸载体在脑缺血神经元死亡中的作用。方法用原位杂交和免疫组织化学方法观察大鼠脑缺血后脑内谷氨酸载体ＧＬＡＳＴｍＲＮＡ和胶质纤维酸性蛋白（ＧＦＡＰ）表达的变化，用药理分析手段观察谷氨酸载体摄取抑制剂Ｌ反式吡咯烷２，４二羧酸（ＬｔｒａｎｓＰＤＣ）对脑缺血致神经元损伤的影响。结果脑缺血后２４小时，大脑皮层缺血周边区ＧＬＡＳＴｍＲＮＡ和ＧＦＡＰ表达无显著变化；而缺血后７２小时，缺血周边区ＧＬＡＳＴｍＲＮＡ和ＧＦＡＰ表达都显著增加，脑缺血后２４小时和７２小时，缺血侧海马ＣＡ１区ＧＬＡＳＴｍＲＮＡ表达则无显著变化；与侧脑室注射生理盐水组相比，侧脑室注射１μｇ或２μｇＬｔｒａｎｓＰＤＣ有使脑梗塞体积增大的趋势，但差异无显著意义；侧脑室注射５μｇＬｔｒａｎｓＰＤＣ使脑梗塞体积显著增大。结论脑缺血后谷氨酸载体功能可能呈代偿性增强，提示谷氨酸载体在限制脑缺血时谷氨酸神经毒中起重要作用。     

脑缺血再灌流大鼠海马egr-1及bcl-2基因的表达

目的探讨脑缺血后再灌流海马结构选择性易损伤的分子机制。方法采用阻断大鼠两侧颈总动脉结合放血制备前脑缺血再灌流损伤动物模型,应用Ｎｏｒｔｈｅｒｎ杂交、原位杂交及免疫组织化学技术,检测了ｅｇｒ－１及ｂｃｌ－２基因的表达和组织学分布。结果发现易损伤的海马ＣＡ１区锥体细胞的ｅｇｒ－１及ｂｃｌ－２基因表达受抑制,而耐受缺血的海马ＣＡ３区锥体细胞则明显表达这两种蛋白,并且这两种基因表达的组织学分布具有惊人的一致性。结论提示ＥＧＲ－１及ＢＣＬ－２蛋白参与损伤神经元的修复,对神经元有保护作用;ＥＧＲ－１蛋白可能参与ｂｃｌ－２基因表达的调控。     

大鼠脑缺血和再灌流过程脑组织一氧化氮合成酶表达的变化

目的　探讨大鼠脑缺血再灌流海马及皮层一氧化氮合酶（ＮＯＳ） 的变化。方法　用线栓法建立大脑中动脉梗死（ＭＣＡＯ） 模型,用烟酰胺腺嘌呤二核苷酸磷酸黄递酶（ＮＡＤＰＨｄ） 染色法观察ＮＯＳ阳性细胞的变化。结果　海马及皮层ＮＯＳ阳性细胞在缺血１５ 分明显增多,１ 小时减少,６ 小时有所恢复;再灌流１５ 分又显著减少,１ 小时渐增多,２４ 小时皮层出现较多ＮＯＳ阳性的毛细血管和大量胶质细胞。结论　本实验结果符合ＮＯ 在缺血早期增加和再灌流后期大量增加的变化,支持ＮＯ参与脑缺血再灌流损害的观点。     

大鼠可逆性全脑缺血再灌流过程中几个脑区Na~＋K~＋－ATP酶活性的动态变化

应用大鼠４血管关闭（４ＶＯ）方法制作全脑缺血再灌流模型，观察海马、新皮层及皮层下结构在缺血及再灌流不同时相Ｎａ＋Ｋ＋─ＡＴＰ酶活性变化。缺血时即刻出现海马Ｎａ＋Ｋ＋─ＡＴＰ酶活性降低（Ｐ＜０．０５），再灌流后新皮层及皮层下酶活性有暂时性降低，海马的酶活性在再灌流１６８ｈ时仍未恢复（Ｐ＜０．０５）。Ｎａ＋Ｋ＋─ＡＴＰ酶活性的降低与自由基对酶结构的直接破坏、膜磷脂降解以及不饱和脂肪酸过氧化引起界面脂或膜流动性改变有关。     

Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.     

Hyperbaric oxygen treatment decreases post-ischemic neurotrophin-3 mRNA down-regulation in the rat hippocampus.

The therapeutic effect of hyperbaric oxygen (HBO) on ischemic injury was investigated using in situ hybridization to detect the mRNA expression of neurotrophin-3 (NT-3), which is thought to play a crucial role in protecting against neuronal death induced by brain ischemia. The rats under investigation were subjected to 10 min transient forebrain ischemia, and subsequently exposed to HBO (100% oxygen, 2.5 atm absolute) for 2 h. Levels of NT-3 mRNA in the CA1, CA2 and CA3 regions, and the dentate gyrus of the hippocampus were measured after various reperfusion periods. Neuronal death in the hippocampal CA1 region was also measured by Nissl staining, seven days post ischemia. The results demonstrated that HBO treatment significantly reduced the ischemia-induced down-regulation of the NT-3 mRNA level at 4 h post ischemia, and significantly increased cell survival 7 days after reperfusion. The findings suggest that an HBO treatment maintaining the NT-3 mRNA level in the hippocampus can be beneficial to the ischemic brain within a certain time frame.     

Alteration of serum/glucocorticoid regulated kinase-1 (sgk-1) gene expression in rat hippocampus after transient global ischemia

Expression of the serum/glucocorticoid regulated kinase-1 (sgk-1) gene has been reported to be induced by various stress stimuli such as hyper- or hypo-osmotic stress, UV irradiation, and heat shock stress; however, its association with global ischemia in the brain has not been studied. Using high-density oligonucleotide array analysis, we found that the sgk-1 gene was one of the genes showing alteration of expression in the rat hippocampus during 1-4 h of reperfusion after 10 min of transient global cerebral ischemia. Using TaqMan RT-PCR analysis, we confirmed an increased level of sgk-1 gene expression with statistical significance in the rat hippocampus at 2 h of reperfusion after 10 min of transient global cerebral ischemia. Using in situ hybridization (ISH) analysis, the increased level of sgk-1 gene expression was found to localize in pyramidal cells of CA2 and CA3 regions of the hippocampus after 2 h of reperfusion. These results provide an insight into the alterations of sgk-1 gene expression in the rat hippocampus after transient global cerebral ischemia.     

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.     

Transient upregulation of the AT2 receptor mRNA level after global ischemia in the rat brain

The present study examined changes in angiotensin receptors (AT1 and AT2) and angiotensinogen mRNA level after global ischemia in the rat brain. The AT2 mRNA level increased by three-fold in both the cortex and hippocampus, which are known to be sensitive to ischemic injury, 3 h after ischemia. The increase thus appeared only during the early reperfusion period. In the striatum, amygdala and cerebellum, the level increased moderately 3 h and/or 24 h after ischemia; there was no change in the hypothalamus. On the other hand, the AT1A and AT1B receptor mRNA levels were not altered in the cortex or hippocampus during the early reperfusion period, even 3 h and 24 h after ischemia. There was no significant alteration in angiotensinogen mRNA level 3 h or 24 h after ischemia. These results suggest that the transient upregulation of AT2 receptor mRNA occurs in the cortex and hippocampus after injury and these changes may be in some way related to the molecular events which lead to delayed neuronal cell death.     

Somatostatin type 2 receptor expression in the rat hippocampus following cerebral ischemia.

We examined how transient cerebral ischemia affects the mRNA expression, and the immunoreactive distribution, of the somatostatin type 2 (sst2) receptor in the adult rat hippocampus. Following reperfusion, sst2 mRNA levels increased significantly in the CA1 region by 3 h, and were also increased in the CA3 and CA4/hilus subfields at 6 and 12 h. At 24 h, however, sst2 receptor mRNA levels returned to baseline throughout the hippocampus. At the protein level, we found the regional immunoreactivity of the sst2a receptor was maintained, or slightly elevated, throughout the hippocampus at 6 h, but not different from control at 24 h. These results suggest that sst2 receptors maintain their normal distribution and prevalence in the post-ischemic hippocampus before the deterioration of the vulnerable CA1 neurons. Thus, they represent attractive targets for neuroprotective interventions.     

两种钙通道拮抗剂抑制脑缺血复灌后转录因子c—Jun的表达

目的：研究两种钙离子通道：NMDA受体型和L－型电压门控钙通道（L－VGCC）在短暂全脑缺血复灌后海马c-Jun表达中的作用。方法：采用SD大鼠四动脉结扎全脑缺血模型，取缺血复灌不同时间（0,1,3,6,12,24和72h)以及对照组2D大鼠的海以，应用免疫印迹的方法来研究c-Jun的表达并观察几种钙通道拮抗剂对c-Jun表达的影响。结果：在假手术以及复灌的各个时间点均有表达，并在复灌6h达到高峰。氯胺酮（一种非竞争性NMDA受体拮抗剂）和硝苯吡啶（一种L－VGCC阻滞剂）抑制c-Jun表达的增加。而DNQX（一种AMPA／KA受体拮抗剂）则无抑制作用（数据未显示）。结论：缺血复灌后，c-Jun的表达增加了，这种增加与NMDA受体和L－VGCC这两种钙离子通道的开放有关。     

Expression of Bax and Bcl-2 protein in the gerbil hippocampus following transient forebrain ischemia and its modification by phencyclidine

Abstract

To determine the effect of phencyclidine (a noncompetitive NMDA receptor antagonist) on expression of Bax and Bcl-2 (apoptosis-regulating proteins) in gerbil hippocampus after transient forebrain ischemia, brain sections were immunohistochemically evaluated 48, 72, 96 hand 7 days following ischemia. In ischemic control animals, the expression of Bax in CA 7 neurons was increased with time and peaked at 72 h, then disappeared at 96 h. In the phencyclidine (5 mg kg^-1 , intraperitoneally)-treated animals, the intensity of Bax expression at 72 h was weaker than that of ischemic control animals. Furthermore, at 96 h, Bax expression was still observed in CA1 neurons. No expression of Bcl-2 in the CA1 neurons was detected in either control or phencyclidine-treated animals. From these results, it is possible that NMDA receptor antagonists exert their preventive effect against delayed neuronal death through inhibition of Bax protein expression, although the precise relationship between the function of Bax protein and delayed neuronal death is still unclear. [Neural Res 1997; 19: 629-633]     

Activation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn-PSD95-NR2A complex after transient brain ischemia

Recent studies have indicated that tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) by Src family kinases (Src, Fyn, etc.) up-regulates NMDA receptors activity and postsynaptic density protein 95 kDa (PSD95) may mediate the regulation. To investigate whether the above processes are involved in brain ischemia-induced enhancement of NMDA receptors function, we examined the effects of transient (15 min) brain ischemia followed by reperfusion on interactions involving Fyn, NR2A and PSD95 in rat hippocampus by co-immunoprecipitation. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague-Dawley rats. Association between Fyn and NR2A increased immediately after brain ischemia and the increase was maintained for at least 24 h during followed reperfusion, up to about 1.7-1.8-fold relative to sham-groups. The 15-min reperfusion after brain ischemia induced enhanced co-immunoprecipitation of PSD95, Fyn and NR2A with one another. The associations of PSD95 with Fyn and NR2A increased at 0-24 h, 0-1 h of reperfusion, up to 6.9- and 2.1-fold relative to sham groups, respectively. Inhibiting activation of NMDA receptors or L-type voltage-gated calcium channels (L-VGCC) by ketamine or nifedipine attenuated the above increases of associations. These results suggest that stimulation of NMDA receptors and L-VGCC facilitates formation of a ternary complex: Fyn-PSD95-NR2A during transient brain ischemia followed by reperfusion, which may result in potentiation of NMDA receptor function and contribute to ischemic neuronal cell death.