Molecular and Physiological Bases of Endogenous Cerebral Anti-ischemic Mechanisms and Their Applications to Treatments of Brain Ischemia

在上述项目中，取得了以下成果.用几种预处理方法证实脑抗缺血机制的存在1）虽然在大鼠和沙鼠中有许多种脑缺血预处理模型，但仅报道了小鼠局灶性缺血预处理模型。考虑到大多数遗传改变已在小鼠中进行，利用基因修饰小鼠建立小鼠缺血预适应模型以研究缺血预适应的分子机制是迫切需要的。我们最近在C57 BL/6小鼠上建立了一种前脑缺血预适应模型。用双侧颈总动脉阻断（BCCAO）18 min造成C57 BL/6小鼠前脑缺血，在BCCAO前48 h进行6 min的预处理缺血，在BCCAO后第7天用微管相关蛋白-2免疫组化法检测神经元损伤，并与对照组比较。 ...更多信息 免疫组织化学染色并通过甲酚紫染色定量。缺血18分钟导致纹状体、皮质以及海马的损伤。与海马相比，纹状体神经元损伤更严重且可重复。单纯条件性脑缺血虽然对部分动物造成轻微的纹状体神经元损伤，但明显减轻了随后18 min脑缺血引起的纹状体神经元损伤。该预适应模型可用于利用基因改变的小鼠研究缺血预适应的分子机制。线粒体参与大鼠脑微损伤诱导的缺血耐受与Bcl-XL表达上调的关系基于细胞色素c（Cytc）的丢失，细胞色素c在介导缺血性神经元死亡中起重要作用，我们在大鼠前脑缺血模型中检测了微损伤预处理诱导的缺血耐受是否与线粒体保护有关。在诱导脑缺血前6天，根据实验方案，通过皮质用针插入（25 G）在单侧海马或双侧海马中诱导轻度机械性脑损伤。假手术涉及相同的程序，但没有插入针头。分别于再灌注后第4、7天检测海马CA 1区DNA断裂和缺血性神经元损伤<th><th>，再灌注后6小时用Western blotting法检测胞浆中Cytc的含量。用免疫组化法检测针刺后bcl-XL和bax两种促凋亡相关蛋白的表达。结果表明，微损伤预处理与针刺组织学减少神经元损伤在两个空间模式：保护发生在针道周围的区域或在整个海马损伤部位同侧。此外，微损伤预处理还能减轻缺血10 min引起的DNA断裂程度，并能减轻缺血引起的胞浆Cytc的升高。微损伤可上调损伤侧海马抗凋亡蛋白bcl-XL的表达，提示微损伤预处理诱导的缺血耐受与可保护海马的诱导反应有关.静脉麻醉剂以药物依赖的方式保护神经元免受体外缺血损伤虽然静脉麻醉剂已被认为具有保护脑缺血的作用，但其潜在机制仍不清楚。在体外脑缺血模型中，我们发现静脉麻醉药对缺血性神经传递损伤的保护作用因其对NMDA受体的作用而异，我们的结果表明，抗凋亡蛋白的上调及其相关的线粒体保护参与了内源性脑抗缺血机制。少

In the above mentioned project, following results were obtained.1. Confirmation of the existence of cerebral anti-ischemic mechanisms by using several preconditioning methods1) Although there are many kinds of cerebral ischemic preconditioning models available in the rat and gerbil, only a focal ischemic preconditioning model in mice has been reported Considering that most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning by using gene-altered mice. We have recently developed a forebrain ischemic preconditioning model in C57BL/6 mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO.On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 imm … More unohistochemistry and quantified by cresyl violet staining. Ischemia for 18 min resulted in injury to the striatum, cortex as well as to the hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia alone caused slight striatal neuronal damage in a part of animals, it significantly reduced striatal neuronal damage caused by the subsequent 18-min ischemia Considering many kinds of gene-altered C57BL/6 mice available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning by using gene-altered mice.2) Mitochondria are involved in the ischemic tolerance induced by brain microinjury in relation to an upregulation of Bcl-XL expression in the ratOn the basis of loss of cytochrome c (Cytc) which is important in mediating ischemic neuronal death, we examined if ischemic tolerance induced by microinjury preconditioning is associated with mitochondrial protection in a rat forebrain ischemia model. Six days before induction of cerebral ischemia, mild mechanical brain injury was induced in unilateral hippocampus or in bilateral hippocampi through the cortex with needle insertion (25 G), depending on experiment protocols. Sham surgery involved the same procedures without needle insertion. DNA fragmentation and ischemic neuronal damage in the hippocampal CAl region were examined on the 4^<th> and 7^<th> day after reperfusion, respectively.The levels of Cytc in the cytosolic fraction were examined 6 hours after reperfusion by Western blotting analysis. Expression of bcl-XL and bax, two apoptotic-related proteins after needle insertion was examined by immunohistochemical staining. The results showed that microinjury preconditioning with needle insertion histologically reduced neuronal damage in two spatial patterns : protection occurred in the areas around the needle tract or in the whole hippocampus ipsilateral to the injury site. In addition, microinjury preconditioning was found to attenuate the extent of DNA fragmentation caused by the 10-min ischemia The increased Cytc in the cytosol induced by ischemia was also attenuated by microinjury preconditioning. The expression of bcl-XL, an antiapoptotic protein was upregulaled by microinjury in the hippocampus ipilateral to the injury site.The above results indicate that ischemic tolerance induced by microinjury preconditioning is associated with inducible responses that are capable of protecting mitochondria.2. Intravenous anesthetics protect neurons against ischemic insult in vitro in an agent-dependent mannerAlthough intravenous anesthetics have been known to have protective effed against cerebral ischemia, the underlying mechanisms remain unclear. In an in vitro ischemic model, we found that the effects of intravenous anesthetics in protecting against ischemic neurotransmission damage were different from agent to agent in according to their actions on NMDA receptors.Our results indicate that the upregulation of anti-apoptotic proteins and its related mitochondrial protection are involved in endogenous cerebral anti-ischemic mechanisms. Less