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

在上述项目中，获得了以下结果：几种预处理方法证实脑抗缺血机制的存在1)虽然大鼠和沙鼠的脑缺血预处理模型多种多样，但目前只报道了小鼠的局灶性脑缺血预处理模型，考虑到大多数遗传改变都是在小鼠身上进行的，建立小鼠缺血预处理模型是利用基因改变小鼠研究缺血预处理分子机制的迫切需要。我们最近建立了C57BL/6小鼠前脑缺血预处理模型。双侧颈总动脉闭塞（BCCAO） 18 min诱导8 ~ 10周龄C57BL/6小鼠前脑缺血。在BCCAO 18 min前48 h进行持续6 min的条件性缺血损伤。在BCCAO后第7天，用微管相关蛋白-2 imm法观察神经元损伤，并进行非组织化学染色和甲酚紫染色。缺血18分钟后，纹状体、皮质和海马均出现损伤。与海马相比，纹状体神经元损伤更严重，可重复性更强。虽然单独条件缺血会对部分动物造成轻微纹状体神经元损伤，但可显著减轻后续18min缺血引起的纹状体神经元损伤。考虑到C57BL/6基因改变小鼠的种类众多，该预处理模型可能有助于利用基因改变小鼠研究缺血预处理的分子机制。2)线粒体参与脑微损伤诱导的缺血耐受，其机制与脑内Bcl-XL表达上调有关。基于介导缺血性神经元死亡的细胞色素c （Cytc）的缺失，我们在大鼠前脑缺血模型中研究了微损伤预处理诱导的缺血耐受是否与线粒体保护有关。在脑缺血诱导前6天，根据不同的实验方案，分别在单侧海马或双侧海马通过皮质针插入（25 G）诱导轻度机械脑损伤。假手术涉及相同的手术过程，但没有针头插入。在再灌注后第4天、第7天分别检测海马CAl区DNA断裂和缺血性神经元损伤。再灌注6小时后，采用Western blot检测细胞浆中Cytc的水平。免疫组化染色检测针刺后细胞凋亡相关蛋白bcl-XL、bax的表达。结果表明，针刺微损伤预处理在组织学上以两种空间模式减少了神经元损伤：针束周围区域和损伤部位同侧的整个海马。此外，我们发现微损伤预处理可以减弱10min缺血引起的DNA断裂程度，微损伤预处理也可以减弱缺血引起的细胞质中Cytc的增加。抗凋亡蛋白bcl-XL在损伤部位同侧海马的表达上调。上述结果表明，微损伤预处理诱导的缺血耐受与能够保护线粒体的诱导反应有关。静脉麻醉药在体外以药物依赖的方式保护神经元免受缺血性损伤尽管静脉麻醉药已知对脑缺血有保护作用，但其潜在机制尚不清楚。在体外缺血模型中，我们发现静脉麻醉药对缺血性神经传递损伤的保护作用根据其对NMDA受体的作用不同而不同。我们的研究结果表明，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