A search for the common mechanism underlying cerebral ischemic tolerance induced by different preconditionings

1).在大鼠前脑缺血模型中，我们研究了线粒体细胞色素c （Cyte）的丢失是否与海马CA1神经元死亡相关，以及细胞释放如何影响神经元死亡。再灌注后，海马CA1区出现线粒体细胞早期快速耗竭和晚期弥漫性再分布，而齿状回和CA3区未出现线粒体细胞弥漫性再分布。脑室内给予相对选择性的caspase-3抑制剂z-DEVD-FMK，在再灌注后第7天对缺血性神经元损伤提供有限但显著的保护。在10分钟缺血前48小时进行3分钟缺血预处理（缺血预处理）可减弱细胞重分布的早期和晚期。在另一组用环己亚胺（一种一般蛋白质合成抑制剂）治疗的动物中，晚期细胞再分配受到抑制，而大多数海马神经元从未重新获得线粒体细胞。对神经元存活的检查显示，缺血预处理可防止。而环己亚胺仅延缓缺血性海马CA1神经元死亡。通过末端脱氧转移酶介导的dUTP-nick末端原位标记检测到的DNA片段被缺血预处理大大减弱，环己亚胺适度减少。结果表明，线粒体细胞的丢失与短暂性脑缺血后海马CA1神经元死亡有关，并与caspase依赖性和非依赖性途径相关2)。基于细胞丢失在介导缺血性神经元死亡中的重要作用，我们在相同的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细胞丢失的衰减有关。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CA1区DNA断裂和缺血性神经元损伤。再灌注6小时后，用Western blotting法检测细胞质组分中细胞水平。免疫组化染色检测插入后凋亡相关蛋白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轻10min缺血引起的DNA断裂程度。微损伤预处理也能减弱缺血引起的细胞质中细胞的增加。抗凋亡蛋白bel-XL在损伤部位的同侧海马中表达上调。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。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局灶性脑缺血预处理模型。由于大多数基因改变都是在小鼠身上进行的，因此急需建立小鼠缺血预处理模型，利用基因改变小鼠来研究缺血预处理的分子机制。本课题建立了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。BCCAO后第7天，采用微管相关蛋白-2免疫组化观察神经元损伤，甲酚紫染色定量。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。虽然调节性缺血本身造成部分动物纹状体神经元轻微损伤，但明显减轻后续18min缺血引起的纹状体神经元损伤。考虑到多种基因改变的C57BL/6小鼠可用。该预适应模型可用于研究基因改变小鼠缺血预适应的分子机制。综上所述，我们认为线粒体保护可能是不同条件诱导的缺血耐受的共同特征。少

1). In a rat forebrain ischemia model, we examined whether loss of cytochrome c (Cyte) from mitochondria correlates with hippocampal CA1 neuronal death and how Cyte release may shape neuronal death, Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 min. After reperfusion, an early rapid depletion of mitochondrial Cyte and a late phase of diffuse redistribution of Cyte occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-min ischemia attenuated both the early and late phases of Cyte redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase … More of Cyte redistribution was inhibited, whereas most hippocampal neurons never regained mitochondrial Cyte. Examination of neuronal survival revealed that ischemic preconditioning prevents. whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. The results indicate that the loss of Cyte from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways.2). Based on the loss of Cyte is important in mediating ischemic neuronal death, we examined if ischemic tolerance induced by microinjury preconditioning is associated with an attenuation of Cyte loss from mitochondria in the same cerebral ischemic 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 CA1 region were examined on the 4^<th> and 7^<th> day after reperfusion, respectively. The levels of Cyte 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 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 Cyte in the cytosol induced by ischemia was attenuated by microinjury preconditioning also. The expression of bel-XL, an antiapoptotic protein was upregulated 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.3). Although there are many kinds of rat and gerbil cerebral ischemic preconditioning models available, only a focal ischemic preconditioning model in mice has been reported. Considering that most genetic altertions 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. In this project, we 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 immunohistochemistry 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 itself 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.Taken the above results together, we conclude that mitochondrial protection may be a common feature underlying ischemic tolerance induced by different conditions. Less