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

不同预处理诱导脑缺血耐受的共同机制的探索

• 批准号: 11671484
• 负责人: ZHAN Ren-zhi
• 金额: $ 2.5万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671484/
• 关键词: apoptosis; Bcl-2; caspases; cytochrome c; ischemic preconditioning; ischemic tolerance; mitochondria; C57Black; Crj6マウス; チトクローム c; 微小脳損傷

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

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CA 1神经元死亡相关，以及Cyte的释放如何影响神经元死亡。海马CA 1区出现早期线粒体Cyte快速耗竭和晚期弥漫性Cyte再分布，而齿状回和CA 3区则无此现象。脑室内注射z-DEVD-FMK（一种相对选择性的caspase-3抑制剂），在再灌注后第7天对缺血性神经元损伤提供了有限但显著的保护。在10分钟缺血前48小时进行3分钟缺血处理（缺血预处理）可减弱细胞再分布的早期和晚期。在另一组用放线菌酮（一种通用的蛋白质合成抑制剂）治疗的动物中， ...更多信息 细胞再分布的抑制，而大多数海马神经元从来没有恢复线粒体细胞。神经元存活的检查显示缺血预处理防止。而放线菌酮仅延迟缺血性海马CA 1神经元死亡。通过末端脱氧转移酶介导的dUTP缺口末端标记原位检测到的DNA片段化在很大程度上被缺血预处理减弱，并被放线菌酮适度减少。结果表明，线粒体Cyte的丢失与短暂性脑缺血后海马CA 1区神经元的死亡有关，这与半胱天冬酶依赖性和非依赖性途径有关。基于Cyte丢失在介导缺血性神经元死亡中的重要性，我们在相同的脑缺血模型中检测微损伤预处理诱导的缺血耐受是否与线粒体Cyte丢失的减轻相关。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天和第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，通过Western印迹分析检测胞质组分中的Cyte水平。免疫组织化学染色检测插入后两种促凋亡相关蛋白bcl-XL和bax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，微损伤预处理被发现减轻了10分钟缺血所引起的DNA断裂的程度。微损伤预处理也可减轻缺血引起的胞浆Cyte增多。微损伤预处理可上调损伤侧海马中抗凋亡蛋白bel-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性反应有关。虽然大鼠脑缺血预适应模型种类繁多，但目前仅报道了小鼠局灶性脑缺血预适应模型。鉴于大多数基因改变已在小鼠中进行，迫切需要建立小鼠缺血预适应模型，以利用基因改变的小鼠研究缺血预适应的分子机制。本研究建立了C57 BL/6小鼠前脑缺血预适应模型。采用双侧颈总动脉阻断（BCCAO）18 min诱导C57 BL/6小鼠前脑缺血，于BCCAO前48 h进行6 min的预处理缺血，于BCCAO后第7天采用微管相关蛋白-2免疫组织化学染色观察神经元损伤，并采用甲酚紫染色定量。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。虽然条件性缺血本身造成轻微的纹状体神经元损伤的一部分动物，它显着减少纹状体神经元损伤所造成的随后18分钟的缺血。考虑到现有多种基因改造的C57 BL/6小鼠。该模型为利用基因修饰小鼠研究缺血预适应的分子机制提供了一个新的模型，综合以上结果，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

项目成果

Ren-Zhi Zhan. 他.5名: "Intravenous anesthetics differentially reduce neurontransmission damage induced by oxygen-glucose deprivation in rat hippocampal slices in correlation with N-methyl-D-aspartate receptor inhibition"Critical Care Medicine. 29(4). (2001)

Ren-Zhi Zhan 等 5 人：“静脉麻醉药可显着减少大鼠海马切片中氧-葡萄糖剥夺引起的神经元传递损伤，与 N-甲基-D-天冬氨酸受体抑制相关”，《重症监护医学》2001 年。

Shimoji K, Taga K, Zhan R-Z, Fujihara H, Honda T, Endoh H, Fukuda S: "Current view on CNS resuscitation"Resuscitation. 19(2)(published in Japanese). 93-108 (2000)

Shimoji K、Taga K、Zhan R-Z、Fujihara H、Honda T、Endoh H、Fukuda S：“中枢神经系统复苏的当前观点”复苏。

せん仁知 他.4名: "大脳皮質視覚野の虚血性神経伝達機能障害に対するNMDAレセプター遮断薬の効果"Brain Hypoxia. 13. 25-31 (1999)

Senichi 等人 4：“NMDA 受体阻滞剂对大脑皮层视觉皮层缺血性神经传递功能障碍的影响”Brain Hypoxia。

Zhan R-Z, Fujiwara N, Taga K, Fukuda S, Shimoji K: "The effect of N-methyl-D-aspartate receptor inhibitors on ischemic neurotransmission damage in the sliced rat visual cortex"Brain Hypoxia. 13(published in Japanese). 35-31 (1999)

Zhan R-Z、Fujiwara N、Taga K、Fukuda S、Shimoji K：“N-甲基-D-天冬氨酸受体抑制剂对切片大鼠视觉皮层缺血性神经传递损伤的影响”脑缺氧。