| Co-Investigator(Kenkyū-buntansha) |
FUJIHARA Hideyoshi University Medical Hospital, Niigata University, Lecturer, 医学部・附属病院, 講師 (20251803)
SHIMOJI Koki Niigata Univ., Sch. of Med., Professor, 医学部, 教授 (30040158)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
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
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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
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