Grant-in-Aid for Scientific Research (A).
|Research Institution||Niigata University|
SHIMOJI Koki Niigata Univ., Sch. Of Med. Professor, 医学部, 教授 (30040158)
岡本 学 新潟大学, 医学部附属病院, 助手 (70303146)
TAGA Kiichiro Niigata Univ., Sch. Of Med., Associate Professor, 医学部, 助教授 (00163329)
KUMANISHI Toshiro Niigata Univ., Brain Research Institute, Professor, 脳研究所, 教授 (40018601)
ZHAN Ren-zhi Niigata Univ., Sch. Of Med., Assistant, 医学部, 助手 (20303125)
|Project Fiscal Year
1998 – 2000
Completed(Fiscal Year 2000)
|Budget Amount *help
¥25,400,000 (Direct Cost : ¥25,400,000)
Fiscal Year 2000 : ¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 1999 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Fiscal Year 1998 : ¥17,600,000 (Direct Cost : ¥17,600,000)
|Keywords||ischemic tolerance / ischemic preconditioning / brain microinjury / cyctochrome c / N-methyl-D-aspartate receptor / mitochondria / apoptosis / C57Black / Crj6 mouse / Bcl-XL / intravenous anesthetic / 脳虚血耐性 / 虚血前処置 / 微小脳損傷 / NMDAレセプター / チトクロームc / ミトコンドリア / アポトーシス / Crj6マウス / 静脈麻酔剤 / NMDA レセプター / チトクローム c / 脳虚血 / 抗脳虚血(虚血耐性) / 免疫組織化学 / 3-ニトロプロピオン酸 / NMDA受容体 / spreading depression / サイトカイン|
In the above mentioned project, following results were obtained.
1. Confirmation of the existence of cerebral anti-ischemic mechanisms by using several preconditioning methods
1) 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
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 rat
On 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 manner
Although 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