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Cell biological study of glutamate receptor and gap jujunction in delyed neuronal death

谷氨酸受体和间隙连接在延迟性神经元死亡中的细胞生物学研究

基本信息

批准号：
11671383
负责人：
OGURO Keiji
金额：
$ 2.3万
依托单位：
Jichi Medical School
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-11671383/
关键词：
cerebral ischemia glutamate receptor hippocampus antisense gap junction connexin

项目摘要

1) Certification of GluR2 hypothesis by antisense GluR210nM, 5μl GluR2 antisense oligonucleatide (ODN) was injected into the rat ventricle 4 times by 12hs interval. At 2 days after the last injection, partial cell loss of pyramidal layer of hippocampal CA1 and CA3 could be seen. GluR2 mRNA and protein were reduced prior to the cell loss. CNQX and Naspm could save the cell damage, but AP5 had no effect on neuronal death by GluR2 antisense. GluR2 antisense also accelerate the damage of CA1 pyramidal cells by sublethal (2min) global ishcemia. These findings strongly suggest the GluR2 hypothesis in the field of delayed neuronal death.2) Development of new AMPA receptor antagonists and the determination of therapeutic window of global ischemiaVarious concentration of Naphtyl acetyl spermine (Naspm) was intraventricularly injected at the time of 0,6,24hs after the bilateral common carotid occlusion in gerbil. Only 10mM Naspm injcted at 0 and 6hs afford neueo protection of CA1 pyramidal cells on 7days after the global ischemia. These results suggest that early application of AMPA antagonists are needed for neuroprotection.3) Roles of gap junction in cerebral ischemiaWe examined the changes in connexin 32,36 and 43 mRNA and protein after global ischemia by in situ hybridization and quantitative Western blotting in C57BL/6 mice, which is the backgroud strain of connexin 32 knockout mouse. All of the mRNA and protein of each connexin were the same lavel of slightly increased after global ischemia. Connexin 32 knockout mice showed increased susceptibility to 10min global ischemia compared to the wild type mice. These findings suggest the neuroprotective role of gap junction to the ischemic damage.

1)用GluR 2反义寡核苷酸（ODN）210 nM证明GluR 2假说，将5μl GluR 2反义寡核苷酸（ODN）注射到大鼠心室内，间隔12 h，共注射4次。末次注射后第2天，海马CA 1、CA 3区锥体细胞层可见部分细胞丢失。GluR 2 mRNA和蛋白在细胞损失前减少。CNQX和Naspm可挽救细胞损伤，而AP 5对GluR 2反义引起的神经元死亡无影响。GluR 2反义核酸还可加速亚致死性（2 min）缺血对海马CA 1区锥体细胞的损伤。2）新型AMPA受体拮抗剂的研制及全脑缺血治疗窗的确定在沙土鼠双侧颈总动脉夹闭后0、6、24 h侧脑室注射不同浓度的萘乙酰精胺（Naspm）。全脑缺血后7天，仅在0和6 h注射10 mMNaspm对海马CA 1区锥体细胞有神经保护作用。3）缝隙连接在脑缺血中的作用我们以C57 BL/6小鼠为实验动物，采用原位杂交和定量Western blotting技术，检测了C57 BL/6小鼠全脑缺血后缝隙连接蛋白32、36和43 mRNA和蛋白的表达变化。全脑缺血后各连接蛋白的mRNA和蛋白表达水平均相同或略有升高。与野生型小鼠相比，Connexin 32敲除小鼠对10分钟全脑缺血的易感性增加。提示缝隙连接对缺血性损伤具有神经保护作用。