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

1)用反义GluR210nM验证GluR2假说，将5μl GluR2反义寡核苷酸（ODN）每隔12hs注射大鼠脑室4次。末次注射后2 d，可见海马CA1、CA3锥体层部分细胞丢失。GluR2 mRNA和蛋白含量在细胞丢失前降低。CNQX和Naspm可挽救细胞损伤，而AP5对GluR2反义神经元死亡无影响。GluR2反义蛋白还能加速CA1锥体细胞亚致死性（2min）全身缺血的损伤。这些发现有力地支持了GluR2在延迟性神经元死亡领域的假说。2)新型AMPA受体拮抗剂的研制及全脑缺血治疗窗口期的确定沙鼠双侧颈总动脉闭塞后0、6、24h分别脑室内注射不同浓度的萘酰乙酰精胺（Naspm）。在缺血7天后，仅在0和6h注射10mM Naspm对CA1锥体细胞有新的保护作用。这些结果表明，早期应用AMPA拮抗剂对神经保护是必要的。3)间隙连接在脑缺血中的作用我们采用原位杂交和定量Western blotting检测连接蛋白32敲除小鼠背景菌株C57BL/6小鼠全脑缺血后连接蛋白32、36、43 mRNA和蛋白的变化。全脑缺血后各连接蛋白的mRNA和蛋白均有相同水平的轻度升高。与野生型小鼠相比，Connexin 32敲除小鼠对10min全脑缺血的易感性增加。提示间隙连接对缺血性损伤具有神经保护作用。

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.