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The novel analyzing assay for neuroprotection of the inhalation anesthetics by immuno-precipitation and the protein kinases related cerebral ischemia.

通过免疫沉淀和与脑缺血相关的蛋白激酶对吸入麻醉剂进行神经保护的新型分析方法。

基本信息

批准号：
17591652
负责人：
MATSUMOTO Shohei
金额：
$ 2.37万
依托单位：
Tokyo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Male C57/Black mice were decapitated after inhalation of isoflurane or sevoflurane, and N-methyl-D-aspartate (NMDA) receptor subunits NR2A, NR2B were measured. The interaction between these subunits and calcium-calmodulin dependent protein kinase II (CaMKII) was also measured. Immuno-precipitation and western blot analysis were used for these studies.Method : Mice ischemia model was made by decapitation. After decapitation, the skull was incubated in the 37℃ saline 3 or 5 min, then stored-80℃. Previous to the decapitation, 1MAC or 2MAC isoflurane or sevoflurane were inhalated. The cerebrum was dissected from the skull in the cold globe box (-28℃), then homogenized. Using these samples immuno-precipitation was performed targeting to phosphor-tyrosine. Then the samples were assayed by SDS-PAGE, following western blot analysis was performed with anti-NR2A antibody and anti-NR2B antibody. Likewise, the interaction between phosphor-CaMKII and NR2A, B subunits were also examined.Results : NR2A phosphorylation was decreased when isoflurane and sevoflurane inhalation. The change of NR2B was not obvious. There is a tendency that the change of NR2A phosphorylation is dozing dependent for inhalation anesthetics. The interaction between phosphor-CaMKII and NR2A. B subunits has not been evident so far.Conclusion : Inhalation anesthetics prevent CaMKII translocation to synaptic membrane, subsequently the interaction between phosphor-CaMKII and NR2A is decreased, resulting down regulation of NR2A phosphorylation which reeds to neuroprotecition.

雄性C57/黑鼠吸入异氟醚或七氟醚后断头，测定N-甲基-D-天冬氨酸(NMDA)受体亚单位NR2A、NR2B。此外，还检测了这些亚基与钙调素依赖性蛋白激酶II(CaMKII)的相互作用。采用免疫沉淀和免疫印迹分析。方法：采用断头法制备小鼠脑缺血模型。断头后，将颅骨置于37℃生理盐水中孵育3~5min，然后保存-80℃。断头前分别吸入1MAC或2MAC异氟醚或七氟醚。在冷球盒(-28℃)中将大脑从颅骨中分离出来，然后匀浆。利用这些样品进行了针对磷酸酪氨酸的免疫沉淀。用抗NR2A抗体和抗NR2B抗体进行免疫印迹分析。结果：吸入异氟醚和七氟醚后，NR2A的磷酸化水平明显降低。NR2B变化不明显。吸入麻醉药对NR2A磷酸化的影响有催眠依赖的趋势。磷酸盐-CaMKII与NR2A的相互作用。结论：吸入麻醉药抑制了CaMKII向突触膜的移位，进而减少了磷酸化CaMKII与NR2a的相互作用，导致NR2A磷酸化水平下调，从而影响神经保护作用。