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

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。

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.