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目的 研究大鼠脑局灶性缺血再灌注模型中N 甲基 D天冬氨酸 (NMDA)受体随时间的变化。方法 采用线栓法阻断大鼠大脑中动脉 ,产生局灶性脑缺血再灌注模型 ,快速取脑 ,冰冻切片。以3H MK 80 1作为放射性配基 ,将恒温保温后的标本在室温下氚片上进行放射自显影 ,勾划脑左右两侧感兴趣区 (ROI) ,采用LETCAQ 5 5 0IWL图像分析系统测定并比较缺血侧与对侧ROI内NMDA受体的密度。在受体结合法中 ,分离出缺血区中段皮层 ,匀浆、离心后与放射性配基进行受体结合反应 ,将液闪测得的数值作Scatchard图 ,求出NMDA受体的亲和力 (Kd)及最大结合容量 (Bmax)并进行比较。结果 根据放射自显影图 ,缺血 2h组以及缺血 2h再灌 2h组同假手术组相比 ,相应的NMDA受体密度比值明显升高 ,表明NMDA受体通道大量开放。这主要是兴奋性氨基酸升高所致。再灌注2 4h组及再灌注 72h组相应ROI中NMDA受体开放明显降低 ,且两组无明显差异。各实验组中缺血及再灌注区Kd 值都未发生明显变化 ,表明缺血再灌注前后受体亲和力未发生改变。缺血 2h组及再灌注 2h组Bmax值明显升高 ,再灌 2 4h及 72h组的Bmax明显降低。结论 大鼠脑局灶缺血及再灌注过程中 ,兴奋性氨基酸明显上升并维持数小时 ;NMDA受体在 4h内被大量激活 ,通道开放 ,引起缺血损伤。
Objective To investigate the changes of N-methyl D-aspartate (NMDA) receptor in rats with focal ischemia-reperfusion injury over time. Methods The middle cerebral artery (MCA) was occluded by thread occlusion in rats. A focal cerebral ischemia-reperfusion model was established. Brain and frozen sections were taken quickly. Using 3H MK 80 1 as a radioligand, the isothermally incubated specimens were autoradiographed on trisodium phosphate tablets at room temperature, and the areas of interest (ROI) on both left and right sides of the brain were delineated. The LETCAQ550IWL image analysis system was used to determine and compare Density of NMDA receptors in ischemic and contralateral ROI. In the receptor binding method, the middle cerebral cortex was isolated and homogenized. After centrifugation, the receptor binding reaction with radioactive ligand was carried out. The Scatchard plot was used to determine the affinity of NMDA receptor (Kd ) And the maximum binding capacity (Bmax) and compared. Results According to autoradiograms, the corresponding NMDA receptor density ratio was significantly increased compared with the sham operation group at 2 h ischemia and 2 h ischemia and 2 h reperfusion, indicating that NMDA receptor channels were largely open. This is mainly due to the excitability of amino acids. NMDA receptor opening in the corresponding ROI of 24 h reperfusion group and 72 h reperfusion group was significantly lower, and there was no significant difference between the two groups. There was no significant change in Kd value of ischemia and reperfusion area in each experimental group, indicating that the affinity of receptor did not change before and after ischemia-reperfusion. The Bmax of ischemic 2h group and reperfusion 2h group were significantly increased, while Bmax of 24 h and 72h after reperfusion was significantly decreased. Conclusion During focal cerebral ischemia and reperfusion in rats, the excitatory amino acids significantly increased and maintained for several hours. NMDA receptors were activated in large quantities within 4 hours and the channels were opened to cause ischemic injury.