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目的:采用全脑缺血大鼠模型研究缺血后不同时间神经细胞内Ca2+浓度的动态改变,从单细胞和分子角度探讨缺血性损伤神经元胞内Ca2+代谢的机制。方法:参照改良的Pulsinelli四血管闭塞法制备全脑缺血大鼠模型,缺血后的大鼠分别在存活2、12、24、48及72h后进行皮层神经细胞急性分离、负载荧光指示剂Fura-2/AM,用钙离子成像系统检测单个神经细胞内Ca2+的浓度。实验随机分为7组:正常组,假手术组,缺血再灌模型2h、12h、24h、48h、72h等5个时间点各一组。结果:缺血再灌注不同时点大鼠大脑皮层神经元内Ca2+浓度变化出现两次跃升:全脑缺血再灌注后2h,大鼠大脑皮层神经元内Ca2+浓度达第1次峰值,再灌12h回落至正常水平,再灌24h又升至第2次高峰,甚至远高于第1次峰值,至48h、72h又有所回落,但仍高于正常水平。结论:缺血后不同时间点神经细胞内Ca2+浓度的两次跃升与神经元缺血性损伤有关。
OBJECTIVE: To study the dynamic changes of intracellular Ca2 + concentration in ischemic neurons at different time points after global ischemia in a rat model of global cerebral ischemia, and to explore the mechanism of intracellular Ca2 + metabolism in ischemic injured neurons from single cell and molecular perspectives. Methods: A rat model of global cerebral ischemia was established according to a modified Pulsinelli four-vessel occlusion method. The rats were sacrificed at 2, 12, 24, 48 and 72 hours after ischemia, respectively. Cortical neurons were isolated acutely and loaded with fluorescent indicator Fura -2 / AM, using a calcium ion imaging system to detect the concentration of Ca2 + in a single nerve cell. The experiment was randomly divided into 7 groups: normal group, sham operation group, ischemia-reperfusion model 2h, 12h, 24h, 48h, 72h and other 5 time points. Results: Ca2 + concentration in cerebral cortex neurons increased two times at different time points after ischemia-reperfusion. Ca2 + concentration in cerebral cortex reached the first peak at 2 hours after global ischemia-reperfusion, 12h back to the normal level, reperfusion 24h and then rose to the second peak, or even higher than the first peak, to 48h, 72h and then fell back, but still higher than normal levels. Conclusions: The two levels of Ca2 + concentration in neurons at different time points after ischemia are related to neuronal ischemic injury.