全脑缺血再灌注后海马神经元凋亡及组织NO含量动态变化的研究

目的:观察全脑缺血15 m in后再灌注不同时间点海马神经元凋亡及海马组织NO含量的动态变化。方法:采用4血管闭塞法制备大鼠全脑缺血再灌注模型,采用DNA琼脂糖凝胶电泳和流式细胞仪检测缺血15 m in后再灌注1 d、2 d、3 d、5 d、7 d时海马神经元凋亡情况;硝酸还原酶法检测各时间点海马组织中NO含量。结果:DNA琼脂糖凝胶电泳显示:在再灌注1 d时呈大致正常条带,2 d、3 d呈“梯状条带,”5和7 d出现了代表部分神经元坏死的涂抹状条带;流式细胞仪检测结果显示:在脑缺血再灌注1 d时,海马神经元凋亡率即明显增加,再灌注3 d时凋亡率达高峰,随后凋亡率逐渐下降,7 d时大致达正常水平;海马组织中NO含量于再灌注后2 d明显升高,3 d达峰值,以后逐渐下降,7 d降至接近对照组水平。结论:全脑缺血再灌注后海马神经元凋亡和NO含量均于再灌注3 d时达高峰,7 d大致恢复至正常水平,提示NO增多可能是诱导海马神经元凋亡的机制之一。

Dynamic changes in hippocampus neuronal apoptosis and nitric oxide content of hippocampal tissues after global cerebral ischemia-reperfusion

Objective:To investigate the dynamic changes in apoptosis of hippocampal neurons and nitric oxide content of hippocampal tissues at different time during global cerebral ischemia-reperfusion.Methods:Transient(15 min) global cerebral ischemia was induced by the four-vessel occlusion method invented by Pulsinelli,and reperfusion was instituted for 1,2,3,5,7 d respectively.The neuron apoptosis was identified and analyzed by DNA agarose gel electrophoresis and flow cytometry.Nitrate/nitrite(NO_(3)~-/NO_(2)~-) was assayed to represent nitric oxide content of hippocampal tissues.Results:DNA electrophoresis showed normal bands at 1d reperfusion,ladder pattern band representing apoptosis of neurons at 2 d and 3 d reperfusion.There was no ladder pattern band but bands representing necrosis of some neurons at 5 d and 7 d reperfusion.Flow cytometry showed that apoptotic rate of hippocampal neurons increased as reperfusion time extending and raised to the peak at 3 d reperfusion.Subsequently,apoptotic rate reduced gradually and returned to normal level at 7 d reperfusion.Nitric oxide content of hippocampal tissues increased significantly at 2 d reperfusion and peaked at 3 d reperfusion,then decline and returned to sham group level at(7 d) reperfusion.Conclusion:Both apoptotic rate of hippocampal neurons and nitric oxide content of hippocampal tissues showed dynamic changes after global cerebral ischemia-reperfusion.Two changes were consistent in the course of time.It is suggested that increasing in NO production maybe one of the factors inducing neuronal apoptosis.