脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

Dynamic changes of autophagy activity of cortical neurons in rats with cerebral ischemia and cerebral ischemia/reperfusion

目的:探讨脑缺血和缺血/再灌注不同时间大鼠大脑皮层神经元自噬的变化。方法:健康雄性SD大鼠60只,随机分为:假手术(Sham)组(n=10),脑缺血和缺血/再灌注模型组(n=50).模型组分别在缺血30min、2h,缺血2h再灌注1h、6h、24h五个时间点,随机抽取10只大鼠,测定脑梗死体积和脑含水量,同时采用Western印迹法测定各组大鼠大脑皮层中微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)的水平,透射电镜检测大脑皮层神经细胞自噬情况。结果:脑缺血30min时LC3-Ⅱ/Ⅰ比值未见明显上升,缺血2h时LC3-Ⅱ/Ⅰ比值开始升高,明显高于Sham组(P<0.01);缺血/再灌注1h、6h时LC3-Ⅱ/Ⅰ比值虽较缺血2h组有所下降,但仍明显高于Sham组(P<0.05);缺血/再灌注24h时LC3Ⅱ/Ⅰ比值达高峰,明显高于Sham组(P<0.01)。透射电镜观察进一步证实该现象。缺血/再灌注6h和24h时大鼠脑梗死体积明显增加,与Sham组比较有统计学差异(P<0.01)。缺血/再灌注24h大鼠脑组织含水量明显增加,明显高于Sham组(P<0.05)。HE染色显示:仅在缺血/再灌注24h组大鼠皮层见组织水肿、疏松,部分细胞变性、凋亡,海马区见大量神经元细胞核皱缩、深染呈变性凋亡状。结论:局灶性脑缺血和缺血/再灌注模型中大脑皮层缺血2 h神经元自噬即明显激活,缺血/再灌注1 h、6 h自噬均持续增高,缺血/再灌注24 h自噬达高峰。

Objective： To investigate the changes of autophagy in cerebral cortex neurons at different times after cerebral ischemia and ischemia/reperfusion（ I/R） in rats. Methods： Healthy male SD rats（ n = 60） were randomly assigned into sham-operation group（ sham,n = 10）,cerebral ischemia and ischemia/reperfusion model group（ I/R,n = 50）. In the model group,10 rats were randomly sampled at five different time points： ischemia 30 min,2 h,and reperfusion 1 h,6 h and 24 h after ischemia 2 h. Cerebral infarction volume and cerebral water content were measured. Autophagy of cerebral cortical neurons was examined by transmission electron microscopy（ TEM）. The protein levels of microtubule associated protein light chain 3-II（ LC3-II） in cerebral cortex of the rats in each group were determined by Western blot. Results： LC3-Ⅱ/Ⅰ ratio had no obvious rise at ischemia 30 min,and it began to increase at ischemia 2 h point which was significantly higher than that in sham group（ P 〈0. 01）. LC3-Ⅱ/Ⅰ ratio was still higher at ischemia/reperfusion 1 h and 6 h points than that in sham group（ P 〈0. 05）,although it was decreased comparison with that at ischemia 2 h point. LC3-Ⅱ/Ⅰ ratio reached a peak at ischemia/reperfusion 24 h point,which was significantly higher than that in sham group（ P 〈0. 01）. This phenomenon was further confirmed by the transmission electron microscopy. The infarction volume at ischemia/reperfusion 6 h and 24 h points was increased significantly,and there was a statistical difference compared with sham group（ P〈 0. 01）. The content of water in the brain tissue of rats at ischemia/reperfusion 24 h was increased obviously,which was significantly higher than that in sham group（ P 〈0. 05）. Edema,rarefaction,some cell degeneration and apoptosis only in the cortical tissue of rats at ischemia/reperfusion 24 h were showed,and a large number of neurons nuclei in the hippocampus was shrunken,hyperchromatic showing degeneration and apoptosis by HE staining. Conclusion： Neurons autophagy in cerebral cortices of rats with focal cerebral ischemia and ischemia/reperfusion was obviously activated at ischemia 2 h point,it was increased continuously at ischemia/reperfusion 1 h and 6 h points and reached the peak at ischemia/reperfusion 24 h point.