脑缺血和缺血再灌注大鼠皮层神经元自噬的动态变化

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。

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

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 30min,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 1h and 6h 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 6h 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.