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目的:探讨缺血缺氧及再灌注损伤后大鼠海马神经元在伤后不同时间的凋亡及死亡特性,阐明神经元损伤发生的时间窗特征。方法:实验于2004-05/06在解放军第三军医大学大坪医院野战外科研究所进行。体外培养胚胎大鼠海马神经元,模拟临床缺血过程致神经元缺氧损伤,采用AnnexinV联合PI标记和流式细胞检测技术观察伤后不同时间点的凋亡与死亡情况。结果:海马神经元缺氧损伤后,其细胞损伤的形式为凋亡和坏死同时存在;模拟缺血15,30min,1.0,1.5,2.0,2.5和3.0h,其坏死率分别为(1.37±0.12)%,(3.71±0.34)%,(16.47±1.27)%,(18.73±2.02)%,(19.77±2.32)%,(22.13±2.12)%和(28.78±2.69)%,相互间差异有显著性意义(P<0.05),各时间点的凋亡率分别为(6.23±1.67)%,(6.56±2.38)%,(6.73±2.42)%,(6.95±1.89)%,(7.16±2.82)%,(6.93±2.56)%和(7.23±2.89)%,凋亡率无明显变化(P>0.05)。各时间点再恢复糖和氧供应后至24h,海马神经元存活率进一步明显下降,坏死率进一步增多,各时间点神经元坏死率分别为(6.98±1.23)%,(8.46±1.37)%,(17.68±2.97)%,(20.79±3.16)%,(21.67±3.05)%,(35.53±3.33)%和(50.22±4.19)%,差异亦有显著性意义(P<0.05);凋亡率亦逐渐增多,模拟缺血15,30min,1.0,1.5和2.0h时间点的再灌注24h,其神经元凋
Objective: To investigate the apoptosis and death characteristics of hippocampal neurons in rat hippocampus after hypoxia-ischemia and reperfusion injury and to elucidate the time-window characteristics of neuronal damage. METHODS: The experiment was performed at the Institute of Field Surgery, Daping Hospital, Third Military Medical University, People’s Liberation Army from May 2004 to June 06. The hippocampal neurons were cultured in vitro to simulate the hypoxia injury of neurons induced by clinical ischemia. The apoptosis and death were observed by Annexin V, PI staining and flow cytometry. Results: After hypoxia injury, the cell injury in hippocampal neurons occurred in the form of apoptosis and necrosis. The rates of necrosis at simulated ischemia of 15, 30, 1.0, 1.5, 2.0, 2.5 and 3.0 h were (1.37 ± 0.12 ), (3.71 ± 0.34)%, (16.47 ± 1.27)%, (18.73 ± 2.02)%, (19.77 ± 2.32)%, (22.13 ± 2.12)% and (28.78 ± 2.69)%, respectively (6.23 ± 1.67)%, (6.56 ± 2.38)%, (6.73 ± 2.42)%, (6.95 ± 1.89)% and (7.16 ± 2.82), respectively %, (6.93 ± 2.56)% and (7.23 ± 2.89)%, respectively. There was no significant change in apoptosis rate (P> 0.05). The survival rate of neurons in hippocampus decreased further and the necrosis rate further increased after resuming the supply of glucose and oxygen at all time points. The neuronal necrosis rates were (6.98 ± 1.23)% and (8.46 ± 1.37)% at all time points, (17.68 ± 2.97)%, (20.79 ± 3.16)%, (21.67 ± 3.05)%, (35.53 ± 3.33)% and (50.22 ± 4.19)% respectively. The difference was also significant (P <0.05) Also gradually increased to simulate ischemia 15,30 min, 1.0,1.5 and 2.0 h point of reperfusion 24h, the neuronal apoptosis