氯化锂-匹罗卡品诱发大鼠癫痫持续状态神经元凋亡反应的年龄特征

Age Difference of the Activation of Apoptotic Cascade Reaction Following LiCl-pilocarpine Status Epilepticus

目的探讨氯化锂-匹罗卡品诱发癫痫持续状态(statusepilepticus,SE)后大鼠脑内神经元凋亡过程及Caspase3活性的年龄特征。方法氯化锂-匹罗卡品诱发幼年及成年Wistar鼠持续惊厥发作,比较惊厥发作严重程度的年龄差异,应用原位末端标记、流式细胞仪和荧光分光光度法测定惊厥后脑组织神经细胞凋亡的动态变化及Caspase3活性变化,比较研究其年龄差异特征。结果幼年鼠从给药至惊厥发作的时间为(13.3±5.63)min,而成年鼠为(22.5±5.66)min;幼年鼠的首发SE中4～5级者占68%,而成年鼠仅18%;尽管SE前幼年鼠脑内凋亡细胞数较成年鼠“生理性”增多,但SE后30min,成年鼠脑内(海马CA3区、齿状回、颞叶皮层)TUNEL阳性细胞总数即已明显增多并反超幼龄鼠,分别达524±26和465±26(P<0.05)。即使连续观察到SE后8h,成年鼠脑内凋亡细胞计数也始终明显高于幼年鼠。经流式细胞仪检测的凋亡早期神经细胞也呈现相似变化。与幼龄鼠相比,成年鼠不仅Caspase3活性增高的幅度大,且启动时间早。SE后仅30min,成年鼠海马中Caspase3活性增加的比例已明显超过幼年鼠,分别为0.1±0.07和0.003±0.04。SE后2h,两者差别更大,分别为0.39±0.2和0.1±0.2。结论由氯化锂-匹罗卡品诱发的惊厥持续状态模型再次证实SE发作中未成熟脑内呈现一个主动抑制细胞凋亡进程、对抗惊厥性脑损伤的保护性机制,并提示该保护机制作用于Caspase3级联反应被激活前的细胞凋亡早期事件中。

Objective To explore the age character of the activity of Caspase 3 and neuron death induced by LiCl-pilocarpine status epilepticus. Methods LiCl-pilocarpine was injected into healthy infant rats (19 days) and adult rats (2-3 months) subcutaneously and intra-abdominally to evoke status epilepticus(SE). First, the age difference of the seizure was used to measure the sensitivity of seizure. Second, the dynamic features of the apoptotic neurons and the activity of Caspase 3 at 15, 30 min and 1, 2, 4, 8 hours after SE respectively were investigated by TUNEL, flow cytometry and fluorospectrophotometry. Results ① The average duration from the injection to seizure was (13.3±5.63) min in infant rats, and (22.5±5.66) min in adult rats. ② The proportion of the 4th or 5th degree of severity at onset of seizure was 68% in infant rats and 18% in adult rats. ③Although the count of died neurons(in the CA3 of hippocampus, dentate gyrus and cortex of temporal lobe) was physiologically higher in normal infant rats than in adult rats, the count of positive neurons by TUNEL stain in mature brain(524±26) remarkably increased and exceeded that in premature brain(465±26) at 30 min after SE. Although continuously observed until 8 hours after SE, the count of apoptotic neurons in mature brain was also remarkably higher than that in infant brain. Change of neurons in apoptotic early events detected by flow cytometry was the same as the result of TUNEL. ④The increasing proportion of activity of Caspase 3 after SE for 30 min in adult rats remarkably exceeded that in infant rats; it was 0.10±0.07 in adult rats and 0.003±0.04 in infant rats. The difference between the infant rats(0.39±0.20) and adult rats(0.10±0.20) increased after SE for 2 hours. Conclusion A mechanism of inhibiting apoptotic process in premature brain during SE for the protection against brain damage was well reconfirmed by different animal SE models induced by lithium-pilocarpine. It was indicated that the protective mechanism against brain damage in premature brain could be presented in most severe seizures of different types. This protective mechanism could act on the apoptotic occurrence in the earlier period before the activation of Caspase cascade reaction.