不同时间异氟醚麻醉对幼龄大鼠海马神经元凋亡及自噬的影响

摘要

Studies have shown that isoflurane could cause autotrophy and apoptosis in the develo ping rat brain.Based on the preliminary experiment,forty 7 day Sprague-Dawley rat pups were selected and divided into control group and groups of 1.5 ％ isoflurane anesthesia for 2,3,4 and 6 h randomly.After anesthesia,the rats were euthanized,then hippocampus were collected for TUNEL-staining,LC3-Ⅱ immunohistochemistry and Western blotting,to investigate the level of autophagy and apoptosis.The results showed that with the increasing time exposed to isoflurane,the percent of positive cells were significantly increased at 3 h,the results of LC3-Ⅱ immunohistochemistry showed that the percent of positive cells were increased at 3 and 4 h,but decreased at 6 h;and the expression of Caspase-3 was increased gradually compared with control group,although the expression of Bcl 2 was decreased.However,the expression of Beclin-1 and LC3-Ⅱ/I were increased at the first 4 h with the most expressions at 3 h (P＜0.05) and then decreased;In addition,compared with isoflurane anesthesia at 3 and 4 h,the expression amount of Beclin 1 was significantly decreased at 6 h while Caspase-3 significantly increased (P＜0.05).These results showed that isoflurane can not only induce extensive apoptosis of hippocampal neurons in young SD rats,but also induce autophagy in hippocampal neurons.And the level of apoptosis gradually increased with the prolongation of anesthetic time,while the level of autophagy increased first and then decreased.Autophagy may play a protective role in the apoptosis of hippocampal neurons induced by isoflurane.%相关研究发现异氟醚可引起幼龄大鼠广泛的脑神经元凋亡,且近期发现异氟醚还可以介导海马神经元发生自噬.在前期试验的基础上,选取40只7日龄(P7)SD大鼠幼鼠,随机分为对照组和1.5％异氟醚麻醉2、3、4、6h组,建立实验模型.待麻醉结束后将大鼠安乐死,取脑并分离海马组织.用TUNEL染色、LC3-Ⅱ免疫组化及Western blot检测等方法检测海马神经元自噬及凋亡情况.试验结果显示,随着异氟醚作用时间的增加,TUNEL染色结果显示,麻醉4h后海马阳性细胞率明显增多,而LC3 Ⅱ免疫组化结果显示在3、4h时阳性率较高,6h有所下降;与对照组相比,活化的Caspase 3表达量逐渐增加;Bcl-2表达量逐渐降低.Beclin-1和LC3-Ⅱ/I表达量先升高后下降,并且在异氟醚作用3h时表达量明显升高(P＜o.05);此外,与异氟醚麻醉3、4h时相比,Beclin-1在麻醉6h时表达量明显下降,而Caspase-3表达量明显升高(P＜0.05).试验结果表明,异氟醚不仅可以引起幼龄SD大鼠海马神经元广泛凋亡,而且还能诱导海马神经元自噬,凋亡水平随麻醉时间延长逐渐加强,而自噬水平先升高后降低.自噬在异氟醚诱导的海马区神经元凋亡中可能发挥着保护性作用.