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背景:癫痫持续状态可导致神经元损伤。目的:观察两种不同给药途径诱发大鼠海马CA3区神经元线粒体超微结构损伤及Fas、Bax、Caspase-3的表达变化,为减轻癫痫后神经元损伤提供理论依据。设计:随机对照动物实验。单位:山东大学齐鲁医院神经内科和麻醉科。材料:实验于2005-03/2005-07在山东省医学科学院病理实验室完成。选择成年雄性SD大鼠150只,体质量260～300g(由山东大学实验动物中心提供(SCXK20030004),室温,自由进水、进食。方法:选择成年雄性SD大鼠150只,随机数字表法分为海人酸腹腔注射组和尾静脉注射组。分别采用海人酸腹腔注射和尾静脉注射诱发不同潜伏期的大鼠癫痫持续状态。每组再分5个亚组,分别为癫痫持续状态后3,6,24,48及72h组。每小组取12只诱发成功的大鼠,于癫痫持续状态终止后不同时点取海马。2只用于电镜检查,5只用于Fas和Bax的RT-PCR检测,5只用于Caspase-3的免疫组化。另取12只鼠作为正常对照,不做任何处理。对照组大鼠于相应癫痫持续状态后24h点取材,具体分配同各亚组。电镜观察线粒体的超微结构,半定量RT-PCR检测Fas、Bax的mRNA水平,免疫组化检测Caspase-3蛋白的表达。主要观察指标:①超薄切片透射电镜观察结果。②RT-PCR检测结果。③免疫组化检测结果。结果:132只SD大鼠进入结果分析。①电镜观察线粒体的超微结构:海人酸腹腔注射组线粒体肿胀,神经元呈凋亡征;尾静脉注射组线粒体肿胀且伴膜的崩解,神经元呈坏死表现。②对照组及尾静脉注射组未检测到Fas及BaxmRNA;海人酸腹腔注射组Fas及Bax的mRNA表达均于癫痫持续状态后6h出现,24h增加,48h达高峰,并持续至72h。③两组大鼠均在癫痫持续状态后6h出现Caspase-3表达增高(10.27±0.34,15.21±0.34;P<0.001),24h达顶峰(25.36±0.47,28.23±0.47;P<0.001);海人酸腹腔注射组高表达持续至72h,尾静脉注射组在48h点急剧降低。结论:两种不同的诱发方式导致了大鼠不同程度的线粒体损伤和Fas、Bax及Caspase-3在不同水平的表达,进而决定了神经元死亡的分子机制。
Background: Status epilepticus can result in neuronal damage. OBJECTIVE: To observe the ultrastructural damage of mitochondria and the expression of Fas, Bax and Caspase-3 in hippocampal CA3 region of rats induced by two different routes of administration, and provide a theoretical basis for alleviating the neuronal damage after epilepsy. Design: Randomized controlled animal experiments. SETTING: Department of Neurology and Anesthesiology, Qilu Hospital of Shandong University Materials: The experiment was performed in the Pathology Laboratory of Shandong Academy of Medical Sciences from March 2005 to July 2005. 150 adult male Sprague-Dawley rats weighing 260-300 g (SCXK20030004) were fed at room temperature with free water intake.Methods: One hundred and fifty adult male Sprague-Dawley rats were randomly divided into three groups: random number table For the kainic acid intraperitoneal injection group and tail vein injection group were used intraperitoneal injection of hyaluronic acid and tail vein injection of different latency induced status epilepticus in rats each subdivided into 5 subgroups, respectively, after the status epilepticus 3 , 6, 24, 48 and 72h groups.Each group received 12 rats successfully induced hippocampus at different time points after the termination of status epilepticus, 2 were used for electron microscopy, 5 were used for Fas and Bax RT- PCR, 5 were used for the immunohistochemistry of Caspase-3. Another 12 rats were used as normal control without any treatment.The rats in the control group were drawn at 24 hours after the corresponding status epilepticus and assigned to the same subgroup. The ultrastructure of mitochondria was observed by electron microscope, the mRNA levels of Fas and Bax were detected by semi-quantitative RT-PCR and the expression of Caspase-3 protein was detected by immunohistochemistry.MAIN OUTCOME MEASURES: ①Through ultrathin section transmission electron microscopy observation.②RT-PCR detection results . ③ immunohistochemistry test results. Results : 132 SD rats were involved in the result analysis.①The ultrastructure of mitochondria was observed by electron microscopy: mitochondria were swollen in the intraperitoneal injection of kainic acid, the neurons were apoptotic; the mitochondria swelled and disintegrated in the tail vein injection group, the neurons The expression of Fas and Bax mRNA were not detected in the control group and tail vein injection group. The mRNA expressions of Fas and Bax in intraperitoneal injection of kainic acid were all increased at 6h after the status epilepticus, increased at 24h and reached the peak at 48h 72h.③The expression of Caspase-3 increased at 6h after epileptic seizure in both groups (10.27 ± 0.34,15.21 ± 0.34; P <0.001), peaked at 24 h (25.36 ± 0.47,28.23 ± 0.47; P <0.001) .The expression of Fas, Bax and Caspase-3 were significantly different in kainic acid intraperitoneal injection group for 72h, and the tail vein injection group decreased sharply at 48h.Conclusion: Two different inducing methods lead to different degrees of mitochondrial damage and Fas, Bax and Caspase-3 in rats The level of expression, which in turn determines the molecular mechanism of neuronal death.