两种不同给药途径诱发大鼠海马神经元线粒体损伤及Fas、Bax、Caspase-3表达(英文)

Mitochondrial damage and expressions of Fas,Bax and Caspase-3 in hippocampal neurons of rats induced by different ways of administration

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 injury. OBJECTIVE： To observe the mitochondrial ultrastructural damage and the changes of Fas, Bax and Caspase-3 expressions in hippocampal CA3 neurons of rats of different kindling, so as to provide theoretical evidence for the neuronal injury after epilepsy. DESIGN： A randomized c ntrol animal experiment. SETTINGS： Department of Neurology and Department of Anesthesiology, Qilu Hospital of Shandong University. MATERIALS： The experiments were carried out in the pathological laboratory of Shandong Academy of Medical Sciences between March and July 2005. Totally 150 adult male SD rats of 260-300 g were provided by the experimental animal center of Shandong University （SCXK20030004）, they were raised at room temperature and were free to the access of food and water. METHODS： The adult male Sprague Dawley （SD） rats were divided into intraperitoneal injection of kainie acid group and caudal venous injection of kainie acid group respectively according to the method of random number table, and the rats were administrated by kainic acid injected intraperitoneally （12 mg/kg） and v/a caudal vein （10 mg/kg） respectively, Each group was divided into 5 subgroups, which were 3, 6, 24, 48 and 72 hours after status epilepticus groups respectively. Twelve successfully induced rats were selected from each subgroup, hippocampi were removed at different time points after the termination of status epilepticus, 2 were used for examination under electron microscope, 5 for the reverse transcription-polymerase chain reaction （RT-PCR） detection of Fas and Bax, and 5 for the immunohistochemical assay of Caspase-3. Another 12 rats were used as normal controls without any treatment. The materials were taken at 24 hours after corresponding status epilepticus in the control group, and the specific distributions were the same as those in the subgroups. The mitochondrial structure was observed under electron microscope, the levels of Fas and Bax mRNA were detected with semi-quantitative RT-PCR, and the expression of Castmse-3 protein was determined with the immunohistochemical assay. MAIN OUTCOME MEASURES： ① Results of ultrathin section under transmission electron mcroscope; ② RT-PCR results; ③ Immunohisto- chemical results. RESULTS： Totally 132 rats were involved in the analysis of results. ① Mitochondrial structure under electron microscope： In the intraperitoneal injection group, the mitochondria swelled, and the neurons showed characters of apoptosis. In the caudal venous injection group, the mitochondria swelled, and accompanied by the membranous collapse, and the neurons manifested the necrosis.②No expression of Fas and Bax was detected in the control group and caudal venous injection. In the intraperitoneal injection group, Fas expression appeared at 6 hours after status epilepticus, increased at 24 hours, reached the peak value at 48 hours, and lasted till 72 hours. ③ The Caspase-3 expressions began to increase 6 hours after status epilepticus in both the intraperitoneal injection group and caudal venous injection group（10.27±0.34, 15.21±0.34; P 〈 0.001）, and reached the peak values at 24 hours （25.36±0.47, 28.23±0.47; P 〈 0.001）; The higher expression of Caspase-3 lasted till 72 hours in the intraperitoneal injectiongroup, but sharply decreased in the caudal venous injection group. CONCLUSION： Two different methods of administration result in different severity of mitochondrial damage and different expressions of Fas, Bax and Caspase-3, which further determines the molecular mechanisms of neuronal death.