Mitochondrial and nuclear damages and caspase-3 expression in the hippicampal CA3 region of rats with kainic acid induced statuse pilepticus

摘要

BACKGROUND: Some scholars believed that the neuronal injury after status epilepticus is apoptosis,the main evidence is the changes of expressions of various apoptosis releted genes,such as immediate-early gene,p53 gene and genes of bcl-2 family,etc.But there is still no ultrastructural evidence for apoptosis.OBJECTIVE: To observe the ultrastructural damages of mitochondrion and nucleus and the changes of caspase expression in neurons of hippocampal CA3 region in rats with status epilepticus induced by kainic acid.DESIGN: A randomized controlled study.SETTING: Department of Anesthesiology and Department of Neurology,Qilu Hospital of Shandong University.MATERIALS: Seventy-five adult male Wistar rats of 250-300 g.clean degree,were provided by the experimental animal center of Shandong University.Kainic acid was purchased from Sigma Company (USA);rabbit anti-rat polyclonal antibody caspase-3 from Santa Cruz Company(USA).METHODS:The experiments were carried out in the Department of Anesthesiology,Qilu Hospital of Shandong University from October 2005 to February 2006.①The 75 rats were randomly divided into experimental group (n=45)and control group(n=30).②Model establishment,convulsion grading and the judging standards for status epilepticus:Rats in the experimental group were given intraperitoneal injection of kainic acid(10 mg/kg),and those in the control group were injected with saline of the same volume.The time of seizure was recorded and their behavioral manifestations were observed,and the seizure was terminated by intraperitoneal injection of diazepam(10 mg/kg).③Observation under electron microscope:At 3, 12 and 24 hours after status epilepticus respectively,bilateral hippocampal tissues were taken out,semithin sections of about 75 nm were prepared after fixation,dehydration and embedding,and then observed under H-800 transmission electron microscope.④Immunohistochemical detection:Bilateral hippocampi were removed at 3,12 and 24 hours after status epilepticus respectively,the fixation,dehydration,transparence,wax immersion and embedding were performed,then serial sections of CA3 region were immunohistochemically determined by the SABC method.Leica QWinV3 image analytical software was applied,then the average number and average gray value of positive cells were calculated.MAIN OUTCOME MEASURES:Results of observation under electron microscope,that of immunohistochemical staining of neurons in hippocampal CA3 region;Comparison of number of caspase-3 positive cells and gray value.RESULTS: All the 75 Wistar rats were involved in the analysis of results.①Results of observation under electron microscope:At 3 hours after status epilepticus,swelling crista and membranous disintegration were observed under electron microscope.At 24 hours,obvious nuclear changes occurred,and manifested as the side-aggegation of chromatins.②Results of immunohistochemical detection:In the experimental group,the number of caspase-3 positive cells at 3 hours after status epilepticus had no obvious difference as compared with that in the control group(P＞0.05):At 12 hours,the number and gray value of caspase-3 positive cells in the experimental group were higher than those in the control group (10.49±0.68 vs.5.33±0.43;45.57±2.27 vs,19.79±0.33,P＜0.05),the same results were also observed at 24 hours(37.36±0.57 vs.5.12±0.47;115.24±1.22 vs.18.73±0.42,P＜0.01).CONCLUSION:In the rat models of status epilepticus induced by kainic acid,mitochondrial damage was earlier than the increase of caspase-3 expression and nuclear changes,which suggested that mitochondrion was the key link for the neuronal death after status epilepticus.