Changes In Mitochondrial Fission In Hippocampal Neurons Of Lithium-pilocapine-induced Epileptic Rats

BackgroundEpilepsy is a group of chronic brain diseases characterized by recurrent ep isodic central nervous system dysfunction,which was caused by abnormal disc harge of neurons.Long-term seizures following epilepsy not only can affect the life quality of the patients seriously,but also cause heavy burden to the patients and the whole society.Although it has been known that seizures can c ause the neuronal injury,which will remarkably increase the risk of potential se izure,At present,the mechanisms underlying the neuronal injury induced by seiz ures have not been studied well,Moreover,strategies to protect the injury induce d by seizures are still limited.Mitochondria are double–membrane–bound subcellular organelles,which serves a critical role as a control platform for energy metabolism signal transduction and cell apoptosis in eukaryotic cells.Neurons are metabolically active cells with high energy demands in biological body,which are particularly dependent on mitochondrial function.Mitochondrial dysfunction have attracted considerable attention in the epilepsy research in recent years.Mitochondria are highly dynamic organelles that constantly fuse and divide in living cells,Mitochondrial fusion can make mitochondria a reticulum of elongated and branched filaments,while mitochondrial fission can collapse the reticular structure to fragment.In normal conditions,the dynamic balance mitochondrial fusion and fission(collectively termed mitochondrial dynamics)lead to a continuous remodeling of the mitochondrial network and morphological changes to adapt to the different needs of physiological activity.Because mitochondrial network dynamics are highly sensitive to various pathological and physiological stimuli,the abnormal pathological state such as oxidative stress,ischemia and aging can disturb the the balance mitochondrial fusion and fission.Recent discoveries have highlighted that the disfunction of mitochondrial fission and fusion play prominent roles in the pathological processes of neuronal injury,cell apoptosis and mitophagy in many neurodegenerative diseases,such as Parkinson's disease,Alzheimer's disease,Huntington disease,amyotrophic lateral sclerosis.Dynamin-related protein 1 is essential to regulate mitochondrial fusion in eukaryotic cells.Mitochondrial division inhibitor(mdivi-1)acts as a selective inhibitor of a mitochondrial fission protein Drp1 by inhibiting the activity of GTP enzyme.It has been demonstrated that the mitochondrial oxidative stress injury mediated the pathological processes in epilepsy.However,at present,the role of mitochondrial fission in the neuronal injury induced by epileptic seizures is still unknown,and whether Mdivi-1 have neuroprotective effect on the neuronal injury induced by epileptic seizures and its mechanism are little reported.ObjectiveThe changes of The behavior,neuron loss,cell apoptosis and expression of Drp1 and apoptosis related substances(such as caspase-3,cytochrome C,and AIF apo ptosis-inducing factor)in the epileptic rats and the preconditioning rats by Mdivi-1 was observed by the the Lithium-pilocapine-induced epileptic rats model in order to investigate the effect of mitochondrial fission on the epileptic neuron injury and study the the neuroprotective effects of mitochondrial division inhibitor Mdivi-1 on the epileptic neuron injury.Materials and Methods96 SD rats(weight 200-250 g,healthy adult male)were randomly divided into four groups(PILO group,PILO + Mdivi-1 group,PILO + DMSO group and normal control group).The rats wereinjected intraperitoneally lithium(injection concentrstion 127mg/kg)-pilocapine(injection concentrstion 30mg/kg)to induce SE in PILO group,PILO + DMSO group and PILO+ Mdivi-1 group,and the rats in PILO+DMSO group and PILO+Mdivi-1 group were respectively given DMSO(0.1%)and Mdivi-1(injection concentrstion 1.2mg/kg)intraperiton-eally 30 min before the injection of pilocarpine;the rats of CON group were given equal volume of normal saline intraperitoneally instead of pilocarpine,DMSO and Mdivi-1.When Status epilepticus lasted 1h,the seizures were terminated by the diazepam(injection concentrstion 10mg/kg)intraperitoneal injection.The incubation period of seizures and epileptogenic rate were observed and recorded in the Lithium-pilocapine-induced epileptic rats according to the Racine seizure grade.After the EEGs of antemortem rats were recorded,The rats were anesthetized and decapitated at 24 h after SE,and the bilateral hippocampus of rats were rapidly isolated.The Drp1 m RNA expression levels in hippocampus of rats was detected with the RT-PCR assay.The expression of Drp1,caspase-3,cytochrome C and AIF in rat hippocampus were detected with Western blot assay.After anaesthetized at 72 h after SE,the remaining rats were perfused to death.The neuron injury was detected with the Nissl staining and the neuronal cell apoptosis was detected with the TUNEL assay in hippocampus of rats.We used the mean ± standard deviation(x_± s)to expresse the statistical data,and used SPSS 17.0 satatistical software the analyze statistical data in the study.The the successful epileptogenic rate was analyzed with Chi-Square Tests.one-way analysis of variance(one-way ANOVA)was used to Compare among many groups.the data between two groups was analyzed with LSD-t(Least—Significant Difference).?=0.05 is regarded as the significant level.Results1: The behavior and EEGs of rats:The behavior and EEGs of rats were normal in CON group.After intraperitoneal injection by pilocarpine,The seizures grade of rats reached ?/?or SE acording to the Racine criterion in PILO group,PILO + DMSO group and PILO + Mdivi-1 group.The incubation period of the seizures and successful epileptogenic rate in there groups had no statistically significant difference(p> 0.05).After the intraperitoneal injection by pilocarpine,more high-amplitude sharp waves,spike waves and sharp(spike)and slow wave complex discharge in the EEGs of three groups rats was recorded2:Pathological results2.1 Nissl staining resultsCompared with CON group,the obvious neuronal loss and the neuron number reduction were observed in the CA3 area of rat hippocampus in PILO group,PILO+DMSO group and PILO+Mdivi-1 group at 72 h after SE(p<0.05).Compared with PILO group,the neuron number was reduced in the CA3 area of rat hippocampus in PILO+DMSO group(p>0.05)at 72 h after SE.Compared with PILO group,the neuron number were observed to increase significantly in the hippocampal CA3 area of in PILO+Mdivi-1 group rats at 72 h after SE(p<0.05)2.2 TUNEL assay resultsCompared with CON group,the neuronal apoptosis cell number were observed to increase significantly in the CA3 region of rat hippocampus in PILO group,PILO+DMSO group and PILO+Mdivi-1 group(p<0.05)at 72 h after SE.Compared with PILO group,the neuronal apoptosis cell number was no significant reduction in the CA3 region of rat hippocampus in PILO+DMSO group(p>0.05).Compared with PILO group,the neuronal apoptosis cell number were observed to increase significantly in the CA3 region of rat hippocampus in PILO+Mdivi-1 group at 72 h after SE(p<0.05).3.Western blot analysis resultsCompared with CON group,the expression of Drp1,caspase-3 activation,Cyt C release and AIF translocation were observed significantly in rat hippocampus in PILO group,PILO+DMSO group and PILO+Mdivi-1group at 24 h after SE(p<0.05).Compared with PILO group,t the expression of Drp1,caspase-3 activation,Cyt C release and AIF translocation were observed significantly in the rat hippocampus in PILO+DMSO group at 24 h after SE(p>0.05).Compared with PILO group,the expression level of Drp1 was reduced in the hippocampus of rats of PILO+Mdivi-1 group at 24 h after SE(p>0.05),and Cyt C release,AIF translocation and caspase-3 activation were significantly reduced in the hippocampus of rats of PILO+Mdivi-1 group at 24 h after SE(p<0.05).4.RT-PCR assay resultsCompared with CON group,the Drp1 m RNA expression was observed to increase significantly in the rat hippocampus in PILO group,PILO+DMSO group and PILO+Mdivi-1 group at 24 h after SE(p<0.05).Compared with PILO group,the expression of Drp1 m RNA was reduced significantly in the hippocampus of rats of PILO+DMSO group(p>0.05)and PILO+Mdivi-1 group at 24 h after SE(p>0.05).Conclusion1: The Drp1 and Drp1 m RNA expression were observed to increase significantly in the rats hippocampus after SE,and mitochondrial fission could be involved in the pathologically epileptogenic processes2: Mdivi-1 might reduce neuronal apoptosis in the hippocampus of rats by inhibiting the Cyt C release,AIF translocation and caspase-3 activation for neuroprotection of the epileptogenic rats.The inhibition of mitochondrial fission is hopeful to be a new strategies for neuroprotection in neuronal injury after seizures.