The Effect Of Mitochondrial Division Inhibitor On Hippocampal Neurons In Pilocarpine-induced Seizures And The Mechanism

Background and objectiveEpilepsy (epilepsy, EP) is a clinical syndrome which caused by excessive oroversynchronized discharges of cerebral neurons, and be characteristic with transient,stereotyped, repetitive and episodic. Long-term and repeated seizures not only canseriously degrade the quality of patients’ life, but also bring heavy economic burdenTo the patient’s family and society, so it has important practical significance toinvestigate the pathogenesis of epilepsy, mastered the pathogenesis of epilepsy canprovide a new therapeutic target. The pathogenesis of epilepsy is very complicated.Many reports explore the possibility mechanisms of pathogenesis of epilepsy fromthe neurotransmitters, ion channels, glial cells, gap junctions, synaptic transmission,immunology and molecular biology for a long time. Mitochondria are a major placefor oxidative phosphorylation in eukaryotes, so have an important role in providingenergy for activities in cells. Therefore the research of mitochondria has become anew hotspot in the research of the epilepsy in recent years. Numerous studies havedemonstrated that seizures can cause the disorder of mitochondrial dynamics anddysfunction, resulting in cell necrosis and apoptosis. These changes increase thesensitivity of neurons to the neuronal injury cause by epileptic, resulting in the formof the vicious cycle, which is "seizure-mitochondrial dynamics change- mitochondrial dysfunction-increased neuronal injury after seizures ".Mitochondrialdivision inhibitor (Mdivi-1) is a kind of derivatives of quinazoline. It has the effect toinhibit the activity of GTP enzyme, consequently it can inhibit the function ofDrp1.The latest research reports that because of Mdivi-1can inhibit the function ofDrp1, Mdivi-1can mitigate the disorder of mitochondrial dynamics caused by avariety of obstacles, which leading Mdivi-1can significantly reduce cell apoptosis inthe model of myocardial ischemia-reperfusion injury and acute kidney injury. DoesMdivi-1have neuroprotective effect on hippocampal neuron damage caused byepileptic seizures? What is the mechanism? Currently, we only know a little about it.By lithium chloride-pilocarpine induced epilepsy rats model, this study observedthe change of behavior of each group of rats, hippocampal neuronal injury andapoptosis, the levels of cytochrome C, apoptosis-inducing factor and caspase-3, inorder to find out the effect of Mdivi-1on hippocampal neurons in epilepsy modelsand explore its possible mechanism.Marerials and MethodsEighty-eight healthy adult male SD rats,6-8weeks, about200-250g. They wererandomly divided into four groups: CON group, PILO group, PILO+DMSO groupand PILO+Mdivi-1groups, each group has twenty-two rats. Everyone of the SD ratsis preprocessed by intraperitoneal inject of lithium chloride(127mg/kg), theintraperitoneal injection of pilocarpine (30mg/kg)twenty hours later, the subcutaneousinjection of scopolamine hydrobromide (1mg/kg)should30min before the injectionof pilocarpine, used to antagonize the peripheral cholinergic response of pilocarpine.PILO+Mdivi-1group also need to intraperitoneal injected of Mdivi-1(1.2mg/kg)30min before the injection of pilocarpine, PILO+DMSO group need tointraperitoneal injected of DMSO (0.1%)30min before the injection of pilocarpine.The control group intraperitoneal inject of equal volume of normal saline instead ofpilocarpine. When the Status epilepticus lasts60min diazepam (10mg/kg) beintraperitoneal injection to terminate seizures. Take the Racine seizures gradingcriteria as standard, when seizures up to Ⅳ/Ⅴ grade the rat is model-making success. The levels of seizures, duration, success rate and incubation period of each rat arerecord. Half of the rats were anesthetized SE72h after taking brain perfusion, Nisslstaining and TUNEL assay and apoptosis of neuronal damage, the remaining ratswere anesthetized after24h SE decapitated, isolated bilateral hippocampus; Westernblot assay the levels of Drp1, cytochrome C (Cyt C), apoptosis-inducing factor (AIF)and caspase-3(caspase-3); RT-PCR assay the levels of Drp1mRNA.Results1. Behavior observation of rats: the rats of CON group don’t have any kind ofseizures, the rats of PILO group, PILO+Mdivi-1and PILO+DMSO group haveseizures, and the seizure grade of them up to Ⅳ-Ⅴ grade, there is no significantdifference between the success rate and incubation period of each group(p>0.05);2. Pathology observation of rats: the result of Nissl staining showing thatcompared with CON group, hippocampal neurons have obvious damage of PILOgroup, PILO+Mdivi-1group and PILO+DMSO group, the number of hippocampalneurons are significantly reduce (p<0.05); compared with PILO group, the number ofhippocampal neurons of PILO+Mdivi-1group increase (p<0.05), the number ofhippocampal neurons of PILO+DMSO group reduce (p>0.05). The result of TUNELassay showing that compared with CON group, the number of apoptosis cells ofhippocampal neurons of PILO group, PILO+Mdivi-1group and PILO+DMSO groupincrease(p<0.05); compared with PILO group, the number of apoptosis cells ofPILO+Mdivi-1group reduce(p<0.05), the number of apoptosis cells of PILO+DMSOgroup increase(p>0.05);3. Western blot analysis: Compared with the CON group, the levels of Drp1content, Cyt C release, AIF translocation and caspase-3activation of PILO group,PILO+Mdivi-1group and PILO+DMSO group are significantly increasedtwenty-four hours after status epilepticus(p<0.05); compared with PILO group, thelevel of Drp1content of PILO+Mdivi-1group reduce(p<0.05), the level of Drp1content of PILO+DMSO group increase(p<0.05); the levels of Cyt C release, AIFtranslocation and caspase-3activation of PILO+Mdivi-1group are reduced(p>0.05), the levels of Cyt C release, AIF translocation and caspase-3activation of PILO+DMSO group are increased(p>0.05).4. RT-PCR analysis: Compared with the CON group, the levels of Drp1mRNAcontent of PILO group, PILO+Mdivi-1group and PILO+DMSO group aresignificantly increased twenty-four hours after status epilepticus(p<0.05); comparedwith PILO group, the level of Drp1mRNA content of PILO+Mdivi-1groupreduce(p<0.05), the level of Drp1mRNA content of PILO+DMSO groupincrease(p<0.05).Conclusion1.The levels of Drp1and Drp1mRNA of hippocampal were increased afterstatus epilepticus, so the increase of mitochondrial fission took part in the damagecause by status epilepticus;2. Mdivi-1has neuroprotection on hippocampal neurons of epileptic rats; themechanism is it that Mdivi-1can inhibit the release of Cyt C, translocation of AIFand activation of caspase-3, which caused by mitochondrial permeability transition;3.The inhibition of mitochondrial fission increase could become a new thoughtand method for antiepileptic therapy.