The Effect Of Bcl-2 Overexpression On Hippocampal Damage Induced By Kainic Acid-induced Temporal Lobe Epilepsy In Mice

Background:Epilepsy is a category of neural system disorders with high incidence and complicated mechanisms.Most epileptic conditions are refractory,which causes severe impairment to physical and mental health of the patients,and even social function.As a most common subtype of epilepsy,temporal lobe epilepsy induces damage and death of hippocampal neurons,which is believed to play a vital role in epileptogenesis.There has been evidence from animal and human studies that during temporal lobe epileptic seizures,a series of pro-apoptotic signaling pathways are activated,leading to apoptosis of hippocampal neurons,while simultaneously triggering intracellular anti-apoptotic pathways as a feedback for neuroprotective effect to some extent.B-cell lymphoma-2(Bcl-2)is known as an anti-apoptotic protein up-regulated during seizures of temporal lobe epilepsy,however,little evidence suggests whether artificial up-regulation of bcl-2 gene could alleviate damage to hippocampus induced by temporal lobe epilepsy.Objective:This study aims to discuss whether artificial up-regulation of bcl-2 expression would mitigate hippocampal neuron impairment induced by temporal lobe epilepsy.Design:A lentivirus vector,constructed for bcl-2 gene overexpresssion,was injected to mouse model of temporal lobe epilepsy induced by kainic acid,thus associations among Bcl-2 pathway,hippocampal neuron apoptosis and temporal lobe epilepsy could be discussed.Methods:1.Lentivirus vector construction:Synthesized mouse bcl-2 gene sequence was connected to pLV lentivirus vector after PCR amplification.The vector was subsequently cloned in DH5a E.coli strain.2.Virus packaging:HEK293T cell strain was co-transfected via a three-plasmid lentivirus packaging system.3.Mice hippocampus virus injection:Male C57BL/6J mice at 8-week age were anesthetized by intraperitoneal injection of 5%chloral hydrate of 10 ml/kg body weight.The scalp was cut after 75%ethanol disinfection,and 30%hydrogen peroxide was applied to erode the fascia.The mice were fixed to a stereotaxic apparatus and cranial bone was drilled bilaterally with a mini-electric drill at loci corresponding to hippocampus.1000 nL of virus solution was injected in each side of hippocampus with a microinjector.4.Mice epilepsy model construction:Each mouse was administered intraperitoneal injection of kainic acid of 18 mg/kg body weight one week after virus injection.5.Expression verification:2 hours after kainic acid injection,the mice were sacrificed,the cranial bones were cut and the cerebrums were incised medially.Hippocampus was isolated from one hemisphere for Western blotting to verify Bcl-2 protein level.6.Microscopic slides preparation and examination:The other hemisphere was put into formalin for 48 hours for fixation,and then put into 80%ethanol for dehydration.Paraffin sections of hippocampus was made and hematoxylin staining was subsequently performed after dewaxing.The pyramidal cells in dentate gyrus of hippocampus were examined under optical microscope and dead cells were counted.Results:1.The target gene sequence in cloned plasmids were identical to the original sequence,and the virus was successfully constructed.2.Western blot confirmed significantly higher level of Bcl-2 protein in hippocampal tissue in mice injected with bcl-2 gene up-regulation lentivirus compared to control group.3.The mice with bcl-2 overexpressed showed significantly less hippocampus pyramidal cell deaths in dentate gyrus compared to control group.Conclusion:Neuron apoptosis in hippocampus caused by kainic acid-induced temporal lobe epilepsy is mediated by Bcl-2 pathway;up-regulation of bcl-2 gene would perform further neuroprotective effect in hippocampus.