Temporal And Spatial Expression Of CRMP-2 And Its Phosphorylation Regulation In Rat Hippocampus Of Temporal Lobe Epilepsy

Epilepsy is one of the common chronic diseases of nervous system,about 1/4 are drug-resistant epilepsy,of which 70% of temporal lobe epilepsy.Our previous study,we found that hippocampal neurons were injured by preparing temporal lobe epilepsy in rats.A series of differentially expressed proteins were identified by comparing the hippocampal tissue of TLE rats with normal control group and operation group by proteomics.Collapsion response mediator protein-2 is the most significant down-regulation protein,suggesting that it is closely related to the pathogenesis of TLE.CRMP-2 is one of the 5 subtypes of the CRMP family,mainly expressed in the highly plastic regions of the adult brain,such as hippocampus,olfactory bulb and cerebellum.CRMP-2 can promote axon growth,while phosphorylated CRMP-2 has no effect on axon growth.CRMP-2 has protective effect on neurons.In the process of hippocampal neurons damage or damage in temporal lobe epilepsy,it may be realized by CRMP-2 and its phosphorylation.Objective:The purpose of our study was to observe the temporal and spatial expression and phosphorylation of CRMP-2 in hippocampus of kainic acid kindled rat model,and to explore that CRMP-2 is regulated by its phosphorylation in the process of hippocampal neuron injury or anti-injury in temporal lobe epilepsy.Methods:In this study,kainic acid(KA)was injected into the rat amygdala to induce an epilepsy model.66 adult male Wistar rats were divided into three groups,including the CONT,SHAM,KA.The KA group were divided into six subgroups including the 3 hours,6 hours,24 hours,3 days,7days and 14 days groups to investigate the time dependent changes in expression of CRMP-2 and p CRMP-2 via Western blot and immuno-histochemical examination,to investigate pathogenesis of hippocampal via HE staining.Results:(1)HE staining was used to observe the pathological changes of hippocampus after epilepsy in rats of CONT group and SHAM group.The morphology of pyramidal cells and granular cells in dentate gyrus of hippocampus were normal,well-arranged and intact.Compared with group KA,the damage of hippocampal CA3 neurons was the most serious.In the 3h group,the CA3 area of hippocampus was dominated by neuron degeneration,and there was obvious loss or death of the main neuronal cells in the 6 h group.In the group of 24 h,3 d,7 d 14 d,the condition was further aggravated,obvious neurons disappeared,the remaining neurons were disordered,the glial cells proliferated and the capillaries increased in varying degrees.(2)The Western blot method was used to detect the expression of CRMP-2 in hippocampus of rats after KA-induced epilepsy.The changes of CRMP-2 expression in hippocampus of rats after KA-induced epilepsy were observed at 3 hours,6 hours,24 hours and 7 days and 14 days.The characteristics of the changes were that the expression of CRMP-2 in hippocampus of rats increased first and then decreased gradually.The expression of CRMP-2 in hippocampus of rats after 3 hours of epilepsy increased rapidly to the peak.The lowest was on the 7th day,but on the 14 th day it was higher than that on the 7th day,but lower than that in the SHAM group for 3 hours and 6 hours,which was higher than that in the SHAM group.There was a significant difference in the expression of CRMP-2 between the SHAM group and the groups at 3 hours,6 hours,3 hours,7 days and 14 days after KA-induced epilepsy(P < 0.05).(3)Expression of p CRMP-2 in hippocampus of rats after KA-induced epilepsy was decreasesd at first and then increased gradually.After KA induced epilepsy,the expression of p CRMP-2 in the hippocampus began to decline for 3 hours,the lowest in 6 hours,and then gradually increased.The expression of p CRMP-2 at different time points after KA-induced epilepsy was significantly different from that of SHAM group(p < 0.05).(4)Immunohistochemical staining was used to observe the expression of CRMP-2 and p CRMP-2 in the control group and SHAM operation group,3 hours,6 hours,24 hours,7 days and 14 days after epilepsy.The number of CRMP-2 and p CRMP-2 positive cells in the CA3 region was significantly different from that in the SHAM group,but there was no significant difference in the CA1 and DG area of the hippocampus.Conclusions:(1)In the epileptic group,the hippocampal neuronal injury was mainly in the CA3 area.The cells are regular in CA1 and DG region.Therefore,neuronal hyperstimulation and neuronal death are most likely to occur in the CA3 region.(2)After kainic acid induced epilepsy,the expression of CRMP-2 in hippocampus of rats was increased firstly and then decreased gradually,while the expression of p CRMP-2(Thr514)was decreased firstly and then increased slightly.(3)The expression of CRMP-2 and phosphorylated protein is different in time and space in hippocampus of rats after kainic acid induced epilepsy.CRMP-2 and its phosphorylation may be involved in the regulation of neuronal axon growth after epilepsy.They may be closely related to neuronal injury in the course of epilepsy.