The Expression Of KCC2 And GABA_ARα1 Protein In Rat Hippocampus After Status Epilepsy And The Effect Of BDNF On Them

Objective To observe the expression of KCC2 and GABA_ARα1 protein in rat hippocampus 2 weeks after status epilepsy(SE)and the effect of using of sheep BDNF neutralization antiserum(anti-BDNF). Investigate the role of KCC2 in the formation of SE and SRS,and the relationship between KCC2 and GABA_ARα1,demonstrate the effect of BDNF on modulating the action and expression of KCC2 protein by inhibiting BDNF,and the neuronal protective effect of anti-BDNF in SE.Method 25 SD male rats are divided into 5 groups randomly,5 each group.Sheep BDNF neutralization antiserum are injected through lateral ventricle by stereo taxis,and rats are sacrificed after 90min, 180min,270min and 360min respectively,rats of sham group are injected with normal sheep serum,and sacrificed after 2h.Western Blot is used to evaluate the changes of BDNF protein in hippocampus,to decide the proper time point of intervention.130 SD male rats are randomly divided into N.S.control group(10 rats),SE group(60 rats) and experiment group(60 rats).Rats of SE group are induced SE by LiCl-PILO i.p.Rats of experiment group received the same treatment as SE group,except for being injected with anti-BDNF 3h before PILO i.p. Rats of both SE group and experiment group are randomly divided into 6 subgroups respectively(10 rats each),and are sacrificed 2h,6h,24h, 3d,7d and 14d after SE ended.The changes of ethology of two groups during and after SE induced are observed and evaluated.Nissl stain is used to estimate the change of pathobiology of hippocampus.The expression and distribution of KCC2 and GABA_ARα1 in hippocampus are detected with immunofluoresence and Western Blot.The expression of BDNF protein is detected with Western Blot.Results1.Expression of BDNF protein is decreased to perigee 3h after anti-BDNF used;2.Results of animal model:The achievement ratio of experiment group(respectively 90.0%and 75.6%),and mortality rate are lower than SE group(respectively 16.67%and 7.69%).Compared with SE group,the differences of mean dosage of PILO of experiment group(respectively 24.62±5.49 mg/kg and 34.56±9.01mg/kg),mean time needed to induce SE(respectively 27.90±13.59min and 55.86±17.48min),mean persistent time of severe SE(respectively 28.13±4.66 min and 12.77±5.06min) and mean dosage of 10%Chloral Hydrate for ending SE(respectively 0.38±0.63 ml/100g and 0.32±0.03 ml/100g)have significant statistic differences(p＜0.01).3.Result of Nissl stain:There are progressive,selective numerous cell loss and necrotic in hippocampus during acute phase after SE, especially CA1 area,hilus area emerges ectopic granule cells and abnormal pyramid neurons,while DG area present with marked proliferation within first week in SE groups.The pathologic change of experiment group is similar with SE group,but the degree of cell loss and necrotic is much milder and recover faster(p＜0.05 or p＜0.01).4.Expression of KCC2 is acute substantially down-regulated in SE group within 6h after SE ended,it is then gradually and slowly recover, but still lower than control at 14d.There are significant statistic differences in all six time points when compared with control(p＜0.01). Down-regulation of KCC2 firstly and significantly appears in CA1 at 2h, then CA3 and DG.Compared with SE group,that of experiment group is with similar distribution but less down-regulated and almost rebound to control at 7d after SE.There are significant statistic differences between these two groups in all detected time points(p＜0.01).5.The GABA_ARα1 expression of SE group is down-regulated within 6h,but greatly up-regulated since 24h,and arrives peak at 14d after SE,with significant statistic differences compared with control(p＜0.01).The space distribute pattern of GABA_ARα1 is great distinct between different areas.It is acute substantially decreased in CA1 and CA3 area since 2h,but greatly increased in DG area since 24h till 14d.Compared with control,the GABA_ARα1 expression of experiment group is down-regulated at 2h,but up-regulated from 6h to 3d(p＜0.01), then rebounds to normal at 14d.There are significant statistic difference when GABA_ARα1 expression of experiment group is compared with SE group at 6h-14d after SE(p＜0.01).The distribution pattern of GABA_ARα1 of experiment group is similar with SE group.6.Expression of BDNF of SE group is acute substantially increased since 6h after SE,achieves peak at 3d,then decreases gradually.There are significant statistic difference when SE group compared with control from 6h to 7d(p＜0.01).BDNF expression of experiment group is marked lower than control at 2h after SE ended(p＜0.01),but it is quickly increased and arrives peak at 6h(p＜0.01),then decreased slowly and rebound to normal at 3d.Compared with SE group,BDNF expression of experiment group of all six time points are much lower than SE group, with significant statistic differences(p＜0.01).Conclusion1.The long-term down-regulation of KCC2 protein may play an important role in GABA_AR-induced excitation after SE,and may involved the formation of re-establish of hippocampus synapse and abnormal mossy fiber sprouting,and then causes spontaneous recurrent seizures.2.The action of BDNF reducing GABA_AR inhibitory action by down-regulating the expression and function of KCC2 may play an important role in the acute phase of modern of PILO-induced SE.3.The treatment of anti-BDNF can decrease the action of BDNF protein and reduce the down-regulation of KCC2 and GABA_AR inhibitory action,delay the onset of SE and reduce the degree and duration of severe SE,has neural protective effect.