Study About Expression Characteristics Of NMDAR1 In Hippocampus Of Epileptic Rats Induced By Penicillin And About The Mechanism Of Seizure

Background and objective:It has become a consensus that epileptic discharge is generated when the balance of excitation and inhibition is impaired.Previous studies have showed the inhibition barrier existed in central nervous system to prevent the hyperexcitability of neurons.It is suggested that inhibition barrier was broken through by the excitative discharge is the prerequisite of the clinical seizure.In this study, the expression of N-methyl-D-aspartate receptor subunit1(NMDAR1)in hippocampus of the epileptic rats induced by penicillin was observed to explore the mechanism of seizure.Methods:Rats were microinjected with penicillin in right hippocampus to establish acute epileptic model.Thirty-six S.D.rats were divided into 6 groups randomly.Each group included 6 rats:1.Group A(normal group):normal S.D.rats2.Group B(normal saline control group):injected with normal saline instead of penicillin3.Group C(pre-convulsion phase group):penicillin was micro-injected into right hippocampus of rats to make epileptic model,then recorded the brain discharge synchronously with EEG.The rats were perfused as soon as there was epileptic discharge in EEG and no behavioral epileptic seizures4.Group D(convulsion phase group at 2 hour after epileptic seizures onset):penicillin was micro-injected into right hippocampus of rats to make epileptic model.The rats were regarded at convulsion phase when there were behavioral epileptic seizures and Racine grade was 2 or above 2.They were perfused at 2 hour after epileptic seizures onset.5.Group E(convulsion phase group at 6 hour after epileptic seizures onset):penicillin was micro-injected into right hippocampus of rats to make epileptic model.The rats were regarded at convulsion phase when there were behavioral epileptic seizures and Racine grade was 2 or above 2.They were perfused at 6 hour after epileptic seizures onset.6.Group F(post-convulsion phase group):penicillin induced epileptic model,in which there were behavioral epileptic seizures and Racine grade was 2 or above 2.The rats were considered at post-convulsion phase after seizures ended.They were perfused at 24 hour after epileptic onset,which was considered as a time point of observation of post-convulsion because most of rats have no epileptic seizures at that time.EEG and the frequencies of seizure were recorded after the models were established.Group C rats were anaesthetized and perfused before seizure occurred while epileptic wave could be recorded in EEG.For group D,E,F rats,EEG and the frequencies of seizure were recorded again at 0h,2h,6h,24h after epileptic seizure onset.All the rats were anaesthetized and perfused at corresponding time point.Then made coronary paraffin slice.Tested the NMDAR1 subunit expression level in each region(CA1,CA3,dentate gyrus)of hippocampus of rats with immunohistochemical method and statistically analyzed the gray value by AVONA.Results:1.The acute epileptic model was successfully induced with penicillin micro-injection into hippocampus.The probability of success is 87.5%.2.the behaviors of seizure:①The main attack was at Racine grade 4,except group A,B,C②The frequencies of seizure were different at different time point.The peak was at 2h after penicillin injection then decreased at 6h.It indicated peak attack was at about 1～2h after penicillin injection in right hippocampus.3.EEG:There were multiform epileptic waves in EEG after penicillin injection, including scattering distribution of share waves and spike waves, paroxysmal or persistent epileptic waves.Persistent epileptic waves with medium-high amplitude such as sharp wave,spike wave,sharp-slow wave complex and spike-slow wave complex could be observed in EEG at the second hour time point.The frequencies and amplitude of epileptic waves decreased at the sixth hour time point.Epileptic waves with low amplitude could be observed at 24th hour time point.4.the expression of NMDAR1: ①The expression level of NMDAR1 increased significantly in dentate gurus,CA1 and CA3 regions in epileptic models induced by penicillin.②The expression level of NMDAR1 began to increase in pre-convulsion phase.Further increase was observed at 2h.The peak was at 6h then decreased gradually.The expression level was higher than normal in post-convulsion phase.Conclusions:1.Local injection of penicillin into hippocampus of rats could induce and establish acute epileptic model.2.The expression level of NMDAR1 in dentate gyrus,CA1,CA3 regions began to increase in pre-convulsion phase.Further increase was observed at 2h.The peak was at 6h then decreased gradually.The expression level was higher than normal in post-convulsion phase.As an index of excitatory factor,NMDAR1 might take part in seizure onset. The expression of NMDAR1 during the course of seizure supported the hypothesis about breakthrough of inhibition barrier.