Relationship Between The Features Of GAD Expression In Hippocampus And Epilepsy Pathogenesy In Penicillin-induced Acute Seizure Rats

Background and Objective:Epilepsy is a common disease in nervous system.The domestic epidemiological study showed the incidence of epilepsy in our country is about 5‰.The pathgenesis of epilepsy is very complex.Right now,many scientists in epileptology agreed the opinion that mechanism of epilepsy is caused by the imbalance between excitation and inhibition.Recent study showed that there is an inhibitory system to prevent the neural overexcitation not only in structral and also in functional aspects.It is supposed that there is a "mechanism of inhibitory barrier breakthrough" during epileptic seizures attack. GABA is an important inhibitory neurotransmitter which is enzymed by glutamic acid decarboxylase(GAD).In this study,the features of the changes of GAD65 and GAD67 expression are studied in each phase and each region of hippocampus of penicillin-induced acute seizure rats to prove the hypothesis of "the mechanism of inhibitory barrier breakthrough" in seizure.Materials and methods:36 SD rats are divided into 6 groups.Each group is manipulated as following:(1)Normal control group:not induce epileptic model with penicillin, and not inject NS into epileptic locus.(2)NS control group:not induce epileptic model with penicillin, only micro-injected the same volume NS into the same site in epileptic locus.(3)Pre-seizure group:penicillin is micro-injected into right hippocampus of rats to make epileptic model,then record the brain discharge synchronously with EEG.The rats are sacrificed as soon as there is epileptic discharge in EEG and no behavioral epileptic seizures.(4)2-hour-seizure group:penicillin is micro-injected into right hippocampus of rats to make epileptic model.The rats are regarded at seizure phase when there are behavioral epileptic seizures and Racine grade is above 2.They are sacrificed at 2 hour after epileptic seizures onset.(5)6-hour-seizure group:penicillin is micro-injected into right hippocampus of rats to make epileptic model.The rats are regarded at seizure phase when there are behavioral epileptic seizures and Racine grade was above 2.They are sacrificed at 6 hour after epileptic seizures onset.(6)Post-seizure group:penicillin induced epileptic model,in which there are behavioral epileptic seizures and Racine grade is above 2.The rats are considered at post-seizure phase after seizures ended.They are sacrificed at 24 hour after epileptic onset,which is considered as a time point of observation of post-seizure because most of rats have no epileptic seizures at that time.Each group rats are assessed respectively with Racine grade at zero hour,an hour,2 hour,6 hour after epileptic seizure onset and at post-seizure phase.At the same time,record the times of seizures above Racine grade 2,then calculate the times of seizures per minutes,that is the frequency of seizures,and then make statistic comparation of each time point.Synchronousely recorded the EEG of each group rats at pre-seizure phase,seizure phase and post-seizure phase.All the rats are sacrificed at the corresponding time point,and then make coronary paraffin slice.Test the GAD expression level in each region (CA1,CA3,dentate gyrns)of hippocampus of rats with immunohistochemical method,and then statistically analyze the relative gray value(|the gray value of positive cell-the gray value of background|,RGV)by ANOVA.Results:1.87.5%of experimental rats are successfully induced to seizure with penicillin micro-injection into hippocampus.2.Behavioral changesThere are no seizures in normal control and NS control group. Epileptic seizures above Racine grade 2 emerge at about 24.88±19.28 minutes after injection of penicillin.Intensity of seizures:at zero hour after epileptic seizure onset,the intensity of seizures is at Racine Grade 2～3 in most of rats.At 1～2 hour after seizure onset,there is an enhancement with Racine Grade at 4～5.At 6 hours after seizure onset,the intensity of seizures return to Racine Grade 2～3.The epileptic seizures end at 24 hour after seizure onset in all 6 post-seizure group rats.3.Changes of EEGMost waves of normal rats EEG areα,βwaves,occasionallyθwaves.The wave amplitudes are lower than 75uv.There are no significant differences in EEG between normal control groups and NS control groups.Many diverse epileptic discharge waves are seen in EEG at about 14.17±5.92 after injection.Most of them are paroxysmal sharp waves and spike waves.At 2 hours after seizure onset,EEG mainly show persistent sharp waves,spike waves,sharp and slow wave complexes, with middle and high wave amplitude.At 6 hours after seizure onset,the frequency and amplitude of waves gradually become lower.At 24 hours after seizure onset,low amplitude epileptic waves can still be seen in EEG,although there are no behavioral epileptic seizures.4.Results of immunohistochemical test4.1 Results of GAD67GAD67 expression in each region of hippocampus of normal rats are respectively(showed with RGV):CA3 111.12±2.20,CA1 101.72±1.89,dentate gyrus(DG)82.52±1.94,CA3＞CA1＞DG(P＜0.05).There are no significant differences between NS control group and normal control group.GAD67 expression in CA3 and CA1:at pre-seizure phase significantly increasing,then at 2 hours after epileptic seizures significantly decreasing,finally at 6 hours and 24 hours after epileptic seizures gradually return to relative high level.GAD67 expression in DG:no significant changes at pre-seizure, 2-hour-seizure,6-hour-seizure phase;significantly increase at post-seizure phase.4.2Rresults of GAD65GAD65 subunit expression in each region of hippocampus of normal rats are respectively:CA3 98.64±0.72,CA1 96.44±0.52,dentate gyrus(DG)81.07±1.03,CA3＞CA1＞DG(P＜0.05).There is no significant differences between NS control group and normal control group.GAD65 expression in CA1,CA3 and DG:no significant changes at pre-seizure,2-hour-seizure phase and 6hour-seizure phase;significantly increase at post-seizure phase.Conclusions:1.Acute epileptic seizure model could be established by local injection of penicillin into hippocampus.2.Praxiology and electrophysiology support the existence of mechanism of inhibitory barrier breakthrough in penicillin-induced acute epileptic seizure model.3.The features of GAD67 expression in penicillin-induced acute epileptic model are increasing at pre-seizure phase,and then decreasing at pre-seizure,finally gradually recover at post-seizure phase.It indicates that mechanism of inhibitory barrier breakthrough would be important mechanism in epileptic seizure.