Study On Features Of GABA_ARa1 Subunit Expression In Hippocampus Of Penicillin-Induced Acute Seizure Rats

Background and Objective: Epilepsy is a common disease in nervous system. It was reported by WHO (Word Health Organization) that the incidence of epilepsy in the world was about 8‰in 2001. Seizure onset not only decreased patients' quality of life, but also increased the economic burden of medical health care. The epileptogeneis was very complex. Right now, the view that epilepsy is caused by the imbalance between excitation and inhibition, is agreed by the majority of specialists in epileptology. In the past, the causes of epileptic seizure were poorly understood. Failure of inhibitory function may underlie spontaneous seizures. However, recent studies showed that there was an augmented effect of GABAergic inhibition during epileptic seizures. From macroscopic view, inspired by "flood discharge phenomenon", we suppose that there would be a "mechanism of inhibitory barrier breakthrough" during epileptic seizures. GABA is the most important inhibitory neurotransmitter in mammalian central nervous system, GABA_AR is the most important iontotropic receptor, andα1 subunit is the combining site of benzodiazepine(BZ),barbiturate(BB) and GABA, so the GABA_ARα1 subunit is the most important component of inhibitory system. To prove "mechanism of inhibitory barrier breakthrough" and explore the possible molecular mechanism, we observed the changes of GABA_ARα1 subunit expression in each phase and each region of hippocampus of penicillin-induced acute seizure rats.Materials and methods: 36 SD rats were divided into 6 groups. Each group was manipulated as following:(1) Normal control group: not induce seizure model with penicillin, and not inject NS into epileptic focus;(2) NS control group: not induce seizure model with penicillin, only micro-injected the same volume NS into the same site in epileptic focus;(3) Pre-seizure group: penicillin was micro-injected into right hippocampus of rats to make seizure model, then recorded the brain discharge synchronously with EEG. The rats were sacrificed as soon as there were epileptic discharge in EEG and no behavioral seizures;(4) 2-hour-seizure group: penicillin was micro-injected into right hippocampus of rats to make seizure model. The rats were regarded at seizure phase when there were behavioral seizures and Racine grade was 2 or above. They were sacrificed at 2 hour after seizures onset;(5) 6-hour-seizure group: penicillin was micro-injected into right hippocampus of rats to make seizure model. The rats were regarded at seizure phase when there were behavioral seizures and Racine grade was 2 or above. They were sacrificed at 6 hour after seizures onset;(6) Post-seizure group: penicillin induced seizure model, in which there was behavioral seizures and Racine grade was 2 or above. The rats were considered at post-seizure phase after seizures ended. They were sacrificed at 24 hour after epileptic onset, which was considered as a time point of observation of post-seizure phase because most of rats have no seizures at that time in preliminary experiments.Each group rats were assessed respectively with Racine grade at zero hour, an hour, 2 hour, and 6 hour after epileptic seizure onset and at post-seizure phase. At the same time, recorded the times of seizures above Racine grade 2, and then calculate the times of seizures per minutes, that are the frequency of seizures, and then make statistic comparison of each time point. Synchronously recorded the EEG of each group rats at pre-seizure phase, seizure phase and post-seizure phase. All the rats were sacrificed at the corresponding time point, and then make coronary paraffin slice. Test the GABA_ARα1 subunit expression level in each region (CA1,CA3, dentate gyrus) 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.Result1. The acute seizure model was successfully induced with penicillin micro-injection into hippocampus. The probability of success was 87.5%.2. Behavioral changesThere were no seizures in normal control and NS control group. Seizures above Racine grade 2 emerged at about 24.88±19.28 minutes after injection of penicillin. Intensity of seizures: at zero hour after seizure onset, the intensity of seizures was at Racine Grade 2～3 in most of rats. At 1～2 hour after seizure onset, there was a enhancement with Racine Grade at 4～5. At 6 hours after seizure onset ,the intensity of seizures return to Racine Grade 2～3. The seizures ended at 24 hour after seizure onset in all 6 post-seizure group rats.3. Changes of EEGMost waves of normal rats EEG wereα,βwaves ,occasionallyθwaves. The wave amplitudes were lower than 75uv. The EEG between normal control group and NS control group has no significant differences. Many diverse epileptic discharge waves were seen in EEG at about 14.17±5.92 after injection. Most of them were paroxysmal sharp waves or spike waves. At 2 hours after seizure onset, EEG mainly showed persistent sharp waves, spike waves, sharp and slow wave complexes,spike and slow wave complexes with middle and high wave amplitude. At 6 hours after seizure onset, the frequency and amplitude of waves gradually became lower. At 24 hours after seizure onset, low amplitude epileptic waves can still be seen in EEG, although there were no behavioral seizures.4. Results of immunohistochemical testGABA_ARα1 subunit expression in each region of hippocampus of normal rats were respectively(show with RGV): CA3 111.28±6.04, CA1 104.80±2.93, dentate gyrus(DG) 92.11±4.77, CA3 > CA1 >DG (P < 0.05) .There was no significant differences between NS control group and normal control group.GABA_ARα1 expression in CA3: At pre-seizure phase significantly increased, and then at 2 hours after seizure onset significantly decreased, finally at 6 hours and 24 hours after seizure onset gradually returned too relative high level.GABA_ARα1 expression in CA1: At pre-seizure, 2-hour-seizure phase, there were no significantly changes; however, at 6-hour-seizure and post-seizure phase, gradually increased.GABA_ARα1 subunit expression in DG: no significant changes at pre-seizure,2-hour-seizure,6-hour-seizure phase; significantly increased at post seizure phaseConclusion1. Local injection of penicillin into hippocampus of rats can establish acute seizure model.2. the changes of GABA_ARα1 subunit expression in penicillin-induced acute seizure rats, that is increasing at pre-seizure phase, then decreasing at seizure phase, finally gradually recovering at post-seizure phase, might support the hypothesis of the mechanism of inhibitory barrier breakthrough.