The Study On Hippocampal Neuronal Damage And Its Possible Mechanism In Status Epilepticus

BackgroundLithium-pilocarpine induced status epilepticus (status epilepticus, SE) can cause serious disease in several brain regions, of which the most significant damage lie in the hippocampus. Many studies suggest that hippocampal neurons damage may be related to glutamate a-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptor-mediated excitatory neurotoxicity. But adult hippocampus AMPA receptor (AMPAR) mainly made by the second subunit (GluR2) were low calcium permeability and it is difficult to cause overload intracellular calcium which caused neuronal injury. How activation AMPAR cause neuronal damage is unclear.Recent studies have found that a protein complex made by GluR2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) formed in cerebral ischemia model increases, causing neuronal degeneration. Use peptides which can destroy the complex coupling can effectively reduce the neuronal injury in cerebral ischemia model.ObjectiveGluR2/GAPDH complexes and changes has not been reported in SE, therefore, the present study through the establishment of lithium chloride-pilocarpine-induced SE model to study the damage of hippocampal neurons, GluR2 protein expression and GluR2/GAPDH protein complex coupled change, and then study the hippocampal neuronal damage mechanism which may exist in SE. Thereby providing a new target for the treatment of patients with status epilepticus.Methods1.Experimental Animals and research programs:eighty-two healthy male Wistar rats,6-8 weeks old, weighing 200-240 g were used in the present study. All of the rats were keeping in the temperature 23～25℃, free access to water and diet. They were randomly divided into 2 groups:normal control group (n= 20), SE group (n= 62). SE group was randomly divided into SE 1 h,6 h,24 h,72 h,7 d group. Randomly selected 6 rats through healthy controls and different time points rats after SE respectively, then make Nissl and in situ end labeling (TUNEL) staining after hippocampus paraffin sections prepared. Randomly selection control and SE 1 h,6 h, 24 h,72 h,7 d of each six rats, then extracting the hippocampus tissue protein make Western blot experiments. According to the analysis of hippocampal neuronal damage and GluR2 expression changes, choose the most obvious changes time point in SE rats and conduct co-immunoprecipitation experiments.2.SE model preparation and inclusion criteria:Wistar rats were treated with intraperitoneal injection of lithium chloride, after 18-24 h, intraperitoneal administration of pilocarpine hydrochloride. After 60 to 90 minutes of status epilepticus, given intraperitoneal injection of diazepam relieve convulsions. Seizure events were graded according to Racine’s standard classification (Racine,1972). Level 4 and level 5 is generalized tonic-clonic seizures. Only level 4 or above were used in this research.3.Histopathological assessment:Normal and every time points rats after status epileptics sacrificed. Each group randomly selected six and making cardiac perfusion. 10% chloral hydrate anesthetized rats were perfused, fixed, dehydrated, embedded in paraffin, and then using continuous coronal paraffin sections to make Nissl staining and TUNEL staining in hippocampal CA1, CA3 region. therefore, the morphological changes of apoptosis can be analysised.4.Nissl staining:conventional dewaxing paraffin sections to water,37 ℃ water bath to 0.5% sulfur Jin and incubated 10 min,95% ethanol to differentiate background colorless, conventional dehydration, transparent, sealing piece. Take a slice from every 5 slice, for a total of three for staining. Staining count 100μm×100μm area of CA1 and CA3 region at ×400 magnification microscope.5.TUNEL staining:Paraffin sections were dewaxed to water, to remove endogenous peroxidase, proteinase K treatment. TUNEL No.1 and No.2 mixture made a reaction at 37 ℃ for 1 h, the negative control group was treated with PBS in place of No.1, the others are same. After PBS washing every group plus 50 ul TUNEL No.3 at 37 ℃ and incubated for 30 min. After washing PBS plus DAB staining fluid, hematoxylin dyeing again. And then make conventional dehydration, transparent, mounted with neutral gum. And then analyze the results.6.Western blot:Extracted hippocampus protein qiuickly on ice. After total protein was extracted and determine the protein concentration. Electrophoresis, transferred to a membrane, blocking, next making primary antibody incubation, and then make the second antibody incubation. Adjusting internal error using β-action. the interest protein Gray Value/the reference protein Gray Value were the relative protein expression.7.Co-immunoprecipitation:Extracted hippocampus protein qiuickly on ice like operated in western blot. Using Protein A/G plus agarose to remove non-specific binding protein. Then the primary antibody and control antibody was used to incubated in the extracted hippocampus protein. Centrifuged to remove proteins which are not researched, steped by Western blot prosess. Analyse the SE group compared with the control group.8.The SPSS 17.0 computer program was used for all analysis and statistical evaluations. Measurement data were expressed as means ± standard deviation(SD). T test was used to compare between the two sets of data. Differences between each groups were analysised by one-factor analysis of variance(ANOVA). The level of significance was set to p< 0.05.Results1.Compared with the control group, the number of nerve cells in the hippocampus significantly reduced at all studied time points (P< 0.05).2.Apoptotic cells in hippocampus were significantly increased at 24 h,72 h and 7d after status epilepticus (P< 0.01).3.GluR2 at 1 h,6 h after status epilepticus was equal to that of test control group(P> 0.05), but it was shown to be significantly down-regulated at other studied time points (F=76.51, P< 0.01).4.when compared with the control group, the GluR2/GAPDH interaction is significantly up-regulated in the hippocampus at 72 h after status epilepticus (t=7.03, P<0.05).Conclusions1.Status epilepticus can lead to neuronal damage in the hippocampus.2.Down-regulation of GluR2 and increase of the GluR2/GAPDH complex formation might be one of the mechanisms involved in hippocampal neuronal damage.