Study On Anti-epilepsy Effect And Mechanism Of Resverotral Against Kainate-induced Temporal Lobe Epilepsy In Rats

ObjectiveThe rats were induced temporal lobe epilepsy (TLE) by Kainate injected into the center site of CA3 region of right hippocampus with the stereotaxic technology. The following experiments were carried on this model. Three levels experiments (behavior monitoring, histological analysis, and Western blot analysis) were used to evaluate the efficiency and analyze the mechanism of Resveratrol (Res) in anti-epilepsy.Methods1. Kainate-induced TLE. Adult male Wistar rats (220~250 g and clean stage) were used in the experiments. Under chloral hydrate (350 mg/kg, i.p.) anesthesia, rats were placed on the stereotaxic apparatus and 2.5μl kainate (0.4μg/μl) was slowly injected (about 10 min) to CA3 region of right hippocampus (4.0 mm posterior to bregma, 4.4 mm lateral to the midline, 3.8 mm below dura). The microsyringe was removed 3~5 min later and the rat scalp was sutured. The behavioral progression of kainate-induced seizures was scored according to Racine's standard classification. Only those rats that could reach at least the class 4 seizures were used in subsequent pharmacological studies. The equivalent volume of normal saline to the same site was injected in the control rats.2. Grouping. All the rats in the experiments were divided randomly into the following three groups: Normal Saline (NS) group, Epilepsy (kainate) group, Kainate and Res (Res) group. The Res were applied intragastrically at a dose of 15 mg/kg body weight once a day for ten days after the first onset of seizure in acute stage.3. Behavior monitoring. After kainate injection, all the rats were maintained under standard laboratory conditions (23°C±1°C) with a natural light–dark cycle and were allowed free access to standard dry rat diet and tap water. And all rats ware monitored under a video capture system for 2 months (8 h/d, 5 d/week) to record the spontaneous seizures.4. Nissl and Timm staining. After Behavior monitoring, rats were deeply anesthetized with chloral hydrate and perfused transaortically with the modified fixation procedure. Then, the brain was removed and postfixed overnight in 4% paraformaldehyde at 4°C, then immersed in 30% sucrose phosphate buffer at 4°C until they sank to the bottom of the vial. Twenty-micrometer thick cryostat slices were serially cut in coronal through the hippocampus for histological analysis. Nissl staining was used to evaluate the degenerating neurons of CA1, CA3 and dentate gyrus (DG) and Timm staining to evaluate mossy fiber reorganization in the inner molecular layer of DG that accompanied epileptogenesis.5. Western Blotting. Rats were deeply anesthetized with chloral hydrate, then the brains were dissected on ice, and the hippocampi of both hemispheres isolated and stored at - 80°C until use. Total proteins were isolated from the whole hippocampus of each rat. Protein concentration was determined by the BCA Protein Assay kit. After sodium laurylsulfate–polyacrylamide gel electrophoresis on a 10% resolving gel, proteins were transferred onto a BioTrace PVDF membrane. The membrane was blocked in freshly prepared PBS with 5% nonfat dry milk for 30 min at room temperature, incubated with primary antibodies for GluR6/7-containg KARs overnight at 4°C, followed by washes in PBS-0.05% Tween 20 (PBS-T), 3×10 min, and incubation with secondary antibody and HRP-conjuncted antibody for GAPDH for 90 min at room temperature. Immunodetection of proteins by chemiluminescence (ECL kit) was followed by exposure to X-ray film.6. Statistical Analysis. All statistical analysis was performed using SPSS software (version 13.0). Values are expressed as mean±SEM. The data among the groups were compared using one-way ANOVA. Between groups, variance was determined by a Fisher's least significant difference post hoc test after ANOVA, except the percentage of rats with spontaneous seizure, which is examined by x2 test. Statistical significance was defined as p < 0.05.Results1. Behavior observation. The seizures reached class 5 during the acute period in 97% (31 of 32) of rats treated with kainate. In rats treated with kainate and kainate + Res, 75.0% (9 of 12) and 14.3% (1 of 7) of animals developed spontaneous seizure 8 weeks after status epilepticus, respectively.2. Morphological analysis. From the Nissl stanining, a large number of neurons were lost in the CA1, CA3, and hilar regions in the kainate group. However, there was no apparent neuronal cell death in the NS group. Res markedly protected against kainate-induced death of neurons in CA1 as well as CA3a regions, but not in CA3b and hilar regions (p < 0.05). From the Timm staining, a dense broad band of aberrant MFS appeared in the upper granule cell layer in the kainate group, but there was no such sprouting in the NS group. The degree of MFS in the Res group was significantly lower than that in the kainate group (p < 0.05).3. Western blot analysis. The hippocampal KARs protein level in kainate-treated rats was significantly higher compared to their controls. The upregulation of the KARs protein by kainate was almost completely inhibited by Res treatment (p < 0.05).Conclusion1. Resveratrol decreased the frequency of spontaneous seizures, and Res inhibited the epilepsy generation and progression of kainate-induced TLE animal model.2. Res markedly reduced the severity of Kainate-induced neuronal cell loss in the CA1 and CA3a regions, and the MFS is also inhibited in TLE animal model.3. Res significantly inhibited the upregulation of KAR protein by kainate treatment, and the anti-epiletic effect of Res may through that it can inhibit the expression of KAR in the hippocampous.