| ObjectiveThe temporal lobe epilepsy (TLE) model was induced by kainic acid (KA) injected into the center site of right hippocampus CA3 region with the stereotaxic technology in rats. The following experiments were carried on this model. Behavior monitoring and Western Blot analysis were used to observe the seizures and the expressive levels of GluR6 and GABAR-α1 during different periods of TLE, respectively. The objection is to evaluate the epileptogenesis and progression of TLE and analyze the anti-epilepsy effect and mechanism of Resveratrol (Res).Methods1. KA-induced TLE model. Adult male Wistar rats (220 - 260 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 KA (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 KA-induced seizures was scored according to Racine's standard classification. Only those rats that could reach at least the classⅣseizures 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 by different phases of TLE: the acute period (3 d), the quiet period (15 d), the chronic period (60 d). The experimental animals were divided randomly into three groups: Normal Saline group (NS), Epilepsy group (KA), KA and Res treatment group (Res, 15 mg/kg/d). In the acute period, Res were applied intragastrically once a day for three days after the first onset of seizure. In the quiet and chronic period, Res were applied intragastrically once a day for ten days after the first onset of seizure.3. Behavior monitoring. After KA injection, all the rats were allowed free access to standard dry rat diet and tap water under standard laboratory conditions (23°C±1°C) with a natural light–dark cycle. All rats ware monitored under a video capture system (8 h/d, 5 d/week) to record the seizures.4. Western blot analysis. Rats were deeply anesthetized with chloral hydrate, then the brains were dissected on ice, and the hippocampus 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 PBS with 5% nonfat dry milk for 30 min at room temperature, then incubated with primary antibodies for GluR6/7-containg KARs overnight at 4°C, washed in PBS-0.05% Tween 20 (3×10 min ), and incubated with secondary antibody and HRP- GAPDH antibody for 90 min at room temperature. Proteins were detected by chemiluminescence (ECL kit) and exposured to X-ray.5. Statistical Analysis. All statistical analysis was performed using SPSS 13.0 software. The data among the groups were compared by one-way ANOVA. Between groups, variance was determined by LSD test after ANOVA. The spontaneous seizure percentages of rats were examined by x2 test. Values are expressed as mean±SEM. Statistical significance was defined as p < 0.05. Results1. Behavior observation. The rate of seizures reaching at least the classⅣduring the acute period was about 97% (62 / 64) after treated with KA in rats. The NS group had no seizures. In acute period, the rate of seizures was about 87.5% (7/8) and 75.0% (6/8) in the KA group and Res group, respectively. In quiet period, the rate of seizures was about 0 (0/8) in both the KA group and Res group. In chronic period, the rate of spontaneous seizures was about 87.5% (7/8) and 12.5% (1/8) in the KA group and Res group, respectively.2. Western blot. Compared with the NS group, during the acute period, the expression of GluR6 and GABAAR-α1 in hippocampus neurons decreased significantly in the KA group (n = 8, p < 0.05), however, the expression of GluR6 and GABAAR-α1 were somewhat restored in Res group (n = 8, p < 0.05), which were less than that in NS group. Compared with the NS group, during the quiet period, the expression of GluR6 and GABAAR-α1 in hippocampus neurons were hardly detected (n = 8, p < 0.05), however, the expression of GluR6 and GABAAR-α1 were also somewhat restored in Res group (n = 8, p < 0.05), but were significantly less than that in NS group. Compared with the NS group, during the chronic period, the expression of GluR6 and GABAAR-α1 in hippocampus neurons were increased significantly in the KA group (n = 8,p < 0.05), and GABAAR-α1 was also increased in the Res group (n = 8, p < 0.05), however GluR6 was somewhat restored (n = 8, p > 0.05) .Conclusion1. Resveratrol markedly decreased the frequency of spontaneous seizures, and Res inhibited the epileptogenesis and progression of kainite-induced TLE model in rats.2. In three different periods of KA-induced temporal lobe epilepsy (TEL) rats, the expressions of GluR6 and GABAAR-α1 in hippocampus neurons could been come out up-regulated or down-regulated, which could been modulated or restored by Res to achieve antiepileptic effect. |
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