Effects Of Resveratrol On Variations Of The Amino Acids In Hippocampus And Cortex In Different Periods Of Temporal Lobe Epilepsy

ObjectiveThe rats were induced temporal lobe epilepsy (TLE) by kainic acid (KA) injected into the center site of right hippocampus CA3 region with the stereotaxic technology. The following experiments were carried on this model. Behavior monitoring and high performance liquid chromatography analysis were used to observe the seizures and the levels of amino acids release during different periods of TLE, respectively. The objection is to evaluate the generation and progression of TLE and analyze the mechanism of Resveratrol (Res) in anti-epilepsy.Methods1. KA-induced TLE. 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 (14 d), the chronic periodⅠ(60 d) and chronic periodⅡ(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 chronicⅠperiod, Res were applied intragastrically once a day for ten days after the first onset of seizure. In the chronicⅡp eriod, Res were applied intragastrically once a day for ten days after sixty days of 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 and were maintained under standard laboratory conditions (23°C±1°C) with a natural light–dark cycle. And all rats ware monitored under a video capture system (8 h/d, 5 d/week) to record the seizures.4. High performance liquid chromatography analysis. Rats were quickly decapitated, removed the hippocampus and cortex on ice before electric homogenated, later plus 3 times volume of acetonitrile to remove protein, then centrifuged at 4°C and obtained the supernatant. The diluted sample were pre-column derivatizated, then injected to the system to get chromatographic analysis, according to the peak area and standard curve to calculate the contents of glutamic acid (Glu), glycine (Gly) andγ-aminobutyric acid (GABA) in hippocampus and cortex.5. Statistical Analysis. All statistical analysis was performed using SPSS 13.0 software. The data among the groups were compared using one-way ANOVA. Between groups, variance was determined by LSD test after ANOVA. The spontaneous seizure percentage of rats is examined by x2 test. Values are expressed as mean±SEM. Statistical significance was defined as p < 0.05.Results1. Behavior observation results. The rate of seizures reaching at least the classⅣduring the acute period was about 97% (62 / 64) treated with KA in rats. The NS group had no seizures. In the 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 the quiet period: the rates of seizures were about 0 (0 / 8) in both the KA group and Res group; In chronic periodⅠ: the rate of spontaneous seizures was about 87.5% (7 / 8) and 12.5% (1 / 8) in the KA group and Res group, respectively; In chronic periodⅡ: the rate of spontaneous seizures was about 85.7% (6 / 7) and 71.4 % (5 / 7) in the KA group and Res group, respectively.2. High performance liquid chromatography analysis results. Compared with the NS group during the acute period, the ratio of Glu/GABA in hippocampus increased significantly in the KA group and Res group (n = 8, p < 0.05); the ratio of Glu/Gly in hippocampus or the ratios of Glu/GABA and Glu/Gly in cortex were no significant difference in the KA group and Res group (n = 8). Compared with the NS group during the quiet period, there was no significant difference between the ratios of Glu/GABA and Glu/Gly in hippocampus and cortex in the KA group or Res group (n = 8). However, compared with the NS group during the chronic periodⅠ, the ratios of Glu/GABA and Glu/Gly in hippocampus and cortex increased significantly in the KA group (n = 8, P < 0.05), and the ratios of Glu/GABA and Glu/Gly in hippocampus was decreased in Res group (n = 8, p < 0.05), but not in cortex (n = 8, P < 0.05); Compared with the NS group during the chronic phaseⅡ, the ratios of Glu/GABA and Glu/Gly in cortex increased significantly in the KA group and Res group (n = 7, p < 0.05); the ratios of Glu/GABA and Glu/Gly in hippocampus had no significant difference in the KA group and Res group (n = 7).Conclusion1. Res decreased the frequency of spontaneous seizures in the KA-induced chronic TLE animal model. 2. Res plays a certain reverse role on the imbalance between the excitatory-inhibitory amino acid neurotransmitter in hippocampus in the TLE model rats which induced by KA.