| Part One Establishment and evaluation of mouse pilocarpine model of temporal lobe epilepsyObjective To evaluate the application of pilocarpine model of temporal lobe epilepsy (TLE) established on C57BL/6 mice and to investigate an optimized scheme of this model.Method Adult C57BL/6 male mice (6-8 weeks of age) were divided randomly into experimental group and control group. Status epilepticus (SE) was induced by intraperitoneal injection of pilocarpine in experimental group, while the controls were injected with equal dose of saline. According to the SE criteria, experimental group was further divided into SE group and no-SE group. Behavior was observed and spontaneous seizures were recorded for consecutive 50 days after SE. After then, mice’s hippocampus was analyzed with Nissl staining for histological lesions and with Timm stain for MFS.Results1. In experimental group, the successful rate of the model was 40% and mortality rate was 47.5%.2.87.5%of mice with pilocarpine-induced SE developed SS. The average latent period was 12.8±8.7 days. SS occurred 2.18±0.45 times per week on average, usually during the light on/off period and appeared in clusters. One seizure often lasted for 10～40 seconds, usually less than 1 minute.3. In SE group, significant cell loss in area CA1-3 and hilus was observed compared with the control (P<0.01), but no obvious cell loss was observed in the dentate gyrus. In no-SE group, cell morphology and arrangement was almost normal in different regions of hippocampus, and number of neurons was not significantly different with the control.4. All the mice in SE group showed prominent mossy fiber sprouting in the inner molecular layer of the dentate gyrus, while some of them showed occasional Timm granules in CA3 area. Timm scores of SE group were significantly higher than control group(IML, P<0.01; CA3, P<0.05). There was no obvious difference in Timm scores between no-SE group and control group.Conclusion The pilocarpine model of C57BL/6 mice is an ideal temporal lobe epilepsy model. Part Two The role of TRPC3 and TRPC6 channels in regulating seizures of pilocarpine-induced epileptic miceObjective To investigate the role of TRPC3 and TRPC6 channels in regulating acute seizure induced by pilocarpine and spontaneous seizures in chronic phrase.Method 30 adult C57BL/6 male mice (6-8 weeks of age) were divided randomly into three groups:anti-TRPC3 group、anti-TRPC6 group and control group. Each group was treated respectively with intracerebroventricular injection of anti-TRPC3、anti-TRPC6 or saline.5 hours after the surgery, all the mice received examination of seizure susceptibility:after intraperitoneal injection of pilocarpine, mice were observed for 2 hours for the emergence of first seizure attack (stage≥3), the proportion of seizures each grade and the mortality rate. Immunohistochemistry was performed to examine the expression of TRPC3 and TRPC6 protein in different regions of hippocampus at each time point after SE. Another 18 C57BL/6 mice underwent pilocarpine-induced SE were divided randomly into 3 groups: anti-TRPC3 group、anti-TRPC6 group and control group. Each group was treated respectively with intracerebroventricular injection of anti-TRPC3、anti-TRPC6 or saline at 1 day、15 days after SE. Their behavior was observed for continuous 60 days and spontaneous seizures were recorded.Results 1. The results of seizures susceptibility:the latency to the first stage 3 seizure in anti-TRPC3 group and anti-TRPC6 group were 63.4±43.5 min and 53.8±40.4 min, significantly longer than control group. The ratio of seizure (stage≥5) in control group was 50%, but only 20%and 18%in anti-TRPC3 group and anti-TRPC6 group. The mortality rates within 2 hours were 10%(anti-TRPC3 group),20%(anti-TRPC6 group) and 20% (control group).2. Immunohistochemistry showed no significant difference in the expression of TRPC3 protein at each time point after SE compared with control. While the expression of TRPC6 protein in CA3 region markedly increased from 2h after SE (p<0.01), reached the peak twice on 1 day and 5 days after SE (p<0.01), and kept high level at the rest time points (SE 7d, p<0.05; SE 60d, p<0.01). There was no significant difference in any other region at each time point except that the expression of TRPC6 protein in CA1 region on 15days was significantly increased compared with control group.3. In anti-TRPC6 group, the total number of spontaneous seizures within 60 days (3.83±1.47) was significant less than control (6.83±2.31) and anti-TRPC3 group (6.00±2.00) (p<0.05). The latent period of three groups was 19.8±8.98 days,13.3±4.84 days and 14.1±4.12 days respectively.Conclusion 1. TRPC3 and TRPC6 channels may mediate pilocarpine-induced acute seizures.2. TRPC6 channel may be related to the severity of spontaneous seizures in temporal lobe epilepsy. Part Three The role of TRPC3 and TRPC6 channels in regulating hippocampal neuronal plasticity of pilocarpine-induced epileptic miceObjective To explore the changes of hippocampal neuronal plasticity in mice of temporal lobe epilepsy and the role of TRPC3 and TRPC6 channels in this procedure.Method C57BL/6 mice underwent pilocarpine-induced SE were divided randomly into three groups:TRPC3 intervention group (n=12)、TRPC6 intervention group (n=12) and control group (n=12). Each group was treated respectively with intracerebroventricular injection of anti-TRPC3、anti-TRPC6 or saline at 1 day、15 days after SE. At 60 days after SE, mice were executed for Timm stain and Golgi stain. Timm score was analyzed for MFS. Dendritic branches and spine intensity were calculated in dentate granule cells and CA3 pyramidal cells by Golgi stain. 12 control mice received saline instead of pilocarpine and antibodies.Results1.Timm stain:in sham group、anti-TRPC3 group and anti-TRPC6 group, there was prominent mossy fiber sprouting in the inner molecular layer of the dentate gyrus and occasional Timm granules in CA3 area. Timm scores in these three groups were not significantly different but were all higher than control group. 2. Golgi stain:in sham group、anti-TRPC3 group and anti-TRPC6 group, the number of dendritic branches and spine intensity of dentate granule cells were not significantly different. The number of dendritic branches in CA3 pyramidal cells of sham group was more than the control, without significant difference. The number of dendritic branches and the spine intensity of 2nd branches in CA3 pyramidal cells of anti-TRPC6 group were significantly less than control (p<0.05)Conclusion TRPC6 channel may mediate hippocampal neuronal plasticity in temporal lobe epilepsy. |
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