| Objective To observe the dynamic changes of TRPC expression in the rat hippocampus after pilocarpine-induced seizures, and to investigate its involvement in synapse remodeling.Method 108 healthy male Sprague-Dawley (SD) rats were divided randomly into experimental group (n=90) and control group (n=18). The temporal lobe epilepsy model was established by intraperitoneal injection of lithium and pilocarpine, while the controls were injected with equal dose of saline (NS). The experimental rats were equally divided into 5 subgroups at time points 1 day,7 days,15 days,30 days and 60 days after status epileptics (SE). Each subgroup was subsequently divided into 3 panels (6 rats in each panel) for the following tests respectively:①expression of TRPC and Synaptophysin protein in rats'hippocampus by Western blot;②Timm staining and score;③co-expression of TRPC and BDNF in rats'hippocampus by double immunofluorescence and hippocampal pathology by Nissl staining. The control rats were equally divided into 3 subgroups and received the aforementioned tests. Results1. The lithium-pilocarpine model of TLE:SE rate reached 88.9%, total mortality rate was 22.2% and the succeful rate was 66.7%.2. The expression of TRPC protein in the hippocampus of experimental rats compared with the control rats:①The expression of TRPC1 protein markedly decreased from 1d after SE (P＜0.01), and reached a minimum on 7d. On 15d, it rebounded gradually yet was still lower than normal. Until 30d, it significantly increased and reached the peak on 60d (P<0.01).②The expression of TRPC3 protein gradually decreased from 7d after SE (P<0.01) and reached a minimum on 60d.③The expression of TRPC4 protein was markedly up-regulated and reached the peak on 1d after SE. Thereafter, it gradually decreased but maintained higher than in control on 7d(P<0.01), but the protein level on 15d after SE was lower than in control group(P<0.05), and lowest on 60d after SE.④The expression of TRPC5 was markedly decreased from the time point of 1d after SE (P<0.01), then gradually decreased, reaching its lowest on 60d after SE.⑤The expression of TRPC6 was markedly increased and reached its peak on 1d after SE (P<0.01), and it was higher than normal at all the other time points (P<0.01).3. The expression of Synaptophysin in the experimental groups rats compared with the control group rats:The expression of Synaptophysin was markedly up-regulated on 15d,30d,60d after SE (P＜0.05 or P<0.01), and reached the peak on 30d after SE. 4. The loss of hippocampal neurons in the experimental group was most evident on 7d and 60d after SE. Significant loss of hilar neurons and pyramidal neurons was present in area CA1 (P<0.01), while the loss of granular cells in dentate gyrus was relatively slight (P＜0.05).5. There were Timm granules in molecular layer of gyrus dentatus since 7d after SE in the hippocampus of experimental rats, then they gradually increased.6. In the experimental group, BDNF and TRPC1,TRPC3,TRPC4,TRPC5,TRPC6 co-expression was observed all the time in rat's hippocampus.ConclusionTRPC may play a potential role in mossy fiber sprouting, in which BDNF may involve. Objective To observe the effect of intervention in BDNF on TRPC expression and mossy fiber sprouting in rat's hippocampus after pilocarpine-induced seizures.Method 270 healthy male Sprague-Dawley (SD) rats were divided randomly into 3 groups:the K252a+Pilo group (n=90), the NS+Pilo group (n=90) and the K252a+NS group (n=90). Rats in K252a+Pilo group were treated by intraperitoneal injection of lithium and pilocarpine, with intracerebroventricular injection of K252a 3h before pilocarpine injection; the NS+Pilo group rats received intracerebroventricular injection of sterile saline 3h before pilocarpine injection; and the K252a+NS group rats received intraperitoneal injection of sterile saline 3h after intracerebroventricular injection of K252a. These three groups all selected 5 time points (1d,7d,15d,30d,60d after intraperitoneal injection) for investigation. The rats of each time point (n=18) received the detection below respectively:①expression of TRPC and Synaptophysin protein in rats'hippocampus by Western blot;②Timm staining and score;③hippocampal pathology by Nissl staining.Results1. SE ratio of the K252a+Pilo group (83.3%) was lower than that of the NS+Pilo group (90.7%), and mortality rate was lower as well (8% and 19.3%, respectively). The mean time required for inducing SE in the K252a+Pilo group (55.84±17.44min) was longer than that in the NS+Pilo group (27.80±13.58min). The dosage of pilocarpine required for inducing SE in the K252a+Pilo group (34.84±9.44 mg/kg) was larger than that in the NS+Pilo group (24.80±5.58 mg/kg). The mean duration of grade III-V seizure in the K252a+Pilo group (12.84±5.44 min) was shorter than in the NS+Pilo group (28.20±4.58 min). And the average total amount of chloral hydrate required for ending seizures in the K252a+Pilo group (3.24±0.44 ml/kg) was lower than that in the NS+Pilo group (3.80±0.54 ml/kg). All the differences above were of statistic significance (P<0.01).2. The expression of TRPC protein in the K252a+Pilo group rats compared with the K252a+NS groups:①The expression of TRPC1 protein was markedly up-regulated since 1d after SE(P<0.01), then gradually increased, and reached the peak on 60d after SE.②The expression of TRPC3 protein markedly decreased since 1d after SE (P＜0.05), then gradually decreased and reached the lowest on 60d.③The expression of TRPC4 protein markedly decreased on 7d,15d,30d,60d after SE (P<0.01), and reached the lowest on 15d.④The expression of TRPC5 protein was markedly up-regulated since 1d after SE (P<0.01), then gradually increased and reached the peak on 60d.⑤The expression of TRPC6 protein was markedly up-regulated since 1d after SE (P<0.01), but the extent of increase declined, on 30d it was still higher than that of the K252a+NS group (P<0.01), but became lower than normal on 60d (P<0.01). Compared with the K252a+NS group:At each time point, the expression of TRPC1,TRPC4 and TRPC5 protein were all down-regulated greatly (P<0.01); The expression of TRPC3 protein was significantly down-regulated since 1d after SE and reached the lowest (P<0.01); Thereafter, it gradually increased but was still lower on 7d,15d,30d (P<0.01 or P<0.05), until it was significantly up-regulated on 60d (P＜0.01).However, the expression of TRPC6 protein was markedly up-regulated since 1d after SE(P<0.05), then gradually increased, and reached the peak on 30d after SE(P<0.01), but markedly decreased on 60d.3. The expression of Synaptophysin protein in the K252a+Pilo group compared with that in the K252a+NS group:it was markedly up-regulated on 15d after SE (P<0.01), inceased gradually, and then reached the peak on 60d (P＜0.01). Compared with that in the NS+Pilo group:it was markedly up-regulated since 1d after SE (P<0.01), inceased gradually, and then reached the peak on 60d (P<0.01).4. In the K252a+Pilo group, hippocamapal neuron loss can be observed, extremely obvious on 15d and 60d after SE. Compared with the K252a+NS group, significant hilar neuron loss and pyramidal neuron loss was present in area CA1-3 (P＜0.01), while the loss of granular cells in dentate gyrus was relatively slight (P<0.05). Compared with the NS+Pilo group, the degree of hippocampal neuron loss in the K252a+Pilo group was significantly sligter, especially since 7d after SE, with significant statistic differences (P<0.05).5. There were Timm granules in molecular layer of gyrus dentatus since 15d after SE in the K252a+Pilo group. The Timm score gradually increased and reached the peak on 60d. Compared with the NS+Pilo group, the degree of sprouting in the K252a+Pilo group was markedly slighter (P＜0.01).Conclusion1. Intervention in BDNF can decrease the severity of seizure.2. Intervention in BDNF can inhibit mossy fiber sprouting, possibly through regulating the expression of TRPC. |
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