| Epilepsy is a common disease in the central nervous system, whose prevalence rate is second to stroke, the symptoms of transient cerebral dysfunction are caused by repeated epileptic discharge. However, antiepileptic drugs could only control the seizures in the 80% of patients,33.5% of which would have various side effects due to long-term drug treatment, and the others may develop refractory epilepsy because of drug resistance. Transcranial magnetic stimulation is a new neural electrophysiological technique which employs induced current formed by a rapidly changing magnetic field to stimulate brain tissue across the scalp and skull. A great number of experimental and clinical studies demonstrated that low-frequency repetitive transcranial magnetic stimulation (rTMS) had antiepileptic effect, but its mechanism hasn’t been clear yet. As we all know, neurogenesis persists in some areas of adult mammalian brain, and it has the potential to repair cerebral injury. Meanwhile neurogenesis is changed in animal models of epilepsy. Therefore, the present study is to investigate the antiepileptic mechanism of rTMS, and provide experimental datas for the treatment to epilepsy.Objective:To investigate the effects of low-frequency rTMS on the mice model of status epilepticus (SE) and hippocampal neurogenesis in the the dentate gyrus (DG), aiming to disclose the possible antiepileptic mechanisms of rTMS.Methods:Fifty SPF male KM mice were selected to prepare SE models. SE models were established with intraperitoneally injecting scopolamine 1 mg/kg, and then intraperitoneally injecting pilocarpine 280 mg/kg thirty minutes later. After injection of pilocarpine, we need to observe the general behavior changes of mice. When a mouse seizured for at least sixty minutes, it suggested this one was a successful model. Another fifteen mice were selected as control group injected with normal saline at the same time. All the mice were randomly divided into three groups:Group A (control group, NS+sham-rTMS), Group B (model group, SE+sham-rTMS) and Group C (treatment group, SE+rTMS). While there were 15 mice in Group A,18 ones were in each Group B and Group C. Every mouse in each group would be handled correspondingly after injection of NS or pilocarpine. The mice in rTMS intervention group received 500 pulses with "8" shaped coil,0.5 Hz,40% of the maximum stimulation intensity and 30 seconds interval daily for 7 days(stimulation at relatively fixed time everyday). The two sham-rTMS groups received the same magnetic stimulation without energy output, these mice were stimulated mainly by sound. All the experimental mice were monitored by video surveillance system each 24 hours before they were sacrificed, the number of epileptic convulsions and the period of the first spontaneous seizure were recorded everyday. Then these mice in every group were sacrificed at definite time point (1 day,1 week and 8 week) after injecting pilocarpine or normal saline. They were perfused and fixed, and then these brains were removed to do frozen sections. We detected the positive expression of ki67 and doublecortin (DCX) in the dentate gyrus of hippocampus by immunohistochemistry, and did semi-quantitative analysis by counting positive neurons.Results:(1) Thirty-six mice were successfully established into SE model with pilocarpine and survived in good condition, the success rate was about 72%. (2) After the low-frequency rTMS stimulation, the period of the first spontaneous seizure in Group C (SE+rTMS) was significantly longer than that in Group B (SE+sham-rTMS) (P<0.05). (3) Immunohistochemistry of ki67:Compared with Group A (NS+ sham-rTMS), the number of ki67 immunoreactive positive cells was increased significantly in the dentate gyrus of hippocampus at 1 d and 1 w after injecting pilocarpine in Group B (SE+sham-rTMS) (P<0.05), especially at the time point of 1 w. Although the number of ki67 positive cells increased at 8 w, there was no significant difference. Besides, compared with Group B (SE+sham-rTMS), the number of ki67 immunoreactive positive cells was reduced in Group C (SE+rTMS), but there was no significant difference (P>0.05). (4) Immunohistochemistry of DCX:Compared with Group A (NS+sham-rTMS), the number of DCX immunoreactive positive cells was increased significantly in the dentate gyrus of hippocampus at 1 d,1 w and 8 w after injecting pilocarpine in Group B (SE+sham-rTMS) (P<0.05), especially at the time point of 1 w. Besides, compared with Group B (SE+sham-rTMS), the number of DCX immunoreactive positive cells was significantly reduced in Group C (SE+rTMS) (P<0.05).Conclusions:(1) It was well to establish mice SE models with intraperitoneally injection of pilocarpine (280 mg/kg),30 min after injection of scopolamine (1 mg/kg). (2) The expressions of ki67 and DCX in the dentate gyrus of mice hippocampus increased remarkably following pilocarpine-induced seizures, but the trend was that it increased first and then decreased over time. These results suggested ki67 and DCX might play an importmant role in the occurrence and development of epilepsy. (3) The low-frequency rTMS could prolong the incubation period of spontaneous seizures following pilocarpine-induced seizures, it suggested the low frequency rTMS had antiepileptic effect. (4) The low-frequency rTMS could significantly reduce the expressions of ki67 and DCX in the dentate gyrus of hippocampus, it suggested the low-frequency rTMS could inhibit hippocampus neurogenesis in SE models, which may be one of the antiepileptic mechanisms of rTMS. |
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