어린 생쥐에서 Pilocarpine-유발 발작 후 해마의 신경 발생 및 표현형 분화
Hippocampal Neurogenesis and Phenotypic Differentiation after Pilocarpine-Induced Seizures in Young Mice
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목 적 : 본 연구는 3주령의 어린 ICR 생쥐에서 pilocarpine-유발 발작 후 해마에서의 신경발생이 증가하는지 및 이 때 새로 생성된 세포가 신경세포로 분화하는지를 조사하고자 하였다. 방 법 : 3주령의 어린 ICR 생쥐에서 pilocarpine을 투여하여 발작을 유발한 후 BrdU를 사용하여 새로이 생성되는 세포를 표지하고 동물을 희생하여 얻은 뇌조직으로 면역조직염색을 시행하였다. 새로이 생성된 BrdU 표지세포가 신경세포로 분화하는지 혹은 별아교세포로 분화하는지 확인하기 위하여 마지막 BrdU가 주입된 이후 28일째 동물을 희생하여 얻은뇌조직으로 이중면역형광염색을 하였다. 결 과 : 해마 치아이랑에서 BrdU로 표지된 세포의 수는 pilocarpine 처리군 동물에서 단면당 230.5±59.5으로 대조군의 148.6±40.0보다 유의하게 많았다(P<0.001). Pilocarpine 처리 후 BrdU-양성 세포는 대부분 신경세포로 분화하였고 대조군과 비교할 때 표현형 분율의 차이는 없었다(P=0.20). 결 론 : 본 연구 결과를 통하여, 해마 치아이랑에서 BrdU-양성 세포의 수는 대조군과 비교하여 pilocarpine 처리군에서 현저하게 증가하였으며 새로이 생성된 세포의 대부분이 신경세포로 분화함을 확인할 수 있었다.
Abstract
Purpose : Proliferation, differentiation, and survival of hippocampal dentate granule cells have been reported to be influenced by epileptic seizures in rodent epilepsy models. However, most studies have been done in adult rat models. This study was designed to investigate hippocampal dentate granule cell neurogenesis after pilocarpine-induced seizures in young mice. Methods : Fifteen male ICR mice at postnatal day 21 were divided into pilocarpine-treated (n=7) and control (n=8) groups. Seizures were chemically induced by intraperitoneal injection of pilocarpine (300 mg/kg). Bromodeoxyuridine (BrdU, 50 mg/kg) was subsequently administered once a day for 6 consecutive days, starting at 24 hours after pilocarpine or saline treatment. We then examined BrdU-positive cells in the hippocampal dentate gyrus by immunohistochemistry and by double-labeled immunofluorescence with confocal microscopy. Results : After pilocarpine administration, every seizure behavior was grade 3 or more. Quantitative analysis revealed that BrdU-positive cells were significantly increased in the pilocarpine-treated group compared to control (230.5±59.5 vs. 148.6±40.0, P<0.001). The majority of these mitotic cells were differentiated into neurons. Conclusion : Our results indicated that mitotic activity in the hippocampal dentate gyrus was enhanced after pilocarpine-induced seizures in young mice, and the majority of BrdUpositive cells showed the phenotypic differentiation to neuronal cells.
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