흰쥐 뇌의 냉동 손상 후 발현되는 중간세사단백
Expression of Intermediate Filament Proteins in Rat Brain after Cryoinjury
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목 적 : 뇌의 외상은 직접적인 기계적 손상, 실질내출혈 및 거미막 밑 출혈, 혈액 뇌 장벽 파괴, 흥분독성, 그리고 허혈 등을 일으키는 종합적인 손상이다. 지금까지 다양한 동물모델을 이용한 연구에도 불구하고 그 기전에 대한 정보가 부족한 실정이다. 그러므로 이 연구는 새로운 냉동 손상법을 이용하여 흰쥐 뇌에 외상을 유발시킨 후 신경아교흉터형성에 관여하는 중간세사단백인 신경교 섬유산 단백(glial fibrillary acidic protein; GFAP), 비멘틴 및 네스틴의 발현 양상을 밝히기 위한 목적으로 시도하였다. 방 법 : 실험동물로는 생후 2개월(평균 300 g)된 웅성 흰쥐(Sprague-Dawley)를 사용하였다. 흰쥐를 3.5% chloral hydrate(1 mL/100 g)로 복강내주사하여 마취시킨 후 두피를 절개하여 머리뼈를 노출시킨 다음 정수리점과 시옷점 사이에서 머리뼈 절제를 시행하여 왼쪽 전두엽을 노출시킨 뒤 액체 질소로 미리 얼려 두었던 직경 3 mm 철막대로 1분간 전두엽을 냉동 손상시켰다. 실험군은 냉동손상 후 경과시간에 따라 6시간, 24시간, 4일, 7일 및 14일군으로 분류하였고, 정상군을 실험군에 대한 대조군으로 사용하였다. 뇌 조직을 연속동결절편으로 제작한 다음 GFAP에 대한 단세포군항체인 mouse anti-rat GFAP 항체와 비멘틴에 대한 mouse anti-rat vimentin, 그리고 네스틴 중간세사단백에 대한 mouse anti-rat nestin항체를 각각 적용시켜 면역조직화학염색을 시행하였다. 결 과 : 실험결과는 다음과 같다. GFAP, 비멘틴 및 네스틴에 양성반응을 보이는 반응성 성상교 세포들이 냉동손상 후 6시간째에 출현하였다. GFAP 및 네스틴 양성반응세포들이 냉동손상 후 24시간째부터 증식을 시작하여 손상 후 4일째 최고치에 도달하였고, 손상 후 7일째에 정상으로 회복되었다. 비멘틴 양성반응세포들이 냉동손상 후 24시간째부터 증식을 시작하여 손상 후 4일째 최고치에 도달하였고, 손상 후 14일째에 정상으로 회복되었다. 반응성 성상교 세포는 냉동손상 후 4일째에 특징적인 형태학적 변화를 나타내었다. 결 론 : 위의 실험결과로 미루어보아 GFAP, 비멘틴 및 네스틴 양성반응세포들은 신경아교흉터를 형성하는데 관여할 것으로 생각된다.
Abstract
Purpose : Traumatic brain injury is a multifaceted injury that involves direct mechanical damage, intraparenchymal and subarachnoid hemorrhage, breakdown of the blood-brain barrier, excitotoxicity, and ischemia. Even though much investigations were performed, acceptable mechanical informations were rare. The aim of this study was to reveal the expression pattern of intermediate filament proteins associated with gliotic scars in cerebral cortex of rats after cryoinjury. Methods : A total of 18 male Sprague-Dawley rats weighing 300 g, 2 months old, were used throughout the experiments. To injure the brain, rats were anesthetized for surgery with 3.5% chloral hydrate(1 mL/100 g, intraperitoneally); the frontal bones were exposed by elevating the skin; and craniectomies were performed adjacent to the central suture, midway between lambda and bregma. A cryoinjury was then created by applying a cold probe(3-mm-diameter steel rod chilled in liquid nitrogen) to the left frontal cortex(ipsilateral cortex) for 1 min. Rats were sacrificed at 1, 4, 7 and 14 days postsurgery(n=3, per time point), and three rats were sacrificed as normal controls. Serial brain cryosections were made by cryostat. For immunohistochemistry, brain tissue sections were allowed to react with mouse anti-rat GFAP antibody(1:200), mouse anti-rat vimentin antibody(1: 200), and mouse anti-rat nestin antibody(1:200). Results : Reactive astrocytes expressing GFAP, vimentin and nestin appeared for the first time at 6 hours after cryoinjury. Proliferation of GFAP and nestin positive cells started at 1 day after cryoinjury, reached its maximum on day 4, and returned to normal level after the 7th post-injured day. Proliferation of vimentin positive cells started at 1 day after cryoinjury, reached its maximum on day 4, and returned to normal level after the 14th post-injured day. Characteristic morphological changes in reactive astrocytes were seen at 4 days after cryoinjury. Conclusion : The above results suggest that GFAP, vimentin and nestin positive cells attend in the formation of gliotic scars.
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