Clinical and Experimental Pediatrics

The Korean Pediatric Society (대한소아청소년과학회)
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Injury of Neurons by Oxygen-Glucose Deprivation in Organotypic Hippocampal Slice Culture

뇌 해마조직 절편 배양에서 산소와 당 박탈에 의한 뇌신경세포 손상

  • Chung, David Chanwook (Department of Pediatrics, College of Medicine, Dankook University) ;
  • Hong, Kyung Sik (Department of Pediatrics, College of Medicine, Dankook University) ;
  • Kang, Jihui (Department of Pediatrics, College of Medicine, Dankook University) ;
  • Chang, Young Pyo (Department of Pediatrics, College of Medicine, Dankook University)
  • 정찬욱 (단국대학교 의과대학 소아청소년과학교실) ;
  • 홍경식 (단국대학교 의과대학 소아청소년과학교실) ;
  • 강지희 (단국대학교 의과대학 소아청소년과학교실) ;
  • 장영표 (단국대학교 의과대학 소아청소년과학교실)
  • Received : 2008.06.11
  • Accepted : 2008.08.18
  • Published : 2008.10.15
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Abstract

Purpose : We intended to observe cell death and apoptotic changes in neurons in organotypic hippocampal slice cultures following oxygen-glucose deprivation (OGD), using propidium iodide (PI) uptake, Fluoro-Jade (FJ) staining, TUNEL staining and immunofluorescent staining for caspase-3. Methods : The hippocampus of 7-day-old rats was cut into $350{\mu}m$ slices. The slices were cultured for 10 d (date in vitro, DIV 10) and and exposed to OGD for 60 min at DIV 10. They were then incubated for reperfusion under normoxic conditions for an additional 48 h. Fluorescence of PI uptake was observed at predetermined intervals, and the cell death percentage was recorded. At 24 h following OGD, the slices were Cryo-cut into $15{\mu}m$ thicknesses, and Fluoro-Jade staining, TUNEL staining, and immunofluorescence staining for caspase-3 were performed. Results : 1) PI uptake was restricted to the pyramidal cell layer and DG in the slices after OGD. The fluorescent intensities of PI increased from 6 to 48 h during the reperfusion stage. The cell death percentage significantly increased time-dependently in CA1 and DG following OGD (P<0.05). 2) At 24 h after OGD, many FJ positive cells were detected in CA1 and DG. Some neurons had distinct nuclei and processes while others had fragmented nuclei and disrupted processes in CA1. TUNEL and immunofluorescent staining for caspase-3 showed increased expression of TUNEL labeling and caspase-3 in CA1 and DG at 24 h after OGD. Conclusion : The numerous dead cells in the slice cultures after OGD tended to display apoptotic changes mediated by the activation of caspase-3.

목 적: 해마 절편 배양에서 산소-포도당 박탈(oxygen-glucose deprivation, OGD)에 의한 세포 사망과 신경 세포 사멸을 propidium iodide(PI) 섭취, Fluoro-Jade(FJ) 염색, TUNEL 염색, caspase-3 면역형광염색 방법으로 관찰하고자 하였다. 방 법: 생후 7일된 Sprague-Dawley 흰쥐의 해마를 MacIlwain chopper로 $350{\mu}m$ 두께의 절편으로 절단하였다. 해마 절편을 6-well plate의 insert 내의 반 유공(sem-porous) 막 위에서 membrane-interface technique으로 10일 동안 배양하였다. 배양된 해마 절편에 산소-포도당 박탈을 60분 동안 가한 후 재산소-재관류하에 기초 배양액에서 48시간 배양하였다. 재산소-재관류 동안 PI 섭취 형광 정도를 시간에 따라 형광 현미경으로 관찰하고 세포사망 백분율(percent cell death)을 측정하였다. 산소-포도당 박탈 직전과 24 시간 후에 해마 절편을 $15{\mu}m$ 두께로 냉동 절단 후 FJ 염색, TUNEL 염색, caspase-3 면역형광염색을 시행하여 세포 사망을 관찰하였다. 결과: OGD 후 PI 섭취 는 해마 절편의 CA1과 DG에 한정되어있었다. OGD 후 재산소-재관류 동안 6시간에서 48시간까지 PI 섭취 형광 강도는 시간이 증가함에 따라 증가하였다. 세포 사망 백분율은 CA1과 DG에서 모두 OGD 후 재산소-재관류 시간이 증가함에 따라 의미 있게 증가하였다(P<0.05). OGD 후 24시간에 세포 변성을 의미하는 많은 FJ 염색 양성 신경 세포 들이 CA1과 DG에서 관찰되었다. 고배율 confocal laser 현미경으로 관찰한 CA1에서의 신경 세포들 중 일부는 명확한 핵과 돌기를 가지고 있는 것을 보여 주었으며, 다른 신경 세포들은 핵의 분절화, 돌기의 손실 등을 보여 주었다. TUNEL 염색과 caspase-3 염색은 OGD 후 24시간에 CA1과 DA에서 TUNEL 양성 발현을 증가시키고 caspase-3 발현을 증가시켰다. 결 론: 해마 절편 배양에서 산소-포도당 박탈 에 의한 다수의 세포 사망을 관찰할 수 있었다. 사망한 세포 들은 주로 신경 세포의 caspase-3 활성화에 의해 매개된 사멸을 보였다.

Keywords

Acknowledgement

Supported by : 단국대학교

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