All issues > Volume 51(10); 2008

Original Article

Korean J Pediatr. 2008;51(10):1112-1117. Published online October 15, 2008.

Injury of neurons by oxygen-glucose deprivation in organotypic hippocampal slice culture

David Chanwook CW Chung¹, Kyung Sik KS Hong¹, Jihui JH Kang¹, Young Pyo YP Chang¹

¹Department of Pediatrics, College of Medicine, Dankook University, Cheonan, Korea

Correspondence Young Pyo YP Chang ,Email: ychang@dankook.ac.kr

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 µm 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 µm 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.

Keywords :Hippocampus, Oxygen glucose deprivation, Hypoxia, Ischemia, Apoptosis, Immature, Rat, Brain