Characterisation of neuronal cell death in acute and delayed in vitro ischemia (oxygen-glucose deprivation) models

Using 96 well microtitre plate sized glass wells we have established and characterised two in vitro ischemia (oxygen-glucose deprivation) models that induce acute or delayed neuronal cell death. In vitro ischemia was induced by washing cortical neuronal cultures with a balanced salt solution either with (acute model) or without (delayed model) 25 mM 2-deoxy-D-glucose, and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and adding glucose containing media (delayed model) or removing the balanced salt solution/2-deoxy-D-glucose medium (acute model) and adding glucose containing media. The models were characterised with respect to in vitro ischemia dose duration, cell death time course and for necrosis, apoptosis, autophagy and necroptosis biomarkers. To this end, biomarkers for all four modes of cell death were detected in both in vitro ischemia models, although the time of onset and relative proportion of each cell death mode differed between models. While it is likely that different modes of cell death were activated in the same cell, autophagy appeared to be a prominent cell death mode, especially in the delayed model. Together these models will provide valuable tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput screening system to evaluate potential neuroprotective agents. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.