Excessive Autophagy Contributes to Neuron Death in Cerebral Ischemia

Abstract

AIMS: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. METHODS: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo. RESULTS: In both primary cortical neurons and SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3-II to LC3-I and Beclin-1 (BECN1) expression. Using Fluoro-Jade C and monodansylcadaverine double-staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3-methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3-7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. CONCLUSION: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.