Article Text

ENHANCEMENT OF AUTOPHAGIC CELL DEATH IN A NEONATAL MODEL OF CEREBRAL HYPOXIA-ISCHEMIA
  1. V Ginet1,
  2. J Puyal2,
  3. P G H Clarke2,
  4. G Magnin1,
  5. A C Truttmann1
  1. 1Division of Neonatology, Department of Pediatrics, University Hospital Center and University of Lausanne, Lausanne, Switzerland
  2. 2Department of Biology and Cellular Morphology, University of Lausanne, Lausanne, Switzerland

Abstract

Background Moderate autophagy occurs in healthy cells to maintain cellular homeostasis, but excessive autophagy has been involved in cell death mechanisms. Enhanced autophagy has been shown to occur in different excitotoxicity-related conditions, but its function remains unclear.

Objectives The present study investigated the effect of cerebral hypoxia-ischemia on autophagy and autophagic cell death in a neonatal rodent model.

Methods P7 rats underwent permanent carotid occlusion followed by 2 h of hypoxia (8%) with subsequent cortical, striatal, hippocampal and thalamic lesions. Using acid phosphatase histochemistry, immunocytochemistry and Western blots against lysosomal and autophagic markers, autophagy was studied and confirmed by electron microscopy.

Results There was a significant increase in LC3-II expression in all four studied areas at 6 and 24 h after HI, mainly in neurons. Lysosomal activities were also increased following HI. Many neurons were strongly labeled for acid phophatase, and cathepsin B and D. These increases were confirmed using enzymatic assays and the presence of both autophagosomes and autolysosomes was detected using colocalization studies in the same neuronal cells. Electron microscopy confirmed the presence of numerous autophagosomes in the cytoplasm of dying neurons in the lesion (at 6 h) and also autolysosomes (at 24 h). Preliminary in vitro results using siRNA for Beclin 1 (Atg 8) was protective in a serum deprivation model.

Conclusions HI strongly enhanced autophagy by the presence of increased numbers of autophagosomes and autolysosomes mainly in neuronal cells and suggests that autophagic cell death mediates neuronal cell death in hypoxia-ischemia, opening new neuroprotective approaches.

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