Postischemic treatment of neonatal cerebral ischemia should target autophagy.
OBJECTIVE
To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy.


METHODS
Rats were exposed to transient focal cerebral ischemia on postnatal day 12. Some rats were treated by postischemic administration of pan-caspase or autophagy inhibitors. The ischemic brain tissue was studied histologically, biochemically, and ultrastructurally for autophagic, apoptotic, and necrotic markers.


RESULTS
Lysosomal and autophagic activities were increased in neurons in the ischemic area from 6 to 24 hours postinjury, as shown by immunohistochemistry against lysosomal-associated membrane protein 1 and cathepsin D, by acid phosphatase histochemistry, by increased expression of autophagosome-specific LC3-II and by punctate LC3 staining. Electron microscopy confirmed the presence of large autolysosomes and putative autophagosomes in neurons. The increases in lysosomal activity and autophagosome formation together demonstrate increased autophagy, which occurred mainly in the border of the lesion, suggesting its involvement in delayed cell death. We also provide evidence for necrosis near the center of the lesion and apoptotic-like cell death in its border, but in nonautophagic cells. Postischemic intracerebroventricular injections of autophagy inhibitor 3-methyladenine strongly reduced the lesion volume (by 46%) even when given >4 hours after the beginning of the ischemia, whereas pan-caspase inhibitors, carbobenzoxy-valyl-alanyl-aspartyl(OMe)-fluoromethylketone and quinoline-val-asp(OMe)-Ch2-O-phenoxy, provided no protection.


CONCLUSIONS
The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.
Puyal
Julien
J
Department of Cellular Biology and Morphology, University of Lausanne, Lausanne, Switzerland. JulienPierre.Puyal@unil.ch
Vaslin
Anne
A
Mottier
Vincent
V
Clarke
Peter G H
PG
D000225:Adenine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D002545:Brain Ischemia / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D007150:Immunohistochemistry; D007276:Injections, Intraventricular; D002546:Ischemic Attack, Transient / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000187:drug effects / Q000473:pathology; D008297:Male; D008854:Microscopy, Electron; D009336:Necrosis / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D009474:Neurons / Q000187:drug effects / Q000473:pathology*; D018696:Neuroprotective Agents; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley
D061945:Caspase Inhibitors; D018696:Neuroprotective Agents; C025946:3-methyladenine; D020169:Caspases; D000225:Adenine