Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices.
Cellular injury was evaluated in superfused cerebrocortical slices (350 micro m) from 7-day-old Sprague-Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase-9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase-9; (ii) localization of cleaved caspase-9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase-9 returned to control values, while whole cell caspase-9 stayed approximately the same, suggesting translocation of caspase-9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis.
Hirai
Kiyoshi
K
Department of Anesthesia, University of California, San Francisco, San Francisco, California 94143-0648, USA.
Sugawara
Taku
T
Chan
Pak H
PH
Basus
Vladimir J
VJ
James
Thomas L
TL
Litt
Lawrence
L
D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism*; D003574:Cytochrome c Group / Q000032:analysis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D002534:Hypoxia, Brain / Q000378:metabolism*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D019344:Lactic Acid / Q000032:analysis / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010725:Phosphocreatine / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D003574:Cytochrome c Group; D010725:Phosphocreatine; D019344:Lactic Acid; D000255:Adenosine Triphosphate; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases