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Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices
Author(s) -
Hirai Kiyoshi,
Sugawara Taku,
Chan Pak H.,
Basus Vladimir J.,
James Thomas L.,
Litt Lawrence
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2002.01130.x
Subject(s) - phosphocreatine , cytochrome c , hypoxia (environmental) , apoptosis , tunel assay , cytosol , biology , western blot , microbiology and biotechnology , cytochrome , caspase 3 , dna fragmentation , fragmentation (computing) , immunostaining , endocrinology , medicine , programmed cell death , biochemistry , chemistry , oxygen , immunohistochemistry , enzyme , immunology , ecology , organic chemistry , gene , energy metabolism
Cellular injury was evaluated in superfused cerebrocortical slices (350 µm) 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. 31 P/ 1 H 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.