(Stroke. 2001;32:506.)
© 2001 American Heart Association, Inc.
Original Contributions |
Increased Cytochrome c–Mediated DNA Fragmentation and Cell Death in Manganese–Superoxide Dismutase–Deficient Mice After Exposure to Subarachnoid Hemolysate
From the Department of Neurosurgery (P.G.M., M.F., A.L., Y.M-F., P.H.C.), Departments of Neurology and Neurological Sciences (M.F., A.L., Y.M-F., P.H.C.), and Program in Neurosciences (M.F., A.L., Y.M-F., P.H.C.), Stanford University School of Medicine, and Surgical Service, Palo Alto Veterans Affairs Health Care System (P.G.M.) (Calif).
Correspondence to Paul G. Matz, MD, Surgical Service (112N), Palo Alto VA Medical Center, 3801 Miranda Ave, Palo Alto, CA 94304. E-mail matzpg{at}yahoo.com
Background and Purpose—We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant.
Methods—Injection of 50 µL of subarachnoid hemolysate or saline was performed in CD1 mice (n=75), mutant mice deficient in Mn–superoxide dismutase (Sod2+/-; n=23), and their wild-type littermates (n=23). Subcellular location of cytochrome c was studied by immunocytochemistry, immunofluorescence, and immunoblotting of cellular fractions. DNA fragmentation was assessed though DNA laddering and terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL). Cell death was examined through basic histology.
Results—Cytochrome c immunoreactivity was present in the cytosol of neurons at 2 hours after hemolysate injection and increased by 4 hours compared with saline-injected animals (P<0.02). Cytosolic cytochrome c was more abundant in Sod2+/- mutants. DNA fragmentation was evident at 24 hours, but not 4 hours, after hemolysate injection as determined by DNA laddering and TUNEL staining (P<0.02). DNA fragmentation colocalized to cells with cytosolic cytochrome c and iron. In Sod2+/- mutants, the extent of fragmentation was increased as determined by TUNEL staining (52% increase; P<0.02) and DNA laddering (optical density=0.819 versus 0.391; P<0.01). Cell death was evident on basic histology as early as 4 hours after hemolysate injection. No cell death was evident in controls. In Sod2+/- mutants, cell death was increased by 51% compared with wild-type littermates (P<0.05).
Conclusions—These results demonstrate that subarachnoid blood products are associated with the presence of cytochrome c in the cytosol and subsequent cell death in neurons. It appears that Mn–superoxide dismutase plays a role in preventing cell death after exposure to subarachnoid blood products.
Key Words: hemoglobin • iron • stroke, experimental • subarachnoid hemorrhage • superoxide dismutase • mice
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