Increased cytochrome c-mediated DNA fragmentation and cell death in manganese-superoxide dismutas-deficient mice after exposure to subarachnoid hemolysate

Background and Purpose-We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant. Methods-Injection of 50 muL 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 3 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 14 hours, but not 3 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 3 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.