Increased cytochrome c-mediated DNA fragmentation and cell death in manganese-superoxide dismutase-deficient mice after exposure to subarachnoid hemolysate.
OBJECTIVE
We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant.


METHODS
Injection of 50 microL 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.
Matz
P G
PG
Department of Neurosurgery, Stanford University School of Medicine, and Surgical Service, Palo Alto Veterans Affairs Health Care System, CA 94304, USA. matzpg@yahoo.com
Fujimura
M
M
Lewen
A
A
Morita-Fujimura
Y
Y
Chan
P H
PH
D000818:Animals; D016923:Cell Death; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D053938:DNA Fragmentation / Q000187:drug effects / Q000235:genetics*; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D006454:Hemoglobins / Q000378:metabolism / Q000494:pharmacology; D006579:Heterozygote; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019579:Neocortex / Q000187:drug effects / Q000473:pathology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D018384:Oxidative Stress / Q000235:genetics; D013345:Subarachnoid Hemorrhage / Q000378:metabolism* / Q000473:pathology; D013482:Superoxide Dismutase / Q000172:deficiency* / Q000235:genetics; D015996:Survival Rate
D003574:Cytochrome c Group; D006454:Hemoglobins; D013482:Superoxide Dismutase