Xanthine oxidase-derived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells.
Oxygen free radicals, generated by cerebral ischemia, have been widely implicated in the damage of vascular endothelium. Endothelial cells have been proposed as a significant source of oxygen free radicals. In the present study, we developed an anoxia-reoxygenation (AX/RO) model using pure cultures of cerebral endothelial cells (CECs) isolated from piglet cortex to measure CEC oxygen free radical production and determine its role in AX/RO-induced CEC injury. CEC injury, as measured by lactate dehydrogenase efflux into the culture medium, increased progressively with the duration of anoxic exposure, becoming significant after 10 h. Reoxygenation significantly increased CEC anoxic injury in a time-dependent manner. A 55% increase in oxygen free radical production, determined by fluorescence detection of dihydroethidium oxidation, was measured at the end of 4-h reoxygenation in CECs subjected to AX/RO conditions that killed 40% of the cells. Blockade of oxygen free radical production with superoxide dismutase (SOD; 250 and 1000 U/ml) or oxypurinol (50 and 200 microM), a potent xanthine oxidase inhibitor, reduced this injury by 32-36% and 30-39%, respectively. Results from our in vitro model indicate that CECs produce significant amounts of oxygen free radicals following ischemia, primarily from the xanthine oxidase pathway. These radicals ultimately have a cytotoxic effect on the very cells that produced them. Thus, reductions in oxygen free radical-mediated vascular injury may contribute to improvements in neurophysiologic outcome following treatment with oxygen free radical inhibitors and scavengers.
Beetsch
J W
JW
Department of Neurosurgery, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, MO 63110, USA.
Park
T S
TS
Dugan
L L
LL
Shah
A R
AR
Gidday
J M
JM
D000818:Animals; D002545:Brain Ischemia / Q000473:pathology / Q000503:physiopathology*; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000502:physiology*; D004730:Endothelium, Vascular / Q000473:pathology / Q000503:physiopathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008833:Microcirculation / Q000502:physiology; D010117:Oxypurinol / Q000494:pharmacology; D015427:Reperfusion Injury / Q000473:pathology / Q000503:physiopathology*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D013481:Superoxides; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D010117:Oxypurinol