TY - JOUR
T1 - Xanthine oxidase-derived superoxide causes reoxygenation injury of ischemic cerebral endothelial cells
AU - Beetsch, Joel W.
AU - Park, T. S.
AU - Dugan, Laura L.
AU - Shah, Aarti R.
AU - Gidday, Jeffrey M.
N1 - Funding Information:
The authors thank Chung Y. Hsu, MD, PhD and Jian Xu, MD, PhD for their endothelial cell expertise. The technical assistance of Lori Rubach in these studies was also greatly appreciated. This work is supported by grants awarded to J.W.B. from the American Heart Association, Missouri Affiliate, and to T.S.P. from the National Institutes of Health (NINDS 21045).
PY - 1998/3/9
Y1 - 1998/3/9
N2 - 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 μM), 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.
AB - 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 μM), 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.
KW - Cell culture
KW - Cerebral endothelium
KW - Ischemia-reperfusion
KW - Oxygen free radical
KW - Oxypurinol
KW - Superoxide dismutase
UR - http://www.scopus.com/inward/record.url?scp=0032498922&partnerID=8YFLogxK
U2 - 10.1016/S0006-8993(97)01407-8
DO - 10.1016/S0006-8993(97)01407-8
M3 - Article
C2 - 9554965
AN - SCOPUS:0032498922
SN - 0006-8993
VL - 786
SP - 89
EP - 95
JO - Brain Research
JF - Brain Research
IS - 1-2
ER -