The mechanism of free radical generation in brain capillary endothelial cells after anoxia and reoxygenation.
We studied the mechanism of reoxygenation injury of cerebral microvessels in cultured rat brain capillary endothelial cells (BCECs). BCECs were isolated from rat cerebral cortices by a two step enzymatic treatment. The monolayers of BCECs were subjected to anoxia for 20 minutes followed by reoxygenation for 3 hours. Cell damage was assessed by measuring the leakage of intracellular lactic dehydrogenase (LDH). The control group was anoxia/reoxygenated BCECs without any protective reagents. To study the protective effect of free radical scavengers and antioxidants, superoxide dismutase, catalase, deferoxamine, oxypurinol, indomethacin, or NG-nitro-L-arginine methyl ester (L-NAME) was applied during anoxia/reoxygenation. Thus 7 experimental conditions were established. Lactic dehydrogenase (LDH) leaked from reoxygenated BCECs due to cell membrane damage. This leakage was almost totally suppressed by superoxide dismutase, indicating that reoxygenation injury of BCECs is mediated by superoxide generation. The other scavengers and antioxidants partially suppressed LDH leakage. Reduction of Ca2+ in the culture medium from 1.6 mM to 0.016 mM also suppressed LDH leakage. These results indicate that BCECs subjected to anoxia/reoxygenation become potent generators of superoxide anion, which is thought to be responsible for reoxygenation injury. The superoxide generation partially depends on the xanthine oxidase and cyclooxygenase pathways. As L-NAME partially suppressed LDH leakage peroxynitrite may contribute to reoxygenation injury of BCECs. The extracellular Ca2+ concentration also plays a critical role in the reoxygenation injury of BCECs.
Wu
S
S
Department of Neurosurgery, Kobe University School of Medicine, Japan.
Nagashima
T
T
Ikeda
K
K
Kondoh
T
T
Yamaguchi
M
M
Tamaki
N
N
D000818:Animals; D001921:Brain / Q000098:blood supply*; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005609:Free Radicals; D002534:Hypoxia, Brain / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology
D005609:Free Radicals; D007770:L-Lactate Dehydrogenase; D002118:Calcium