Elsevier

Neuropharmacology

Volume 39, Issue 7, June 2000, Pages 1319-1330
Neuropharmacology

Involvement of peroxynitrite and hydroxyradical generated from nitric oxide in hypoxia/reoxygenation injury in rat cerebrocortical slices

https://doi.org/10.1016/S0028-3908(99)00197-5Get rights and content

Abstract

The changes in nitric oxide (NO) formation during hypoxia and reoxygenation were measured in slices of rat cerebral cortex, and the possible involvement of NO and its decomposition products, including peroxynitrite and hydroxyradical, in the hypoxia/reoxygenation injury was subsequently investigated. NO formation estimated from cGMP accumulation in the extracellular fluids was enhanced during hypoxia and to a lesser extent in the reoxygenation period. The mRNA for inducible NO synthase (NOS) was detected 3–5 h after reoxygenation, although neuronal NOS mRNA decreased after reoxygenation. Several NOS inhibitors such as NG-monomethyl-l-arginine and NG-nitro-l-arginine blocked not only the NO formation but also the hypoxia/reoxygenation injury as determined by lactate dehydrogenase (LDH) leakage. The hypoxia/reoxygenation injury was prevented by peroxynitrite scavengers including deferoxamine and uric acid, or several hydroxyradical scavengers such as dimethylthiourea, 2-mercaptopropionylglycine and d(-) mannitol. In addition, the hypoxia/reoxygenation injury was attenuated by poly(ADP–ribose)synthetase inhibitors such as banzamide, 3-aminobenzamide and 1,5-isoquinolinediol. On the other hand, both N-morpholinosidnonimine, a peroxynitrite generator, and hydroxyradical-liberating solution containing FeCl3–ADP and dihydroxyfumarate caused a marked LDH leakage in normoxic slices. These findings suggest that the enhanced formation of NO causes hypoxia/reoxygenation injury after degradation to peroxynitrite and hydroxyradical and the resultant activation of poly(ADP–ribose)synthetase.

Section snippets

Hypoxia/reoxygenation-induced tissue injury in slices of the rat cerebral cortex

Male 10–14 week-old Sprague-Dawley rats (Japan Slc, Shizuoka, Japan) were used. They were housed in groups of five to six in a room controlled at 21–25°C, with 45–65% humidity and maintained in an alternating 12-h light/dark cycle (lights automatically on at 8:00 am). Food and water were freely given. Experiments were all carried out in accordance with the Guide for the Care and Use of Laboratory Animals, written by the Japanese Pharmacological Society. After immersing the whole brain in

Time course of changes in NO formation in rat cerebrocortical slices after exposure to hypoxia/glucose deprivation followed by reoxygenation

The accumulation of cGMP determined in the extracellular fluids after the addition of GTP, IBMX and crude extract of the rat cerebellum was markedly enhanced during hypoxia/glucose deprivation (Fig. 1). This increase in the cGMP production was totally abolished by an NOS inhibitor L-NAME or by an intracellular Ca2+ chelator BAPTA/AM. Therefore, the enhancement of cGMP during hypoxia/glucose deprivation may result from activation of Ca2+-dependent constitutive NOS. The enhancement of the NO

Discussion

We have recently reported that the exposure of rat cerebrocortical slices to hypoxia/glucose deprivation followed by reoxygenation causes an increase in LDH leakage into extracellular fluids (Tatsumi et al., 1998). This hypoxia/reoxygenation injury was completely blocked by the removal of extracellular Ca2+ or by chelating intracellular free Ca2+ with BAPTA/AM, thereby indicating an involvement of excessive Ca2+ entry in the tissue damage (Tatsumi et al., 1998). In the present study, changes in

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