Cyclooxygenase-Mediated Generation of Free Radicals During Hypoxia in the Cerebral Cortex of Newborn Piglets

Abstract

Previous studies have demonstrated that free radicals are formed under hypoxic conditions in newborn piglet brain. To test the hypothesis that the cyclooxygenase pathway serves as a source of free radical generation during hypoxia studies were performed on 24 piglets divided into four groups. Six saline (group 3) and six indomethacin treated (group 4) were exposed to hypoxia (FiO2 0.05-0.07) for 60 min. Cerebral hypoxia was documented biochemically by determination of ATP and phosphocreatine. Fluorescent compounds and conjugated dienes were determined as indices of lipid peroxidation. Free radical formation was determined by using n-tert butyl phenyl nitrone (PBN) as a spin trap agent and measuring spin adduct formation in duplicate using a Varian E-109 spectrometer. Groups 1 and 2 (normoxic) showed no spin adduct formation. Group 3 showed a significant increase in spin adduct formation compared to normoxia (372+/-125 vs. 63+/-15, P<0.001). Hypoxic animals pretreated with indomethacin had a spin adduct level of 197+/-132 and were similar to normoxic animals. ATP/PCr levels were the same in groups 3 and 4 denoting the same degree of cerebral hypoxia in all hypoxic animals. Conjugated dienes increased significantly during hypoxia as compared to normoxia (0.142+/-0.017 vs. 0.0+/-0.0) and were decreased insignificantly with indomethacin treatment. Fluorescent compounds were not significantly different among the four groups. Na+,K+-ATPase activity decreased during hypoxia but was not preserved in hypoxic animals pretreated with indomethacin. These data provide direct evidence of the presence of free radicals during hypoxia and the contribution of cyclooxygenase metabolism to their formation.