Oxidative stress in the fetal lamb brain following intermittent umbilical cord occlusion: a path analysis Michael Scott Rogers a, * , Henry George Murray b , Chi Chiu Wang a , Craig E Pennell b , Anita Turner b , Ping Yan b , Calvin C. P. Pang c , Allan M. Z. Chang a Objective To evaluate the relative contribution of cord occlusion length intervals between occlusions and experimental duration on oxidative stress in the fetal lamb brain. Design Acute, partially exteriorised fetal lambs with intermittent total cord occlusion. Setting The Vivarium of Westmead Hospital, University of Sydney, Australia and The Chinese University of Hong Kong. Main outcome measures Arterio-venous differences in the concentration of organic hydroperoxides, measured in paired samples of carotid arterial and jugular venous blood, as an index of oxidative stress in the brain. Methods Thirty-two fetal lambs were exposed to graded hypoxia, induced by intermittent total umbilical cord compression of 30 seconds, 60 seconds and 90 seconds duration, occurring every minute for a total of 27 occlusions over 81 minutes. Three sham experiments were also performed. In addition to organic hydroper- oxides, carotid arterial blood samples were also assayed in 15 animals (two sham) for oxygen saturation, pH, hypoxanthine, xanthine and urate concentrations. A causal model for oxidative stress was defined: occlusions leading to hypoxia with a rise in hypoxanthine; reperfusion during intervals between occlusions leading to the accelerated production of xanthine and uric acid and the generation of oxygen free radicals, which in turn, are responsible for the rise in lipid peroxidation. Path analysis was performed to assess the strength of the relationships between these variables and the cord occlusion length, the interval between occlusions and the duration of the experiment. Results Sham experiments showed no change in organic hydroperoxide production. Following 30-second umbilical cord occlusions a sixfold drop in mean organic hydroperoxides was observed between carotid arterial and jugular venous levels. In contrast, following occlusions of 60 seconds duration (or longer) a median 20-fold increase in organic hydroperoxide production was observed. Path analysis revealed a strong indirect pathway from occlusion length ! hypoxanthine ! urate and weak positive pathways from oxygen saturation ! urate and from interval between occlusions ! urate. After accounting for these pathways reflecting oxidative stress, a strong direct path remained from time from first occlusion ! organic hydro- peroxide production. Conclusions Peroxidation of lipids in the brain occurs under conditions of severe hypoxia and reperfusion associated with intermittent umbilical cord occlusions of 60 seconds or longer. The path analysis supported the causal model as originally defined, with the exception that the indirect pathway via pH was found to be trivial. INTRODUCTION Oxygen free radicals are highly reactive oxygen species, which can initiate cell damage by the peroxida- tion of lipid membranes and deoxyribonucleic acid 1–3 . The brain is an important target of free radical damage because of its large lipid content (75% of myelin, 55% of white matter, and 35% of grey matter) and because of the brain’s high rate of oxidative metabolism 4 . In 1988 Clapp et al. 5 demonstrated that intermittent occlusion of the umbilical circulation for 60 seconds out of every three minutes over a period of two hours was associated with a high incidence of confirmed neural damage, confined to the cerebral white matter. Recently Masaoka et al. 6 ,in a similar study of intermittent umbilical cord occlusion for 60 seconds out of every three minutes for two hours, demonstrated the release of a large amount of oxygen free radicals in the fetal circulation and brain perfusate. These free radicals were associated with increased conversion of hypoxanthine to xanthine. Taking these two studies together, it is reasonable to conclude that hypoxia-reperfusion, induced by intermittent cord occlu- q RCOG 2001 British Journal of Obstetrics and Gynaecology PII: S0306-5456(01)00297-2 British Journal of Obstetrics and Gynaecology December 2001, Vol. 108, pp. 1283–1290 www.bjog-elsevier.com * Correspondence: Professor M. S. Rogers, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong at Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR. a Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong b Department of Obstetrics and Gynaecology, Nepean Hospital, University of Sydney, Australia c Department of Opthalmology and Visual Science, Hong Kong Eye Hospital, The Chinese University of Hong Kong, Hong Kong