Necrotic cell injury in the preterm and near-term ovine fetal brain after intermittent umbilical cord occlusion Elizabeth Rocha, MSc, Robert Hammond, MD, Bryan Richardson, MD* CIHR Group in Fetal and Neonatal Health and Development, Department of Obstetrics and Gynaecology, Physiology, Paediatrics and Neurological Sciences, The Lawson Health Research Institute, University of Western Ontario, London, Canada Received for publication October 16, 2003; revised January 19, 2004; accepted January 21, 2004 – Objective: This study was undertaken to determine the extent of necrotic cell death as a measure of neurologic injury in the preterm and near-term ovine fetal brain in response to intermittent umbilical cord occlusion (UCO) with severe, but limited hypoxia and no cumulative acidosis to ensure longer-term survival. Study design: Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with UCOs performed in the experimental group animals by complete infla- tion of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and the fetal brain perfusion-fixed and prepared for subsequent histology and assessment of necrotic cell injury by using standard staining with hematoxylin and eosin (H&E), and with a novel fluorescent marker, Fluoro-Jade B, that targets degenerating neurons. Results: In both preterm and near-term animal groups, UCO caused a large decline in arterial PO 2 (to w7 mm Hg), a modest decline in pH (to w7.30), and a modest rise in PCO 2 (to w61 mm Hg) (all P!.01), but with a return to control values after the occluder release and no cumu- lative acidosis over each day of study. Overall, very low levels of H&E-stained necrotic-appearing cells and Fluoro-Jade Bestained positive cells were observed across all brain regions studied with values not significantly different from zero, excepting that for the gray matter of the preterm con- trol (by H&E staining), preterm and near-term cord occlusion (by H&E and Fluoro-Jade B stain- ing), and the thalamus of the near-term cord occlusion (by H&E staining) animals. Although there were no differences in the levels of H&E-stained necrotic-appearing cells and Fluoro-Jade Bestained positive cells between respective control and cord occlusion group animals for most of the brain regions studied, a significant increase in Fluoro-Jade Bestained positive cells was ob- served in the gray matter of both the preterm and near-term cord occlusion animals (P!.05). Conclusion: Intermittent cord occlusion insult with severe but limited fetal hypoxemia and no cu- mulative acidosis, was generally well tolerated in both the preterm and near-term animal groups as assessed by measures of necrotic cell injury throughout the brain with minimal evidence for KEY WORDS Brain development Necrotic cell injury Hypoxia This work was supported by grants from the Canadian Institute of Health Research (B.R.). Dr Richardson is currently the recipient of the Wyeth Clinical Research Chair for Women’s Health in Perinatology. * Reprint requests: Bryan S. Richardson, MD, Department of Obstetrics and Gynaecology, St. Joseph’s Health Centre, 268 Grosvenor St, London, Ontario, Canada N6A 4V2. E-mail: brichar1@uwo.ca American Journal of Obstetrics and Gynecology (2004) 191, 488e96 www.elsevier.com/locate/ajog 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.01.039