Hypoxia initiates Ductus Arteriosus Remodeling. • 101

Abstract

After birth, the ductus arteriosus actively constricts in response to the rise in PaO2 and undergoes extensive histologic changes that prevent it from subsequent reopening. We used 7 full-term (1-5 d.o.) and 21 preterm (0.67 gestation) newborn baboons to test the hypothesis that following the initial constriction of the ductus, a region of hypoxia develops within the vessel wall that initiates the subsequent histologic changes. Twenty-four hours prior to sacrifice, we injected the baboons with the drug EF5, a nontoxic 2-nitroimadazole, which binds irreversibly to intact cells at a rate that increases 100-fold as the PO2 decreases from air to N2. EF5's plasma half-life is similar in both preterm and full-term baboons. Using a fluorochrome-conjugated mAb to bound EF5, we can detect changes in EF5 accumulation within the tissue by immunohistochemistry. In the full-term baboons, pulsed-Doppler exam on day 1 showed that the ductus lumen was closed. In the 1-day-old ductus, an intense zone of hypoxia (EF5 staining) was detected in the middle of the muscle media. In addition, all the smooth muscle cells in this intense hypoxic zone were undergoing apoptosis (TUNEL technique). By day 2 and 3, vascular endothelial growth factor (VEGF) was expressed in the muscle media on either side of the hypoxic zone; endothelial cells of the ductus lumen and vasa vasorum began to proliferate (began to express proliferating cell nuclear antigen [PCNA]) and invade the muscle media toward the hypoxic zone (which was becoming acellular). Among the 21 preterm baboons, pulsed-Doppler exams found that only 8 had closed their ductus by 6 d after birth. At necropsy on day 6, there was either no or negligible hypoxia(EF5 binding) even in those with a closed ductus on Doppler exam. There was no apoptosis, or staining for VEGF or PCNA. In contrast with the full-term ductus, the preterm ductus, despite Doppler evidence of closure, fails to develop a zone of hypoxia in the muscle media and fails to remodel, making it susceptible to later reopening.