Background Necrotizing enterocolitis (NEC) is a severe intestinal disease of premature infants with high mortality. Studies suggest a causative relationship between red blood cell (RBC) transfusion and NEC, however, whether RBC transfusion leads to worse outcomes in NEC is unknown. We sought to determine whether RBC transfusion was associated with an increased risk of surgical NEC and mortality. Methods In this retrospective study, 115 patients were enrolled with NEC Bell’s Stage 2A or greater from 2010–2015. Patients were classified based on the timing of RBC transfusion prior to NEC: ≤72 hours, >72 hours, and no transfusion. Variables including gestational age (GA), birth weight (BW), feedings and hematocrit levels were analyzed. Outcomes were surgical intervention for NEC following RBC transfusion and mortality. Results 23 (20%) infants developed NEC ≤ 72 hours after RBC transfusion, 16 (69.6%) required surgery with a mortality rate of 21.7% (n=5). 17 (15%) infants developed NEC > 72 hours after RBC transfusion, 12 (70.6%) required surgery with a mortality rate of 23.5% (n=4). 75 (65%) patients developed NEC without RBC transfusion, 17 (22.7%) required surgery with a mortality rate of 4% (n=3). Lower GA and BW were significantly associated with RBC transfusion and the need for surgical intervention. RBC transfusion ≤72 hours prior to NEC was associated with surgical NEC (pairwise adjusted p<0.001) and mortality (pairwise adjusted p=0.048). However, multivariable logistic regression analysis revealed RBC transfusion is not an independent risk factor for surgical NEC. Conclusions Infants of lower GA and BW were more likely to receive an RBC transfusion prior to NEC, which was significantly associated with surgical intervention and an increasing risk of mortality. Judicious use of transfusions in premature infants may improve NEC outcomes.

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The etiology of pulmonary vascular abnormalities in CDH is incompletely understood. Studies have demonstrated improvement in pulmonary vasculature with prenatal therapy in animal models. We hypothesize that prenatal sildenafil may attenuate defective pulmonary vascular development via modulation of vSMC phenotype from undifferentiated, proliferative phenotype to differentiated, contractile phenotype. We utilized the nitrofen model of CDH to examine the effect of IA sildenafil on pulmonary vSMC phenotype during lung development. Timed-pregnant CD-1 mice were gavage fed 25 mg nitrofen or olive oil (control) at E8.5 of gestation. Single IA injections of Sildenafil (Revatio; 10 µL of 4 mg/4 ml solution) or dextrose control were performed at E12.5. Mice were sacrificed on various gestational days for embryonic lung harvest. Markers of vSMC development of undifferentiated and differentiated phenotypes were analyzed by immunostaining and western blot. Across all time points in gestation, nitrofen-treated embryonic lungs demonstrated increased vSMC expression of NOTCH3, Hes-5, PDGFR-β, desmin and α-SMA and decreased expression of calponin and SMMHC, compared to oil controls. IA dextrose treatment had no effect on expression levels. However, IA Sildenafil treatment resulted in down-regulation of NOTCH3, Hes-5, PDGFR-β, desmin and α-SMA and upregulation of calponin and SMMHC, comparable to oil controls. In the nitrofen model, vSMC express markers consistent with more undifferentiated proliferative phenotype, resulting in hypermuscularization of intrapulmonary arterioles in CDH. A single dose of IA Sildenafil treatment early in gestation, results in sustained normalization of vSMC phenotype. Pharmacologic modulation of the vSMC phenotype at key gestational points may have therapeutic potential.

Background: Defective lung development resulting in lung hypoplasia and an attenuated and hypermuscularized pulmonary vasculature contributes to significant postnatal mortality in congenital diaphragmatic hernia (CDH). We hypothesize that deficient embryonic pulmonary blood flow contributes to defective lung development in CDH, which may therefore be ameliorated via enhancement of embryonic pulmonary blood flow. Methods: The mouse nitrofen model of CDH was utilized to measure embryonic pulmonary blood flow by in utero intracardiac injection of FITClabeled tomato lectin and color-flow Doppler ultrasound. The effect of prenatal intra-amniotic treatment with sildenafil on survival, lung growth, and vascular morphology in the nitrofen model was determined. Results: Nitrofen-treated embryos exhibited decreased blood flow in the lung periphery compared to controls, and intra-amniotic sildenafil significantly improved embryonic pulmonary blood flow. Similar to nitrofen alone, pups delivered after nitrofen treatment and intra-amniotic injection of dextrose control exhibited respiratory distress and never survived beyond 6 h. Intra-amniotic sildenafil ameliorated respiratory distress in nitrofen-treated pups and improved postnatal survival to 82%. Following intra-amniotic sildenafil treatment at embryonic day (E)10.5, nitrofen-treated P0 lungs were larger with increased left lobe weight, reduced small pulmonary arterial wall muscularization, and increased airway branching complexity compared to controls. Intra-amniotic sildenafil treatment later at E15.5 also resulted in improved survival, lung growth, and attenuation of vascular remodeling in nitrofen-treated embryos. Conclusions: Defective embryonic pulmonary blood flow may contribute to lung maldevelopment in CDH. Enhancement of embryonic pulmonary blood flow via intra-amniotic sildenafil results in lung growth and attenuation of pulmonary vascular remodeling and may have therapeutic potential for CDH.