Loss of Endothelial Nitric Oxide Synthase Exacerbates Intestinal and Lung Injury in Experimental Necrotizing Enterocolitis

Abstract

Background Necrotizing enterocolitis (NEC) continues to be a devastating condition among preterm infants. Nitric oxide, which is synthesized in the intestine by endothelial nitric oxide synthase (eNOS), acts as a potent vasodilator and antioxidant within the mesentery and may play a role in prevention of NEC. We hypothesized that loss of endothelial nitric oxide would worsen both intestinal and associated lung injury and increase local and systemic inflammation during experimental NEC. Methods NEC was induced in five-day-old wild type (WT) and eNOS-knockout (eNOSKO) mouse pups. Experimental groups (n = 10) were formula fed and subjected to intermittent hypoxic and hypothermic stress, while control groups (n = 10) remained with their mother to breastfeed. Pups were monitored by daily clinical assessment. After sacrifice on day nine, intestine and lung were assessed for injury, and cytokines were measured in tissue homogenates by ELISA. Data were compared with Mann–Whitney, and p < 0.05 was significant. Results Each NEC group was compared to its respective strain’s breastfed control to facilitate comparisons between the groups. Both NEC groups were significantly sicker than their breastfed controls. eNOSKO NEC animals had a median clinical assessment score of 3 (IQR = 1–5), and the WT NEC animal’s median score was 3 (IQR = 2–5). Despite similar clinical scores, intestinal injury was significantly worse in the eNOSKO NEC groups compared to WT NEC groups (median injury scores of 3.25 (IQR = 2.25–3.625) and 2 (IQR = 1–3), respectively (p = 0.0474). Associated lung injury was significantly worse in the eNOSKO NEC group as compared to the WT NEC group (median scores of 8.5 (IQR = 6.75–11.25) and 6.5 (IQR = 5–7.5), respectively, p = 0.0391). Interestingly, cytokines in both tissues were very different between the two groups, with varying effects noted for each cytokine (IL-6, IL-1β, VEGF, and IL-12) in both tissues. Conclusion Nitric oxide from eNOS plays a key role in preventing the development of NEC. Without eNOS function, both intestinal and lung injuries are more severe, and the inflammatory cascade is significantly altered. Further studies are needed to determine how eNOS-derived nitric oxide facilitates these beneficial effects.