Formula feeding and immature gut microcirculation promote intestinal hypoxia, leading to necrotizing enterocolitis

Yong Chen, Yuhki Koike, Lijun Chi, Abdalla Ahmed, Hiromu Miyake, Bo Li, Carol Lee, Paul Delgado-Olguín, Agostino Pierro, Yong Chen, Yuhki Koike, Lijun Chi, Abdalla Ahmed, Hiromu Miyake, Bo Li, Carol Lee, Paul Delgado-Olguín, Agostino Pierro

Abstract

Major risk factors for necrotizing enterocolitis (NEC) are formula feeding and prematurity; however, their pathogenic mechanisms are unknown. Here, we found that insufficient arginine/nitric oxide synthesis limits blood flow in the intestinal microvasculature, leading to hypoxia, mucosal damage and NEC in the premature intestine after formula feeding. Formula feeding led to increased intestinal hypoxia in pups at postnatal day (P)1 and P5, but not in more mature pups at P9. Accordingly, blood flow in the intestinal microvasculature increased after formula feeding in P9 pups only. mRNA profiling revealed that regulators of arginine/nitric oxide synthesis are at higher levels in endothelial cells of the intestine in P9 than in P1 pups. Importantly, arginine supplementation increased intestinal microvasculature blood flow and prevented NEC, whereas an arginine antagonist exacerbated NEC. Our results suggest that balancing intestinal oxygen demand and supply in the premature intestine by modulating arginine/nitric oxide could be used to prevent NEC.This article has an associated First Person interview with the first author of the paper.

Keywords: Hypoxia; Microvascular flow dynamics; Microvasculature dilation; Necrotizing enterocolitis; Premature intestine circulation; Vascular maturation.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

© 2019. Published by The Company of Biologists Ltd.

Figures

Fig. 1.
Fig. 1.
Formula feeding induces mucosal hypoxia in experimental NEC. (A) Haematoxylin and Eosin (HE) and pimonidazole staining in ileum from pups: dam fed (DF; n=9), DF+lipopolysaccharide (LPS) (n=8), formula+LPS (n=9), and formula+LPS+systemic preprandial hypoxia (n=10). Arrowheads point to tips of villi in DF and DF+LPS pups; arrows point to damaged villi in formula+LPS and formula+LPS+hypoxia pups. Samples were collected 90 min after feeding. Scale bars: 100 µm. (B) NEC severity score. (C) Pimonidazole grading. (D) qRT-PCR showing normalized expression levels of Il-6, Il-1b, Il-10 and Tnf-a mRNA in ileum. (E) Western blot analysis of HIF-1α in ileum. (F) qRT-PCR of GLUT-1 and PHD-3 in ileum. (G) qRT-PCR of GLUT-1 and PHD-3 in liver, kidney and heart. *P<0.05, **P<0.01, ***P<0.001. All values are mean±s.d.
Fig. 2.
Fig. 2.
Formula feeding induces mucosal hypoxia in experimental NEC. (A,B) qRT-PCR of GLUT-1 (A) and PHD-3 (B) in proximal (jejunum), mid and distal (ileum) small intestine of P5 pups at different postfeeding time points. (C,D) Pimonidazole staining (C) and pimonidazole grading (D) of ileal tissue from P1, P5 and P9 pups fed with PBS or hyperosmolar formula (n=6). Scale bars: 100 µm. (E-G) qRT-PCR of GLUT-1 (E), PHD-3 (F) and Il-6 (G) on ileum from P1, P5 and P9 pups fed with PBS or hyperosmolar formula. *P<0.05, **P<0.01. All values are mean±s.d.
Fig. 3.
Fig. 3.
Inadequate intestinal haemodynamic response to feeding in the immature intestine in early neonatal life. (A) Set-up for two-photon microscopy. RosamT/mG/+;Tie2-Cre pups under general anaesthesia and laparotomy for visualization of the intestinal microcirculation. (B) Plane of imaging. (C) Representative image of intestinal submucosal flow using in vivo two-photon microscopy. A, arteriole; V, venule. White bar, arteriole diameter; yellow bar, platelet movement trajectory. (D-F) Quantification of arterial diameter (D), arterial velocity (E) and arterial flow (F) at several time points after gavage formula feeding in P1, P5 and P9 pups. (G) Plane of imaging for visualizing the villus microvasculature. (H) Representative micrographs from two-photon microscopy of the capillary network in intestinal villi of the jejunum and ileum in P1, P5 and P9 RosamT/mG/+;Tie2-Cre pups. At least three animals from each age group were analysed. For all graphs, error bars represent ±s.e.m. Scale bars: 100 µM.
Fig. 4.
Fig. 4.
The mature intestine upregulates arginine synthesis and concomitantly downregulates endothelin (ET-1) signalling pathways. (A) Hierarchical clustering of the transcriptome of intestinal endothelial cells in P1 (n=4) and P9 (n=5) RosamT/mG/+;Tie2-Cre pups. (B) Upregulated pathways in P9 compared to P1 intestinal endothelial cells. (C) Volcano plot showing increased expression of genes in the arginine biosynthesis pathway (red), and downregulation of genes in the ET-1 signalling pathway (blue), in P9 versus P1 intestinal endothelial cells. (D) qRT-PCR showing normalized expression of genes in the arginine biosynthesis pathway in ileum from P1 and P9 pups. (E) Immunostaining of CPS1 (green) in P1 and P9 intestine. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm. (F) Downregulated pathways in P9 versus P1 pups. (G) qRT-PCR showing normalized expression of genes in the endothelin pathway in ileum from P1 and P9 pups. *P<0.05, **P<0.01, ***P<0.001. All values are mean±s.d.
Fig. 5.
Fig. 5.
Inhibition of nitric oxide synthesis compromises postprandial hyperaemia and increases NEC severity, whereas arginine supplementation reduces NEC severity. (A) Micrographs from two-photon microscopy were used to quantify arterial diameter at several time points after gavage feeding of P5 pups with formula supplemented with arginine. (B) Quantification of arterial diameter change at several time points after gavage feeding of P9 pups with formula supplemented with L-NAME. (C) HE and pimonidazole staining in ileum of control (n=8), NEC (n=11), NEC+L-NAME (N=8) and NEC+arginine pups (n=9). Arrows point to damaged villi. (D) NEC severity score. (E) Pimonidazole grading. (F) qRT-PCR of Il-6 in ileum. (G) qRT-PCR of Tnf-a in ileum. Scale bars: 100 μm. *P<0.05, **P<0.01, ***P<0.001. All values are mean±s.d.

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