Development of a novel definitive scoring system for an enteral feed-only model of necrotizing enterocolitis in piglets

Abstract

IntroductionNecrotizing enterocolitis (NEC) is a complex inflammatory disorder of the human intestine that most often occurs in premature newborns. Animal models of NEC typically use mice or rats; however, pigs have emerged as a viable alternative given their similar size, intestinal development, and physiology compared to humans. While most piglet NEC models initially administer total parenteral nutrition prior to enteral feeds, here we describe an enteral-feed only piglet model of NEC that recapitulates the microbiome abnormalities present in neonates that develop NEC and introduce a novel multifactorial definitive NEC (D-NEC) scoring system to assess disease severity.MethodsPremature piglets were delivered via Caesarean section. Piglets in the colostrum-fed group received bovine colostrum feeds only throughout the experiment. Piglets in the formula-fed group received colostrum for the first 24 h of life, followed by Neocate Junior to induce intestinal injury. The presence of at least 3 of the following 4 criteria were required to diagnose D-NEC: (1) gross injury score ≥4 of 6; (2) histologic injury score ≥3 of 5; (3) a newly developed clinical sickness score ≥5 of 8 within the last 12 h of life; and (4) bacterial translocation to ≥2 internal organs. Quantitative reverse transcription polymerase chain reaction was performed to confirm intestinal inflammation in the small intestine and colon. 16S rRNA sequencing was performed to evaluate the intestinal microbiome.ResultsCompared to the colostrum-fed group, the formula-fed group had lower survival, higher clinical sickness scores, and more severe gross and histologic intestinal injury. There was significantly increased bacterial translocation, D-NEC, and expression of IL-1α and IL-10 in the colon of formula-fed compared to colostrum-fed piglets. Intestinal microbiome analysis of piglets with D-NEC demonstrated lower microbial diversity and increased Gammaproteobacteria and Enterobacteriaceae.ConclusionsWe have developed a clinical sickness score and a new multifactorial D-NEC scoring system to accurately evaluate an enteral feed-only piglet model of NEC. Piglets with D-NEC had microbiome changes consistent with those seen in preterm infants with NEC. This model can be used to test future novel therapies to treat and prevent this devastating disease.

Superior performance of biofilm versus planktonic Limosilactobacillus reuteri in protection of the intestines and brain in a piglet model of necrotizing enterocolitis

“…Using our enteral feed-only model of NEC in piglets 28 , we now demonstrate that enteral administration of Lr in its biofilm state has a positive impact on the intestine, the microbiome, and the developing brain. In addition, Lr in its biofilm state has a greater effect against NEC compared to Lr in its planktonic state.…”

“…At least 3 of the following 4 criteria were required for a piglet to be diagnosed with definitive NEC (D-NEC): clinical sickness score (CSS) of ≥ 5 out of 8 in the last 12 h of life, gross injury score of ≥ 4 out of 6, histologic injury score of ≥ 3 out of 5, and bacterial translocation to ≥ 2 internal organs (liver, spleen, and mesenteric lymph nodes with ≥ 1 colony-forming unit (CFU)/mg of tissue) 28 .…”

“…The enteral feed-only model of NEC was used as we have previously described 28 . All animal studies were carried out in accordance with and approved by the Research Institute at Nationwide Children’s Hospital Institutional Animal Care and Use Committee (IACUC) (protocol #AR18-00062).…”

“…Furthermore, pigs also share some features of the human microbiome, making these animals a strong model for digestive disease 36 , 37 . We previously demonstrated an abundance of Gammaproteobacteria in piglets exposed to NEC-inducing conditions 28 . Gammaproteobacteria are similarly increased in preterm infants with NEC 38 .…”

“…Towards our goal of administering Lr in its biofilm state to premature babies to prevent NEC, demonstration of efficacy in a large animal model is highly desirable. We have recently developed an enteral feed-only piglet model of NEC 28 . Compared to rat pups, piglets have similar average birthweight to premature human neonates 29 , making them a more ideal model for drug development for this patient population.…”