Branched Chain Fatty Acids Reduce the Incidence of Necrotizing Enterocolitis and Alter Gastrointestinal Microbial Ecology in a Neonatal Rat Model

Abstract

IntroductionBranched chain fatty acids (BCFA) are found in the normal term human newborn's gut, deposited as major components of vernix caseosa ingested during late fetal life. We tested the hypothesis that premature infants' lack of exposure to gastrointestinal (GI) BCFA is associated with their microbiota and risk for necrotizing enterocolitis (NEC) using a neonatal rat model.MethodsPups were collected one day before scheduled birth. The pups were exposed to asphyxia and cold stress to induce NEC. Pups were assigned to one of three experimental treatments. DF (dam-fed) ; Control, hand-fed rat milk substitute ; BCFA, hand-fed rat milk substitute with 20%w/w BCFA. Total fat was equivalent (11%wt) for both the Control and BCFA groups. Cecal microbiota were characterized by 16S rRNA gene pyrosequencing, and intestinal injury, ileal cytokine and mucin gene expression, interleukin-10 (IL-10) peptide immunohistochemistry, and BCFA uptake in ileum phospholipids, serum and liver were assessed.ResultsNEC incidence was reduced by over 50% in the BCFA group compared to the Control group as assessed in ileal tissue; microbiota differed among all groups. BCFA-fed pups harbored greater levels of BCFA-associated Bacillus subtilis and Pseudomonas aeruginosa compared to Controls. Bacillus subtilis levels were five-fold greater in healthy pups compared to pups with NEC. BCFA were selectively incorporated into ileal phospholipids, serum and liver tissue. IL-10 expression increased three-fold in the BCFA group versus Controls and no other inflammatory or mucosal mRNA markers changed.ConclusionAt constant dietary fat level, BCFA reduce NEC incidence and alter microbiota composition. BCFA are also incorporated into pup ileum where they are associated with enhanced IL-10 and may exert other specific effects.

Pomegranate seed oil reduces intestinal damage in a rat model of necrotizing enterocolitis

“…After termination, a 2-cm piece of distal ileum was removed and fixed in 70% ethanol, paraffin embedded, sectioned at 4 -6 m, and stained with hematoxylin and eosin (H&E) for histological evaluation of NEC. Pathological changes in intestinal architecture were evaluated using our previously published NEC scoring system (18,33,50). Histological changes in the ileum were scored by a blinded evaluator and graded as follows: 0 (normal), no damage; 1 (mild), slight submucosal and/or lamina propria separation; 2 (moderate), moderate separation of submucosa and/or lamina propria, and/or edema in submucosal and muscular layers; 3 (severe), severe separation of submucosa and/or lamina propria, and/or severe edema in submucosa and muscular layers, region villous sloughing; 4 (necrosis), loss of villi and necrosis.…”

“…Histological changes in the ileum were scored by a blinded evaluator and graded as follows: 0 (normal), no damage; 1 (mild), slight submucosal and/or lamina propria separation; 2 (moderate), moderate separation of submucosa and/or lamina propria, and/or edema in submucosal and muscular layers; 3 (severe), severe separation of submucosa and/or lamina propria, and/or severe edema in submucosa and muscular layers, region villous sloughing; 4 (necrosis), loss of villi and necrosis. Intermediate scores of 0.5, 1.5, 2.5 and 3.5 were also utilized to more accurately assess levels of ileal damage when necessary (18,33,50). To determine incidence of NEC, animals with histological scores of Ͻ2 have not developed NEC; animals with histological scores of Ն2 have developed NEC (33,50).…”

“…Intermediate scores of 0.5, 1.5, 2.5 and 3.5 were also utilized to more accurately assess levels of ileal damage when necessary (18,33,50). To determine incidence of NEC, animals with histological scores of Ͻ2 have not developed NEC; animals with histological scores of Ն2 have developed NEC (33,50).…”

“…Briefly, after deparaffinization and rehydration, sections were incubated with either rabbit anti-Muc2 polyclonal antibody (Santa Cruz Biotechnology) or rabbit polyclonal Tff3 antibody (54) for 30 min, washed with PBS three times, and incubated with a biotinylated , formula-fed group (FF) (; n ϭ 18), and group that was formula fed with 1.5% pomegranate seed oil added (FF ϩ PSO) (}; n ϭ 19) groups are shown. Ileal damage was assessed using the histological scoring scale of 0 (normal) to 4 (necrosis) as previously described (18,33,50). Animals with scores Ն2 are considered NEC positive.…”

Yeast-Derived ? -Glucan Reduces Intestinal Injury in Rat Model of Necrotizing Enterocolitis

“…Intermediate scores of 0.5, 1.5, 2.5 and 3.5 were also utilized to more accurately assess levels of ileal damage when necessary. To determine incidence of NEC, animals with histological scores of <2 have not developed NEC; animals with histological scores of ≥2 have developed NEC [18].…”