Fig 1.
Experimental model of NEC- a timeline.
Neonates were housed with dams for 3 days before being randomized at postnatal day (PND) 3 into 6 groups as shown. Dam-fed controls were randomized to no stress (DF) or hypoxia / cold stress exposure (DFS), housed with a dam, and allowed ad libitum feeding. Formula-fed neonates were separated from their dams and housed in an incubator. They were randomized to no stress (FF), hypoxia / cold stress exposure (NEC), no stress with CRF administration (CRF), and hypoxia / cold stress exposure after pretreatment with Astressin (AST).
Fig 2.
Mucosal injury and NEC scoring system.
Representative H&E-stained terminal ileum sections to show mucosal injury consistent with NEC. (a) Normal intestinal mucosa: score 0. (b) Mild inflammation: score 1. (c) Moderate inflammation consistent with NEC: score 2. (d) Severe inflammation consistent with NEC: score 3.
Fig 3.
Formula feeding and exposure to acute stressors causes frank NEC-like histopathological damage in rats.
Representative H&E-stained micrographs showing villi damage in NEC. (a-c) Terminal ileum sections of DF, DFS, and FF unstressed pups showed normal gut histology with well-preserved villi structure. (d) Inflammatory changes were present in terminal ileum of FF neonates exposed to stressors (NEC) or (e) FF unstressed pups with CRF administration. (f) Pretreatment with Astressin in FF pups prevented stress-induced changes in ileum histopathology and prevented development of NEC. DF = dam-fed, unstressed; DFS = dam fed, stressed; FF = formula-fed unstressed; NEC = formula-fed, stressed; CRF = formula-fed, unstressed with 30μg/kg of sc CRF administration; AST = formula-fed, stressed with 60μg/kg of sc CRF antagonist, Astressin administration. Scale Bar = 100μM. (g) Stack bar graph summarizing numbers of neonates with 0–3 scores within control or treatment groups.
Fig 4.
Experimental model of necrotizing enterocolitis.
(a) Formula feeding combined with hypoxia and cold stress exposure induced gross changes in the small bowel including erythema, edema, and full thickness necrosis. (b) Gross intra-operative findings representative of complicated NEC in a premature human neonate.
Fig 5.
CRF and CRF receptor immunoreactivity (IR) is increased in the terminal ileum of neonates with NEC.
(a-e) Representative immunostained section from DF, DFS, FF and NEC ileum. CRF-IR (red) and CRFR (CRF1/2-IR, green) was evident in the villi, blood vessels, and neurons within the myenteric plexuses of FF and NEC groups, but only low, diffuse staining was seen in DF and DFS control groups. (f) Higher magnification (63x) confocal images revealed differences in staining pattern in CRF-IR and CRF1/2-IR in DF controls versus NEC groups.
Fig 6.
Incidence of necrotizing enterocolitis among treatment groups.
The incidence of NEC was determined for each treatment group and expressed as percentage ± standard error. Groups were compared using directional Chi square analysis and Fischer’s exact test. *: FF versus NEC groups, p = 0.006; **: FF versus CRF groups, p < 0.001; #: NEC versus AST groups, p = 0.033. DF = dam-fed, unstressed; DFS = dam fed, stressed; FF = formula-fed unstressed; NEC = formula-fed, stressed; CRF = formula-fed, unstressed with 30μg/kg of sc CRF administration; AST = formula-fed, stressed with 60μg/kg of sc CRF antagonist, Astressin administration.
Fig 7.
Bar graph showing increased TLR4 levels in rats with NEC and AST treatment decreased levels to those seen in dam-fed rats. Actin was used as normalization control. P = 0.03 NEC versus AST.