Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC’s main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels—as seen during stress—promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure—NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

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Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model

10.1101/2021.01.20.427423 ◽

2021 ◽

Author(s):

Robert L. Bell ◽

Ginger S. Withers ◽

Frans A. Kuypers ◽

Wolfgang Stehr ◽

Aditi Bhargava

Keyword(s):

Necrotizing Enterocolitis ◽

Rat Model ◽

Stress Responses ◽

Cell Activation ◽

Immune Cell ◽

Corticotropin Releasing Factor ◽

Neonatal Rat ◽

Mucosal Barrier ◽

Mucosal Barrier Function ◽

Neonatal Rat Model

AbstractThe etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC’s main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels—as seen during stress—promote NEC in formula-fed newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure—NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p=0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

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Inhibition of Nuclear Factor-κB Ameliorates Bowel Injury and Prolongs Survival in a Neonatal Rat Model of Necrotizing Enterocolitis

Yearbook of Neonatal and Perinatal Medicine ◽

10.1016/s8756-5005(08)79242-4 ◽

2008 ◽

Vol 2008 ◽

pp. 217-219

Author(s):

J. Neu

Keyword(s):

Necrotizing Enterocolitis ◽

Rat Model ◽

Nuclear Factor Κb ◽

Bowel Injury ◽

Neonatal Rat ◽

Nuclear Factor ◽

Neonatal Rat Model

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Antioxidant effects of N-acetylcysteine in a neonatal rat model of necrotizing enterocolitis

Journal of Pediatric Surgery ◽

10.1016/j.jpedsurg.2012.02.016 ◽

2012 ◽

Vol 47 (9) ◽

pp. 1652-1657 ◽

Cited By ~ 24

Author(s):

Ramazan Ozdemir ◽

Sadık Yurttutan ◽

Fatma Nur Sarı ◽

Bulent Uysal ◽

Hatice Germen Unverdi ◽

...

Keyword(s):

Necrotizing Enterocolitis ◽

Rat Model ◽

Neonatal Rat ◽

Antioxidant Effects ◽

Neonatal Rat Model

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Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00196.2001 ◽

2002 ◽

Vol 282 (1) ◽

pp. G156-G164 ◽

Cited By ~ 178

Author(s):

Bohuslav Dvorak ◽

Melissa D. Halpern ◽

Hana Holubec ◽

Catherine S. Williams ◽

Debra L. McWilliam ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Mrna Expression ◽

Necrotizing Enterocolitis ◽

Rat Model ◽

Transforming Growth Factor ◽

Gastrointestinal Disease ◽

Neonatal Rat ◽

Epidermal Growth ◽

Neonatal Rat Model

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-α mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC.

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