A Murine Model of Experimental Necrotizing Enterocolitis Using Gavage Feeding, Lipopolysaccharide, and Systemic Hypoxia

Summary

This protocol describes how to induce experimental necrotizing enterocolitis (NEC) in newborn rats and mice.

Abstract

This protocol describes a model of experimental necrotizing enterocolitis (NEC) using rats or mice. NEC is a gastrointestinal disease unique to premature infants. Nearly 10% of babies born <1.5 kg develop this disease, and the mortality rate approaches 50%. The pathogenesis remains incompletely understood, but involves feeding, ischemia, inflammation, and infection. Animal models are vital to advancing the collective understanding of NEC. Many laboratories study NEC using the murine model. Other models, including pigs and rabbits, have limitations, including cost, long gestation periods, and smaller litters. Many studies use known risk factors (enteral feeding, infection, inflammation, and ischemia) in NEC research.

One challenge in NEC research is enteral feeds. Pups, normally breastfed by their mother, must be fed by hand. Some methods include syringe or fine-tip applicator feeds. This requires animals to latch and swallow feeds without respiratory compromise. Risks include aspiration, regurgitation, and spilling of feeds. The complications often cause unintended mortality and inconsistent results. Gavage feedings avoid these complications. Feedings are gavaged using a silastic catheter, allowing for safe, efficient feedings. This reduces feeding-related complications and mortality. This method improves reproducibility, as the complete volume is appropriately administered.

The protocol utilizes three interventions associated with clinical NEC: diet, hypoxia, and inflammation. The diet is a high-calorie formula, which is associated with NEC. Pups receive enteral lipopolysaccharide (LPS). LPS, a Toll-Like Receptor 4 (TLR4) agonist, is associated with NEC in animals and humans. Following feeds, animals are subjected to hypoxia. Premature neonates are susceptible to hypoxemia, which, along with decreased intestinal perfusion following feedings, puts the infant at risk for post-prandial ischemia.

Introduction

The field of neonatology has evolved immensely over the last 50 years. Improvements in neonatal care have resulted in an increasing number of premature newborn who survive the first few days of life from respiratory insufficiency¹. However, these infants face the risk of other complications of prematurity. One of these complications is Necrotizing Enterocolitis (NEC), a life threatening GI disease occurring almost exclusively in preterm neonates. The disease occurs in nearly 10% of all infants born less than 1.5 kg. The mortality rate approaches 50% in the most severely affected infants². Despite decades of research, the....

Protocol

Ethical Statement: All protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The Medical College of Wisconsin. All procedures are non-survival surgery. No eye ointment was required. All syringes, utensils and tools were sterile. All chemicals were sterile. Euthanization of adult animals occurred by an overdose of carbon dioxide followed by a thoracotomy/pneumothorax. Euthanization of neonatal pups occurred by a lethal dose of ketamine and xylazine, followed by a thoracotomy/pneumothorax.

Discussion

NEC is a devastating disease among premature infants that desperately requires research to better understand the disease. This murine model of NEC allows researchers to potentially uncover vital knowledge that may benefit these infants. The main advantages of this model include using interventions that are known risk factors for NEC, efficient administration of feedings that reduce mortality, and improved consistency and reproducibility. Additionally, there is limited cost associated with these studies. The catheters can.......

Materials

Name	Company	Catalog Number	Comments
1.9 Fr Argyle Catheter	Coviden-Medrtonic	43309	Can contain human Obtained discarded catheters from NICU at Children's Hospital of Wisconsin
Ethanol	Sigma Aldrich	459844
Bleach	Up and UP/Target	003-07-0058	TOXIC
Incubator	Georgia Quail Farm (Savannah, GA)	1588 Hova-Bator
Puppy Formula	Pet Ag (Hampshire IL)	Esbilac Powder Milk Replacer FG99501
Infant Formula	Mead Johnson	Enfacare Infant Formula
Lipopolysaccharide (LPS)	Sigma Aldrich	L2630	TOXIC.  Dissolved in ddH2O, 2 mg/ml
Hypoxia Chamber	Biospherix (New York)	A-Chamber
Nitrogen Gas	Praxair (Danbury CT)	Compressed Nitrogen Gas	Potentially Harmful
Regulator	Biospherix (New York)	Proox Model 110
Ketamine (Ketaject)	Clipper Distributing Company LLC (St. Joseph, MO)	2010012	Potentially Harmful
Xylazine (Anased)	Lloyd Laboratories		Potentially Harmful
Rats	Jackson Laboratories	Timed-Pregnant Sprague Dawley Pregnant Female Rats
Mice	Jackson Laboratories
L-NAME (N5751 SIGMA Nω-Nitro-L-arginine methyl ester hydrochloride)	Sigma Aldrich	N5751
Lucigenin (N,N′-Dimethyl-9,9′-biacridinium dinitrate)	Sigma Aldrich	M8010	Toxic.  Dissolved in ddH2O, Stock concentration of 5 mg/ml
GP91-ds-tat	Blood Center of Wisconsin	Made from previously described publication (Circ Res. 2001 Aug 31;89(5):408-14.)
FITC-Dextran, 10 kDa	Sigma Aldrich	FD10S
Intestinal Alkaline Phosphatase (IAP)	donated by AM-Pharma (Netherlands)