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

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 collective understanding of the pathophysiology of NEC remains incomplete^{3, 4}.

NEC is a life-threatening gastrointestinal disease affecting neonates resulting in systemic inflammation, primarily affecting the small intestine. Breast milk has been shown to confer some protection⁵. Currently, treatment is largely supportive including bowel rest, antibiotics, the use of ventilators and inotropes to mitigate the effects of shock. Surgery is reserved for failures of medical management and includes resection of dead or perforated bowel. Therefore, the goal of NEC research has been to prevent NEC from occurring by concentrating on preventative factors such as breast milk feedings, growth factors, avoidance of stressful stimuli, and identification of molecular targets with therapeutic potential.

Performing clinical studies and interventions for NEC are difficult, given the uncertainty and complexity of its pathophysiology. Therefore, animal models are required to advance the field. Several models have been used throughout the years⁶. Some animal models, including pigs and rabbits, have limitations, including cost, time, and smaller litters^{7, 8}. To maximize efficiency, many researchers use a murine (rat or mouse) model. Animals subjected to experimental NEC develop histological and biochemical changes similar to human neonates with the disease. However, there are several protocols used that will produce intestinal injury consistent with NEC, but may not resemble the clinical disease. The most significant risk factors for NEC are enteral feeding, ischemia, infection, and inflammation⁹. Many laboratories use some or all of these risk factors in their studies of NEC. The most important benefits of these models are that the findings could accurately represent the clinical disease.

One of the biggest challenges with those models is enteral feedings. The model uses animals that are only a few days old, and would normally be breastfed by their mother. Instead, animals must be fed by hand by the researchers. This is accomplished by using a syringe or a fine-tipped applicator. Animals must be able to adequate take feedings into their mouth, swallow the feed, and still be able to maintain adequate respiratory efforts. However, this is often fraught with complications, including aspiration, regurgitation, and spilling of feeds. Consequently, studies are at risk for unintended mortality and inconsistent results.

One method to reduce these complications is to use gavage feedings. This technique allows for direct administration of feeds into the stomach, significantly reducing the risk of aspiration. In addition, the time required to feed the animals is drastically reduced, allowing for studying several litters simultaneously. The catheters are inexpensive, durable, and can be obtained from a neonatal intensive care unit (NICU). If a laboratory does not have access to these catheters, they can be easily ordered from a commercial vendor.

The induction of experimental NEC is accomplished by using several facets. Animals are fed a high-calorie formula, which is known to be a risk factor for NEC. Lipopolysaccharide (LPS) is also added to the feedings. LPS promotes overwhelming inflammation and is an agonist of the Toll-Like Receptor 4 (TLR4) pathway¹⁰. TLR4 activation is strongly associated with NEC pathogenesis in both animal and clinical studies. After feedings, pups are subjected to systemic hypoxia. Clinically, premature infants are at risk for significant hypoxia. This puts the infant at risk for intestinal ischemia, exacerbated by an increased metabolic demand in the post-prandial state.

The protocol is appropriate for both neonatal rats and mice. Rats are the preferred species, as they can be taken away from their mothers immediately after they are born, and can even be delivered prematurely by cesarean section of the mother. Taking the rats away from their mother immediately after they are born allows for the experimental protocol to occur prior to any effect of the pups receiving breast milk from their mother. However, there are few genetically modified rat strains, so knockout mice are needed. Neonatal mice are much smaller than rats, and must remain with their mother for seven days prior to any experiments.

This model of experimental NEC allows researchers to study the disease with known clinical risk factors. In addition to understanding pathogenesis, it allows for the opportunity to observe how diet modifications, supplements, and other interventions affect the disease.