A Titratable Necrotizing Enterocolitis Mouse Model

Abstract

AbstractNecrotizing enterocolitis (NEC) is an inflammatory gastrointestinal process that afflicts nearly 10% of premature babies born in the United States each year, with a mortality rate of 15 - 30%. NEC severity is graded using the Bell’s classification system based on the presenting symptoms and radiological findings. Animal models are a valuable tool in studying the pathogenesis of this disease; however, prior models cannot control the severity of NEC induced, a critical aspect of the disease in human infants. Here, we aim to bridge this gap in knowledge through development of a murine model of NEC with graded characteristics.Postnatal day 3 (P3) C57BL/6 mice were fed formula containing different concentrations (0.25%, 1%, 2%, and 3%) of dextran sodium sulfate (DSS), an osmotic agent previously shown to induce severe NEC in neonatal mice. P3 mice were fed every 3 hours over a 72-hour period. During feeding, we collected data on weight gain and behavior, which included activity, response, and body color. After feeding, at P6, we dissected the mice to check for bowel features and collect organs for immunohistochemistry and cytokine analysis. Mice fed formula only or 0% DSS were used as a control instead of breastfed mice. Since staying with the dam during the first week of life is vital to normal development in mice, using formula-fed mice as the control accounts for the handling bias that exists during feeding.Comparing breastfed and formula-fed mice alone, weight gain was significantly greater in breastfed mice, and liver cytokine concentrations tended to be higher. In the established graded NEC model, clinical differences were seen among the mice fed different DSS concentrations. Over the course of NEC induction, mice fed higher concentrations of DSS lost weight quicker and exhibited greater clinical severity scores. External bowel scores (dilation, color, and friability) were considerably worse in mice fed higher DSS concentrations; although internal small bowel pathology showed intestinal disarray among mice fed all DSS concentrations. Staining for cellular proliferation marker Ki-67 in intestines shows that mice fed higher concentrations of DSS display less cell proliferation at the base of the small intestine crypts. Pro-inflammatory cytokine levels in the liver, to assess systemic inflammation downstream of the gastrointestinal system, trended higher in mice fed with higher concentrations of DSS.Here, we demonstrate that we have successfully characterized a graded newborn mouse model where NEC severity can be predictably induced. This mouse model will be a valuable tool in gaining greater insight and understanding of the pathophysiology of this devastating disease process and to understand its long0term complications, such as neurodevelopmental impairment.