Validation of protocol of experimental necrotizing enterocolitis in rats and the pitfalls during the procedure

To describe the difficulties of implementing the protocol of experimental necrotizing enterocolitis (NEC) in order to obtain a larger number of newborns affected with the disease and a lower mortality.

Term Sprague-Dawley newborns rats (22 days) were divided into four groups of 12 fetuses each (n = 48): EC--breastfed newborns; IH--breastfed newborns and subjected to a stress protocol by ischemia and hypothermia; ESB--formula-fed newborns (Esbilac®, PetAg, Hampshire, IL, USA) and NEC--formula-fed newborns and subjected to stress protocol. The parameters set for the study protocol were: milk concentration (0.19 g ml or 0.34 g/ml), diet instilled volume (according to body weight--200 kcal/day/Kg--or progressive, according to acceptance), weight (gain, loss or maintenance) and duration of the experiment (72 hours or 96 hours). Data of body weight (BW), intestinal weight (IW) and the IW/BW ratio were obtained. Samples of terminal ileum were collected and analyzed by the degree of injury to the intestinal wall. Statistically significance was set to p<0.05.

The established protocol with less mortality and increased number of NEC was with Esbilac® at a concentration of 0.19 g/ml of diet instilled volume of 0.1 ml, every 3 hours, for 72 hours. All infants fed with artificial milk lost weight. In the degree score of intestinal injury, the ESB, IH and NEC groups were considered positive for NEC with greater histological injury in the latter.

The described NEC protocol in rats allowed a greater survival of puppies with a greater number of animals affected by the disease.