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

Abstract

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

Use of sildenafil and l-arginine in an experimental rat model for the prevention of neonatal necrotizing enterocolitis

“…Perhaps the time of drug administration was inefficient to observe significant improvement of intestinal lesions, although Cintra et al 10 also took a 12-h break between l-arginine administrations made for 3 days, or the NEC induction model was so efficient as to cause a very serious intestinal lesion that could not be prevented by a dose previously tested in other studies. The difference between our study and the experimental model by Jilling et al and further adapted by Gonçalves et al 16,17 it concerns the time of exposure to 100% nitrogen and the administration of the enteral diet. In our study, rats were kept under 100% nitrogen for 70 seconds and fed with 0.1 mL of Esbilac ® every 4h, and the experimental model by Jilling et al and further adapted by Gonçalves et al 16,17 left the rats under 60 seconds in an atmosphere of 100% nitrogen and fed with 0.1 mL of Esbilac ® every 3 h.…”

“…In our study, 0.1 mL of 10% l-arginine solution was used intraperitoneally, as in the study from Cintra et al 10 However, Cintra et al 10 described a protective effect as they show a lower degree of hystologic lesions in the intestine of rats that received l-arginine, contrary to the results found in our study. Our study method differed from the one from Cintra et al 10 , we did not promote reoxygenation (100% O2) and heating (22 °C) after episodes of hypoxia and hypothermia 16,17 . Maybe reoxygenation and rewarming potentiate the protective action of l-arginine in intestinal lesions of NEC.…”

Human placental-derived stem cell therapy ameliorates experimental necrotizing enterocolitis

“…Beginning within 6 h of birth, neonatal rat NEC models utilize formula feeding, LPS administration, and hypoxic stress to induce NEC (33). Previous investigations have established that intestinal damage is initiated within 24 h of NEC induction, while overall mortality limits the study window to 96 h (31)(32)(33)41). To increase intestinal insult before our treatment time point, the NEC rat pup model used in the present study included three doses of LPS within the first 30 h of NEC induction (Fig.…”

Temporal profile of intestinal tissue expression of intestinal fatty acid-binding protein in a rat model of necrotizing enterocolitis

“…The IW/BW ratio was higher in the NEC group than in the C group. This finding could be explained by the nutritional or inflammatory intestinal processes occurring as a result of NEC 10 .…”

“…Experimental NEC was induced according to the protocol described by Jilling et al and adapted by Gonçalves et al 9 , 10 . Newborn rats were fed 0.1 mL of Esbilac® artificial milk (PetAg, Hampshire, IL, USA) every 3 hours beginning at 6:00 am for 24 hours using an adapted, peripherally inserted central catheter (PICC, 1.9 Fr 26 gauge, 1 lumen, BD®, Sandy, UT, USA).…”