Induction of NEC in neonatal mice

Experimental NEC was induced in 7–8 day old (~3.0g body weight) neonatal mouse pups as previously described^{(22), (23; 24)}. Neonatal pups were randomly divided into control and treatment groups and experimental models were repeated at least 3 times with 8 or more mice per treatment group. Neonatal pups were gavage fed (40μL/g) five times/day (7am to 7pm) with formula containing one of three types of fat (described below and illustrated in Figure 1), that was supplemented with bacterial stock that had been cultured from the stool of an infant with severe NEC (12.5μl stool slurry in 1ml formula). The stool mixed formula (50μl per gram of mouse body weight) ⁽²⁵⁾ was administered using a 24-French angiocatheter placed into the mouse esophagus. Mice were exposed to hypoxia (5% O₂, 95% N₂) for 10 minutes in a chamber (Billups-Rothenberg INC. Del Mar, CA), twice daily (7am and 1pm immediately after feeding) for the 4-days, and the bacterial slurry was added to the formula on each day. Additional breast-fed control groups - exposed to hypoxia only, bacteria only and hypoxia + bacteria, and formula fed groups - exposed to formula only, formula + bacteria (no hypoxia), and formula +hypoxia (no bacteria) were also performed to evaluate the role of the individual components of the model of the development of intestinal inflammation. For mice subjected to breast fed/hypoxia alone, mice were given hypoxia (5% O₂, 95% N₂) for 10 minutes, twice daily for 4 days (at 7am and 1pm) and immediately returned with dams similar to NEC treated groups. For the breast fed/bacteria control group, mice were gavage fed bacteria (equal amount of bacteria given to NEC treated mice i.e., 10μL NEC bacterial stock slurry, diluted in 100μL saline/pup) once daily for 4 days. We and others have previously demonstrated that under administration of the standard formula, this experimental model induces significant intestinal inflammation expression of pro-inflammatory cytokines, Interleukin-6 (IL-6), Interleukin 1-beta (IL-1β), and Tumor Necrosis factor-alpha (TNF-α) that closely mimics human NEC ^{(26), (27)}. To determine the effect of antioxidant on ROS generation, NEC formula was supplemented with N-acetylcysteine (NAC) (100mg/kg) and admistered to mice in the NEC model. Control mice were gavage fed once daily with similar dose of 100mg/kg) of NAC.

Experimental scheme and location of tissue sampling used in the current studies.