2.2. Formula NEC Models

NEC was induced in postnatal day P14–P16 C57Bl/6J mice by giving an intraperitoneal injection of 75 mg/kg body weight dithizone (Sigma) dissolved in 25 mM LiCO₃, or an equivalent volume of LiCO₃ buffer alone. Depending on the group being considered, 250 µL of RMS (regular formula NEC), RMS^SALT (high osmolality formula NEC), or saline (control animals) was fed through oral gavage one hour prior to injecting with dithizone and every three hours for a total of four feeds. Mice were sacrificed and tissues were harvested thirteen hours after the first RMS/saline gavage. NEC was induced in P14–P16 PC-DTR mice by giving an intraperitoneal injection of diphtheria toxin at a concentration of 40 ng/g body weight and oral gavage of saline (control animals) or RMS (formula NEC) one hour prior to injection as well as every three hours for a total of four feeds. Mice were sacrificed at sixteen hours after the first oral gavage and tissues were harvested. Similar to above, all experiments were performed on at least two separate occasions using at least two different litters and all controls were littermates from experimental animals.