Pups were randomized to one of the following groups: 1) Sham, 2) NEC, 3) NEC + HA, and 4) HA alone. Sodium hyaluronate (Lifecore Biomedical, LLC, Chaska, MN) with a molecular weight of ~35,000 kDa (HA 35) was given to pups in the NEC+ HA and HA group by gavage at a concentration of 15mg/kg or 30mg/kg body weight once daily for three days prior to induction of NEC and one hour prior to bacterial administration (Figure 1 ). The doses and intervals were based on previous studies done in large intestinal inflammation models [9 (link), 11 (link)].
NEC was induced using the Paneth cell disruption and Klebsiella infection NEC-like model [15 (link), 16 (link)]. Crl:CD1(ICR) pups (Charles River) at P14 to P16 days of age received an intraperitoneal injection of dithizone (33 mg/kg) diluted in ethanol/ammonium hydroxide, followed by gavage administration of 1×108 CFU Klebsiella pneumoniae /kg (ATCC#10031, Manassas, VA). Pups were monitored for 10 hours after gavage for clinical illness and survival [17 (link)]. At the end of the experiment, surviving pups were euthanized, and blood and tissues were harvested for further analysis.
NEC was induced using the Paneth cell disruption and Klebsiella infection NEC-like model [15 (link), 16 (link)]. Crl:CD1(ICR) pups (Charles River) at P14 to P16 days of age received an intraperitoneal injection of dithizone (33 mg/kg) diluted in ethanol/ammonium hydroxide, followed by gavage administration of 1×108 CFU Klebsiella pneumoniae /kg (ATCC#10031, Manassas, VA). Pups were monitored for 10 hours after gavage for clinical illness and survival [17 (link)]. At the end of the experiment, surviving pups were euthanized, and blood and tissues were harvested for further analysis.