Plexiglas chamber

C57BL/6 mice (male, 8 to 12 weeks old; The Jackson Laboratory, Bar Harbor, ME, USA) were used in this study, in accordance with the Institutional Animal Care and Use Committees of St. John’s University. The mice were housed in a specific pathogen-free environment maintained at 22 °C in ≈50% relative humidity with a 12-h light/dark cycle. All mice had ad libitum access to standard rodent food and water. Mice were exposed to hyperoxia as previously described (Patel et al. 2013 (link)). Briefly, animals were placed in micro isolator cages (Allentown Caging Equipment, Allentown, NJ, USA) that were kept in a Plexiglas chamber (BioSpherix, Lacona, NY, USA) and exposed to ≥99% O₂ or remained in room air for 72 h. Mice exposed to hyperoxia were randomized to receive either intraperitoneally administered GTS-21 (0.04, 0.4 and 4 mg/kg) or the control vehicle, saline, every 8 h, starting 32 h after the onset of hyperoxic exposure. At the end of hyperoxic exposure, mice were euthanized with intraperitoneal sodium pentobarbital (120 mg/kg) to obtain bronchoalveolar lavage (BAL) fluid samples or lung tissues, as described below, for further analysis.

The Institutional Animal Care and Use Committee at Northwestern University approved all animal procedures. MISTRG mice were procured from Jackson Lab (JAX 017712), and maintained on 0.27 mg/ml Baytril as previously described (6 (link)). Timed matings were conducted to ensure at least 2 litters of at least 6 pups each litter, and equal numbers of male and female pups, for each experiment using at least 2 patient samples and placebo (PBS) at a time. At p0, litters were examined for the presence of milk spots and to ensure dams were caring for the pups. Cryopreserved monocytes were thawed (see above) and resuspended at a concentration of 1 × 10⁶ monocytes per 30 ul of PBS and administered by intrahepatic injection on p0. An equal volume of PBS was used for placebo injections (PBS group, N = 17 mice). Mice were kept at room air (21% O₂) or at hyperoxia (85% O₂) in a Plexiglas chamber (Biospherix, Lacona, NY) for 14 days. Nursing dams were rotated every 24 h to prevent oxygen toxicity to adult animals.

Litters of vehicle and IUGR pups were placed in either 75% oxygen (hyperoxia) in a Plexiglas chamber (Biospherix) or 21% oxygen (room air) within 24 h after birth for 14 d (Aslam et al., 2009 (link); Lee et al., 2014 (link)). Exposure to hyperoxia was continuous, with brief interruption only for animal care (<10 min/d). The concentration of oxygen was maintained with an oxygen controller (ProOx, Biospherix). Ventilation within the chamber was adjusted to remove CO₂ such that it did not exceed 0.5%. A hygro-thermometer was used in the chamber to monitor temperature and humidity. Temperature in the chamber did not exceed 23°C and humidity level was maintained using dishes of desiccant in the bottom of the chamber. A foster dam was placed in the hyperoxia chamber with each vehicle or IUGR litter, and foster dams were rotated from hyperoxia to room air every 24–48 h to prevent excessive oxygen toxicity to the adult animals. Litters were removed from the hyperoxia chamber at 14 d and euthanized for tissue collection.