Our study was approved by the Animal Care and Use Committee at Taipei Medical University. Time-dated pregnant Sprague-Dawley rats were housed in individual cages with 12-h light-dark cycles. Within 12 hours of birth, litters were pooled and randomly redistributed to the newly delivered mothers, and the pups were then randomly assigned to room air (RA) or oxygen-enriched atmosphere (O2) treatment. The pups in O2 treatment subgroups were reared in an atmosphere containing 85% O2 from postnatal days 1 to 14. The pups in RA control subgroups were reared in normal RA for 14 days. To avoid oxygen toxicity in the nursing mothers, they were rotated between the O2 treatment and RA control litters every 24 hours. An oxygen-rich atmosphere was maintained in a transparent 40 × 50 × 60-cm plexiglass chamber receiving O2 continuously at 4 L/min. Oxygen levels were monitored using a ProOx P110 monitor (Bio-Spherix; Redfield, NY, USA). For intratracheal administration, the rat pups were anesthetized with 2% isoflurane (Halocarbon Laboratories, River Edge, NJ, USA), positioned against an angled restraining stand, and exposed to locate the trachea. Human MSCs (1 × 105 cells) in 30 μl of normal saline (NS); 10 μl of a surfactant (Survanta, Abbvie, North Chicago, IL, USA), corresponding to approximately 35 mg/kg of phospholipids, diluted in 20 μl of NS; and 10 μl of the surfactant and MSCs (1 × 105 cells) diluted in 20 μl of NS were administered into the rat trachea by using a 30-gauge syringe (Hamilton Company, Reno, NY, USA) on postnatal day 5 (Fig. 1). We held the syringe upright and slowly injected the solution into the lungs during inspiratory phase. We examined eight study groups: RA + NS, RA + MSCs (1 × 105 cells), RA + surfactant, RA + surfactant + MSCs (1 × 105 cells), O2 + NS, O2 + MSCs (1 × 105 cells), O2 + surfactant, and O2 + surfactant + MSCs (1 × 105 cells). The surfactant + MSCs mixture was administered within 10 minutes of combination. Thereafter, the rats were allowed to recover from anesthesia and were returned to their mothers. The rats from each group were strongly anesthetized with an overdose of isoflurane on postnatal day 14, and body and lung weights were recorded. Immediately after death, the left lung was ligated and the right lung was fixed by tracheal instillation of 10% buffered formalin at a pressure of 25 cm H2O for 10 minutes.

Fig. 1

Diagrammatic representation of the experimental design showing the study timeline and the treatment groups. MSCs mesenchymal stem cells, NS normal saline