Establishment of hyperoxia-induced BPD neonatal rat models

Twelve specific-pathogen-free (SPF) pregnant Sprague–Dawley (SD) rats (gestational age of 15 days; Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were raised at 22 ± 3 °C with 60 ± 5% humidity and 12 h light–dark cycle. Each pregnant rat was housed individually and was self-delivered after 1 week of acclimation. After delivery, 72 neonatal rats with the birth time 12 h apart were randomly grouped into the hyperoxia treatment group and the control group. The hyperoxia-treated rats with BPD were classified into the miR-214 negative control (NC) (BPD rats infected with adenoviral particles expressing NC for miR-214 agomir), miR-214 agomir (BPD rats infected with adenoviral particles expressing miR-214 agomir), miR-214 NC + PlGF vector (BPD rats infected with adenoviral particles expressing NC for miR-214 agomir and PlGF vector), and miR-214 agomir + PlGF (BPD rats infected with adenoviral particles expressing miR-214 agomir and PlGF) groups (n = 12 for each treatment). The oxygen concentration used in the hyperoxia group was 95%, and that used in the control group was 21%. The above conditions were continued until the end of the experiment. To prevent oxygen poisoning, the rats in the hyperoxia group were housed in a normal environment every 24 h. The neonatal pups were raised to 1 week in age and then injected with pAd-U6-MCS-CMV-GFP adenovirus vector. The recombinant adenovirus was propagated and amplified in HEK293 cells. The virus was injected via the tail vein at the volume of 30 μL at 1 × 10⁸ TU at a time interval of 3 days. miR-214 NC (5′-CGAUCGCAUCAGCAUCGAUUGC-3′), miR-214 agomir (5′-ACAGCAGGCACAGACAGGCAGU-3′), PlGF overexpression vector, and empty vector were purchased from RiboBio (Guangdong, China). The neonatal rats used for BPD modeling were placed in a plexiglas normobaric oxygen box with continuous oxygen input. In the box, the concentration of oxygen was maintained at 95%, and sodium lime absorbed CO₂, with the temperature of 25–27 °C and the humidity of 50–70%. The control rats (air group) were exposed to air (at 21% oxygen concentration), and the remaining experimental conditions and operations were the same as those in the hyperoxia treatment group. The box was routinely opened for 30 min every day, water and food were added, and the litter was replaced. The postpartum rats were exchanged with the control group to avoid a decrease in the feeding ability of postpartum rats because of oxygen toxicity. The rats in the control group were placed in the same room, with similar experimental control factors to those in the hyperoxia treatment group. Three neonatal rats were randomly selected from the two groups by a random number generation method on the 3rd, 7th, and 14th days after the experiment began and then received an intraperitoneal injection of 90 mg/kg pentobarbital sodium for anesthesia. Next, the abdominal cavity was immediately opened, and the right lung was removed and placed in an RNase-free cryovial (Eppendorf, Hamburg, Germany). After rapid freezing with liquid nitrogen, the lungs were stored in a − 80 °C refrigerator for subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Next, 40 g/L paraformaldehyde was slowly injected into the rats through the left bronchus until the apex of the lung was inflated. The lung was placed in an embedding box, and 40 g/L paraformaldehyde solution was added for overnight fixation and subsequent use.