Pro ox 110 chamber controller

OIR was induced in C57BL/6 wild-type mice as previously described.¹³ (link) Briefly, on postnatal day 7 (P7), mouse pups with their nursing dams were exposed to 75% O₂ (Pro-Ox 110 Chamber Controller, Biospherix, Redfield, NY, USA) for 5 days. At P12, mice were returned to room air and they received an intravitreal injection of 1 × 10⁴ ECFCs transfected with miR-130a mimics or control mimics into their right eyes in a 1 μL volume. As a control, 1 μL of phenol red-free DMEM containing no growth factors or serum was injected into the left eye of each pup. Injections were performed under anesthesia induced by intraperitoneal injection of ketamine and xylazine. Cells were administered intravitreally using a 10 μL Flexifil syringe fitted with a 33G needle (WPI, Sarasota, FL). Pups were euthanized at P14 using sodium pentobarbital, and the whole eyes were fixed in 4% PFA for 1 h. Retinal flat mounts were stained with isolectin B4 (Sigma) and streptavidin-Alexa 488 (Thermo Fisher Scientific). Stained retinas were visualized and area quantification was performed using ImageJ software.

The establishment of the mouse OIR model was performed as previously described with modifications (Smith et al., 1994 (link); Fu et al., 2012 (link)). Briefly, neonatal mouse pups and their nursing mothers were placed in an air-tight chamber with 75% oxygen (PRO-OX110 chamber controller; Biospherix Ltd., New York, NY) from P7 to P12 and then returned to the room air 21% oxygen environment on P12 (Figure 1A). Soda lime (Sigma-Aldrich Corporation, St. Louis, MO) was placed in the chamber during the high oxygen treatment to quench excess CO₂. Activated carbon and silica gel desiccants were also placed in the chamber for ammonia and humidity control, respectively. The litter size was restricted to 6 to 7 pups in each litter to make sure all pups in the cage received enough nutrition (Connor et al., 2009 (link)). Retina samples were collected on P12, P17, P21, P25, and P30 and prepared as retinal whole mounts for further evaluations. From P17, only pups with body weight above 6 g were used for examinations (Stahl et al., 2010a (link)). To overcome biological variations, samples in each time point were randomly selected from different litters. Pups of same age and always held in a room air (RA) environment served as control groups. Six to eight pups were included in each group at various time points. Both males and females were used.