Animal care guidelines according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research were followed. All procedures were approved by the institutional animal care and use committee (St. Vincent's Animal Ethics Committee protocol No. 060/09). Wild type (WT) C57BL/6J mice and Nox2 knockout mice (Nox2 KO) on a C57BL/6J background were supplied by Mary Dinauer (Department of Pediatrics, Washington University of School of Medicine, St. Louis, MO),
12 and bred at the EMSU mouse facility (Fitzroy, Victoria, Australia). The Nox2 genotype was confirmed with conventional genotyping (see
Supplementary Fig. S2; Nox2 WT forward primer, 5′AAGAGAAACTCCTCTGCTGTGAA-3′; reverse primer, 5′CGCACTGGAACCCCTGAGAAAGG-3′; Nox2 KO forward primer, 5′AAGAGAAACTCCTCTGCTGTGAA-3′; reverse primer, GTTCTAATTCCATCAGAAGCTTATCG-3′; Sigma-Aldrich, Sydney, New South Wales, Australia). The OIR was induced by exposing 7-day-old neonatal mice (P7) and their mothers to 75% oxygen for 5 days (74.91 ± 0.05% oxygen,
n = 95, P7–P12) followed by a 5-day period in room air (P12–P17).
4,13 Oxygen levels were monitored with an external oxygen analyzer (Model No. 500-AE; Sensidyne, St. Petersburg, FL) and recordings were analyzed. Neonatal mice were sacrificed, and eyes were harvested at P8 and P17, time points corresponding with periods of peak vaso-obliteration and neovascularization, respectively.
4,14 Eyes were fixed in 4% paraformaldehyde (PFA; ProSciTech, Kirwan, Queensland, Australia) for 1 hour, and retinal flat mounts were prepared and stained with fluorophore-conjugated isolectin B4 (5 g/mL, Alexa Fluor 488 No. I21411; Life Technologies, Mulgrave, Victoria, Australia) overnight at 4°C.
4,13 Flat mounts then were washed in PBS and mounted in fluorescent mounting media (Dako, Glostrup, Denmark). Images of each retinal flat mount were captured with a fluorescence microscope (×4 objective magnification, emission 495 nm, excitation 510 nm; Olympus, Victoria, Australia), and the degree of vaso-obliteration and neovascularization then was quantified as described previously.
4 Avascular areas and regions of neovascularization in images of retinal flat mounts were delineated and measured using ImageJ (National Institutes of Health, Bethesda, MD),
15 and expressed as a percentage of the total retinal area. Age-matched neonatal mice exposed to room air were used as normoxic controls. One eye was used for flat mount preparation, while the fellow eye was used either for gene expression analysis or in situ superoxide detection.