Vitreal macrophages express vascular endothelial growth factor in oxygen-induced retinopathy

Purpose: The possibility of vitreal macrophages playing an angiogenic role in oxygen-induced retinopathy (OIR) was investigated. Oxygen-induced retinopathy was produced in newborn animals with the purpose of modeling the proliferative phase of human retinopathy of prematurity (ROP). Materials and Methods: To produce OIR in neonatal mice, litters at postnatal day 7 were placed in 80-90% oxygen for a period of 5 days and then returned to room air. Pups were killed on days 7, 12, 15, 17 and 20 over the postnatal period and were perfusion-fixed using a saline wash-out, followed by 4% paraformaldehyde and then India Ink. Eyes were enucleated and either whole-mounted, or snap-frozen and cryosectioned. Immunostaining procedures were used to visualize macrophages and vascular endothelial growth factor (VEGF) protein. The primary antibodies used were anti-F4/80 and antimouse VEGF, respectively. Vitreal macrophages closely associated with the vitreo-retinal interface (within 25 mum of the inner limiting membrane) were counted. In situ hybridization procedures were used to analyse for the presence of VEGF mRNA transcript in vitreal macrophages. Results: Macrophage numbers were found to significantly increase (P < 0.05) in eyes from oxygen-treated animals compared with those from age-matched controls. A close spatial relationship was observed between macrophages and vitreal neovascular sprouts. In addition, vitreal macrophages were also found to transcribe and express VEGF in the oxygen-treated animals during the vasoproliferative phase. Conclusions: Our results raise the possibility that vitreal macrophages play a role in the pathogenesis of OIR and by inference, ROP.