Objectives/Goals: The objective of this research is to characterize the role of anemia and its treatment with iron in the development of oxygen-induced retinopathy and to identify possible mechanisms of action for future investigation. Methods/Study Population: Newborn rats were exposed to cyclic hyperoxia (50%) hypoxia (10%) for 14 days to induce oxygen-induced retinopathy (OIR). Half of the pups were phlebotomized to induce anemia. Half of anemic and control pups received high-dose iron, while the other half received standard, low-dose iron. Retinas were dissected at postnatal day 20, and vascular outcomes were measured to determine phenotype. Comparisons will be made by two-way ANOVA. Additional future studies are planned: 1) electroretinogram to measure retinal function, 2) measure of retinal hypoxia and angiogenic protein levels to evaluate the effect on hypoxia-sensitive angiogenesis, 3) serum and retinal iron measures to determine iron status, 4) flow cytometry and cytokine array to evaluate immune cell response, and 5) exploratory single-cell RNA sequencing. Results/Anticipated Results: Preliminary vascular outcomes are pending. We anticipate that retinas of anemic pups treated with low-dose (standard of care) iron will exhibit iron deficiency and hypoxia and reduced severity of OIR. Retinas of anemic and non-anemic pups treated with high-dose iron will exhibit iron excess and increased severity of OIR. However, retinal function will be reduced in anemia and rescued with treatment with high-dose iron. We further anticipate that anemia and iron-deficiency will down-regulate overall immune cell fractions, which will correlate with reduced cytokine and chemokine levels, suggesting a reduced immune response. Finally, we expect vascular endothelial cells and pericytes in anemic pup retinas to show increased angiogenic gene expression and neurons to show signs of impaired development. Discussion/Significance of Impact: We expect that anemia has a differential impact on retinal vascular anatomy and neuronal function. While retinopathy severity may be improved, overall retinal function will be dampened. These findings and the mechanistic work of this study will generate targets for intervention to preserve improved retinopathy outcomes but rescue retinal function.