Retinopathy of prematurity (ROP) affects ~16,000 premature infants per year in the US. At preterm birth, there is loss of both ω3 and ω6 long-chain polyunsaturated fatty acid (LCPUFA), normally provided by the maternal/placental interface in utero. prominently contributes to initiation and progression of ROP. Docosahexaenoic acid (DHA, ω3) alone prevents ROP in some but not all studies. If DHA is given alone serum arachidonic acid (AA, ω6) decreases further, and lack of AA also contributes to ROP. We must understand how DHA and AA together prevent ROP to develop potent safe interventions based on physiology. In premature infants developing severe ROP, decreased mitochondrial number and increased peroxisomal activity (cleaving lipids ≥22 carbons) was found. However, knowledge of mitochondrial and peroxisomal lipid oxidation in retinal diseases is limited. Pilot work suggests DHA and AA control peroxisomal activity in a phase 1 ROP model. We will determine DHA/AA effects on retinal metabolism, particularly mitochondrial and peroxisomal fatty acid oxidation in early vessel loss in ROP. DHA/AA controls neurovascular development in phase 1 ROP by improving metabolism. In hyperglycemic mice in phase I ROP (vessel growth suppression and retinal neuronal dysfunction) we will: i) investigate if AA adds to DHA protection against retinal neurovascular abnormalities; ii) determine if DHA/AA alters retinal mitochondrial metabolism, and iii) determine if DHA/AA controls peroxisomal β-fatty acid oxidation. SUMMARY: These studies will determine if AA adds to DHA protection in phase I ROP (improving retinal metabolism) and uncover novel lipid metabolic associations. Supplementing DHA and AA orally will likely help prevent ROP and other retinopathies