Tetrahydrobiopterin is a cofactor of the aromatic amino acid hydroxylases enzymes and also acts as an essential cofactor for all nitric oxide synthase (NOS) isoforms. In Vitro: MicMicroglial cell cultures under hyperoxia are supplemented or not with an effective dose of Tetrahydrobiopterin (BH4) (100 μM). Exposure of microglial cells to hyperoxia-induced oxidative stress for 24 h reveals a robust increase in TSP-1 mRNA expression and protein compare to normoxia (21% O₂). Tetrahydrobiopterin supplementation significantly prevents hyperoxia-induced microglial activation by diminishing Iba-1 and TSP-1 expression and prevents microvascular injury in choroidal explants^[1]. In Vivo: To assess the levels of Tetrahydrobiopterin in the retina, three to five pools of retinas are collected from WT and hph-1mice at postnatal age 7, 14, and 22 and evaluated by LC-MS/MS. LC-MS/MS analysis confirm a significant decrease by approximately 90% in the concentration levels of Tetrahydrobiopterin in retinal tissue from hph-1 mice (0.0009±0.0006; p<0.0001, 0.01±0.001; p<0.0001 and 2.45±0.40; p<0.005) compare to the WT group (0.014±0.001, 0.092±0.01, and 23.13±6.44) at P7, P14, and P22, respectively^[1].