BH4 supplementation reduces retinal cell death in ischaemic retinopathy

Dysregulation of nitric oxide (NO) production can cause ischaemic retinal injury and result in blindness. How this dysregulation occurs is poorly understood but thought to be due to an impairment in NO synthase function (NOS) and nitro-oxidative stress. Here we investigated the possibility of correcting this defective NOS activity by supplementation with the cofactor tetrahydrobiopterin, BH₄. Retinal ischaemia was examined using the oxygen-induced retinopathy model and BH₄ deficient Hph-1 mice used to establish the relationship between NOS activity and BH₄. Mice were treated with the stable BH₄ precursor sepiapterin at the onset of hypoxia and their retinas assessed 48 h later. HPLC analysis confirmed elevated BH₄ levels in all sepiapterin supplemented groups and increased NOS activity. Sepiapterin treatment caused a significant decrease in neuronal cell death in the inner nuclear layer that was most notable in WT animals and was associated with significantly diminished superoxide and local peroxynitrite formation. Interestingly, sepiapterin also increased inflammatory cytokine levels but not microglia cell number. BH₄ supplementation by sepiapterin improved both redox state and neuronal survival during retinal ischaemia, in spite of a paradoxical increase in inflammatory cytokines. This implicates nitro-oxidative stress in retinal neurones as the cytotoxic element in ischaemia, rather than enhanced pro-inflammatory signalling.