The Effect of BH₄ Supplementation in Retinal Ischemia

Abstract

Purpose : Retinopathy of prematurity (ROP) is a serious sight-threatening complication of therapeutic hyperoxia caused by oxidative insult to the immature retinal blood vessels leading to tissue ischemia and hypoxia-induced neovascularisation. Previously we have shown that tissue hypoxia induces the production of the pro-inflammatory mediators TNFα and inducible nitric oxide synthase (iNOS). iNOS is dependent upon the co-factor tetrahydrobiopterin (BH₄) absence of which leads to aberrant production of superoxide. As we have previously shown that BH₄ is depleted by hyperoxia, we investigated the contribution of BH₄ to inflammation-induced damage in the murine model of oxygen-induce retinopathy (OIR) using hph-1 mice that are deficient in GTPCH, the rate limiting enzyme for the production of BH₄.

Methods : Wild type, hph-1 heterozygotic (hph-1^+/-) and hph-1 homozygotic (hph-1^-/-) mice were subjected to oxygen induced retinopathy (OIR) by exposure to hyperoxia from postnatal day (P) 7 to P12. On return to room air at P12, treatment was commenced with the BH₄ precursor sepiapterin or vehicle control (VC) and retinal, lung and brain tissue was collected 48h later at P14. BH₄ levels were determined by HPLC analysis, NOS activity by Activity Assay, apoptosis by TUNEL and superoxide by dihydroethidium (DHE) fluorescence. Gene expression was measured by qPCR. Retinal flat mounts were stained with lectin to visualise vascular area and microglial cell density.

Results : HPLC analysis confirmed a decrease in BH₄ levels from WT, through hph-1^+/- to hph-1^-/-, which was elevated following sepiapterin supplementation. NOS activity was reflective of BH₄ levels, decreasing across genotypes and enhanced in the presence of BH₄. INOS gene expression was unaltered by supplementation. Notably, BH₄ supplementation caused a significant decrease in hypoxia-induced TUNEL staining in the inner nuclear layer (INL) with decreases of 54% in WT, 31% in hph-1^+/- and 20% in hph-1^-/- animals. In addition, BH₄ treated groups had significantly diminished superoxide formation with the WT, hph-1^+/- and hph-1^-/- groups showing decreases in DHE fluorescence of 41%, 40% and 44% respectively. This correlated with a trend towards increased vascular coverage.

Conclusions : Together our results demonstrate that BH₄ supplementation exerts a neuro-protective effect during the ischaemic-hypoxic phase of OIR by modulating the cellular redox state.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.