Dark-Rearing (DR) Mitigates Pre-Retinal Neovascularization and OIR Pathology with no Detriment To Visual Function and Morphology: Compelling Evidence For DR as Non-Invasive Intervention in Retinopathy of Prematurity (ROP)

Abstract

Purpose: ROP is initiated by hyperoxia-induced down-regulation of VEGF expression. We hypothesized that the initiating event in ROP can be blocked by subjecting neonates to total darkness, during the period O₂ exposure is required to protect vital organs, as darkness results in continuous depolarization of photoreceptors, requiring energy (O₂) intensive repolarisation.

Methods: SD rats were raised in: 21% O₂ + dark rearing (DR) from P0-30; hyperoxic conditions (60%/70% O₂ from P0-4, returned to 21% O₂ + normal light) with and without DR & assessed at P7; & under DR/normal light in 50/10 OIR & assessed at P18. Retinae were examined for vascular density (VDI), extent of peripheral vaso-obliteration & neovascularization, pericyte/astrocyte ensheathment and ultrastructural changes via TEM. Flash electroretinograms (fERG) were recorded in both 30 days post 50/10 OIR, & in long term DR rats (P30/60/90). Retinal O₂ profiles were modeled for P14 at 60 & 75% inspired O₂ in light & DR.

Results: We showed DR rats had higher VDI compared to age-matched controls (VDI=43±1.0 vs.38±1.1 p<0.05 - P7). DR protected vessels from O₂-induced peripheral vaso-obliteration (20.64% ± 4.85 v 39.02% ± 4.03 p<0.05) and reduced areas of pathological neovascularization (4.07% ± 0.85 v 15.79% ± 1.26 p<0.05) in 50/10 OIR. DR protects vessels from O₂-induced vaso-obliteration without harmful effects on normal microvascular cell interactions (as evidenced by normal astrocytic and pericytic ensheathement); retinal cell morphology as evidenced by TEM; & function as evidenced by fERG at 30 days post OIR, & after 90 days continuous DR. For all groups, average b-wave amplitudes from DR rats v dark/light cycle rats were not significantly different. O₂ profile modelling under DR at 60% inspired O₂ at P14 showed that the inner retina maintains 'physiological hypoxia', in darkness but not in light whilst 75% inspired O₂ overwhelms the protective effect of DR.

Conclusions: We showed that DR precludes the initiation in experimental models of ROP, offering a viable, non-invasive intervention. DR could be utilized in combinational therapy to minimise the damaging effect of ROP in infants. This is timely, given current clinical use of anti-VEGF therapy for ROP, where long-term systemic effects have not been fully investigated.

Keywords: 700 retinal neovascularization • 698 retinal development • 706 retinopathy of prematurity