The Effects of High Dose Vitamin a Supplementation on the Neurovascular Development of the Retina in an Oxygen Induced Retinopathy Rat Model of Retinopathy of Prematurity.



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PURPOSE: Retinopathy of prematurity (ROP) is a retinal disease in which prematurity and high oxygen therapy disrupt retinal vascular and neural development, leading to peripheral retinal avascularity, neovascularization and neural retinal injury. Current treatments for severe ROP include laser ablation of the peripheral avascular retina, or the off-label use of anti-VEGF therapies; however, these treatments come at the cost of far peripheral vision and adverse off-target effects on other organs, respectively (Heiduschka et al., 2007; Sato et al., 2012; Morin et al., 2016). High dose vitamin A (HDVA) supplementation may be a promising therapeutic for ROP, and the studies presented in this dissertation investigated the effects of HDVA on retinal development in novel rat oxygen-induced retinopathy (OIR) models of ROP.

METHODS: One study was performed entirely at the University of Houston, College of Optometry (UHCO), and the other in collaboration with Baylor College of Medicine (BCM). OIR was induced in albino Fischer 344 rats by exposure to hyperoxia (UHCO: 96±1% O2; BCM: 95% O2) from postnatal day (P) 0 to P7, followed by return to room air. Controls were raised in room air. Pups received daily intraperitoneal injections of vehicle (corn oil, 33ml/kg), or HDVA (UHCO: 2mg/kg or 10mg/kg; BCM: 2mg/kg) from P1 to P5. Retinal function was assessed with dark-adapted electroretinography (UHCO: P20-22; BCM: P20-22, P25-27, P34-37). Retinal vascular coverage and neovascularization was quantitatively assessed in isolectin labeled retinal flat-mounts (UHCO: P5, P7, P14, P20-22), or semi-quantitatively assessed in ADPase histochemically stained retinal flat-mounts (BCM: P20-22, P25-27, P34-37). Retinal thickness was measured in retinal cross-sections (JB-4 embedded, 5μm) stained with toluidine blue (UHCO: P7, P14, P20; BCM: P20 - P35). Retinal VEGF-A and PEDF protein levels were assessed using ELISA (UHCO: P5, P7, P14). Retinal expression of pro- and anti-angiogenic genes was assessed using qRT-PCR, and HIF-1α protein expression was assessed using Western blot (BCM: P7, P14, P20-22, P25-27, P34-37).

RESULTS: UHCO Study: In OIR rats, hyperoxia caused extensive vaso-obliteration (P5, P7), followed by vaso-proliferation and neovascularization after return to room air (P14, P20). OIR stunted inner retinal development, resulting in significantly thinner inner retinal layers, especially at the posterior pole and in avascular retinal regions. Vehicle-treated OIR rats exhibited persistent peripheral avascularity at P20, resulting in a widespread reduction of inner retinal thickness, and consequent severe post-receptoral and inner retinal dysfunction. In contrast, HDVA-treated OIR rats showed greater vascular coverage at P20, and reduced neovascularization at P14 and P20. The increased vascular coverage was associated with greater inner retinal thickness, and HDVA-treated OIR rats had improved post-receptoral and inner retinal function. Retinal VEGF-A expression showed a trend towards lower expression with HDVA-treatment in OIR at P14, coincident with reduced neovascularization. BCM Study: OIR retinas exhibited persistent peripheral retinal avascularity, inner retinal thinning, post-receptoral and inner retinal dysfunction, and dysregulation of pro-angiogenic hypoxia inducible factor 1α (HIF-1α) protein, and of Vegfa mRNA. In contrast, HDVA-treated OIR rats had more fully formed retinal vasculature, and normal retinal function that was no different from room air controls. Inner retinal thickness was reduced in HDVA-treated OIR animals, however, the presence of vasculature was not taken into account in the assessment of retinal thickness in this study. HDVA-treatment reduced anti-angiogenic Serpinf1 mRNA at P7, and reduced Vegfa mRNA at older ages.

CONCLUSIONS: HDVA supplementation during the course of hyperoxic exposure in two novel rat OIR models improved inner retinal thickness and function by promoting retinal revascularization following return to room air. Although the exact mechanism of action for HDVA-treatment in OIR remains unknown, our studies indicate that HDVA-treatment modulates retinal VEGF-A expression.



Retinopathy of prematurity, Oxygen induced retinopathy, Vitamin A, Retina, Vascular development, Neovascularization, Hyperoxia, Hypoxia