# A direct method for imaging gradient levels of retinal hypoxia in a model of retinopathy of prematurity (ROP)

## TL;DR

This study introduces a direct method to image retinal hypoxia in a rat model of retinopathy of prematurity, revealing hypoxia gradients that may help predict disease severity.

## Contribution

The paper presents HYPOX-4 as a novel direct method for imaging retinal hypoxia gradients in a rat model of ROP.

## Key findings

- Retinal hypoxia was minimal near the ciliary bodies and maximal at avascular-vascular transition zones.
- HYPOX-4 and pimonidazole showed similar hypoxia patterns in the peripheral avascular retina.
- The vascularized central retina remained gradient hypoxic, detectable with HYPOX-4.

## Abstract

Retinal hypoxia may contribute to the development of preretinal neovascularization (NV) in patients with retinopathy of prematurity (ROP). We hypothesized that levels of NV may be associated with levels of retinal hypoxia. Imaging retinal hypoxia could be an important diagnostic tool to predict levels of disease severity in ROP patients. In this study, we have investigated a direct method for imaging gradient levels of retinal hypoxia using a model of ROP. We believe that this discovery will help understand the ROP pathogenesis in premature infants.

The rat 50/10 oxygen-induced retinopathy (OIR) model was generated by exposing the newly born Brown-Norway rat pups to a 24 hours alternate cycles of 50% and 10% oxygen for 14 days. HYPOX4 was used as a direct method for imaging gradient levels of retinal hypoxia at the peripheral avascular retina. A separate group of rat OIR pups were used to confirm gradient levels of retinal hypoxia using pimonidazole immunostaining. Gradient levels of retinal hypoxia was analyzed using ImageJ software from fluorescence intensities of HYPOX-4 and Pimonidazole immunostaining. We also confirmed the development of neovascularization in this model.

Retinal hypoxia was observed in the peripheral avascular retinas in rat OIR. Based on fluorescence intensity measurements, retinal hypoxia was at minimal levels near the ciliary bodies. Retinal hypoxia was at its maximum levels towards the avascular-vascular transition zones. Interestingly, we observed hemiretinal avascular retina temporal to the optic nerve in this OIR model, similar to human ROP retinas. In the retinal cross-section, hypoxia was not detectable near the ora serrata in rat OIR may be due to oxygen delivery by the ciliary bodies. Both pimonidazole and HYPOX-4 showed similar patterns of retinal hypoxia at the peripheral avascular retina in this model. As expected, preretinal neovascularization was observed at the avascular-vascular transition zones arising from the existing retinal vascular structures in this OIR model in Brown-Norway rats.

In this study, we have characterized gradient levels of retinal hypoxia in the rat model of 50/10 OIR using a direct method from HYPOX-4 fluorescence. We observed minimal levels of retinal hypoxia near the ciliary bodies in this model and increased towards the avascular-vascular transition zones. In addition, we observed that the central vascularized retina remains gradient hypoxic in this model which could be detected using HYPOX-4. This study may clarify our understanding of persistent mild hypoxia in the vascularized central, mid-peripheral, and increased gradient levels of hypoxia at the avascular retina in the ROP patients.

Imaging gradient levels of retinal hypoxia is an important indicator of severity of ROP pathogenesis including neovascularization. We used HYPOX-4 as a direct method for imaging gradient levels of retinal hypoxia in a model of ROP.We observed minimal levels of retinal hypoxia near the ciliary bodies in this rat 50/10 OIR model and increased levels of hypoxia towards the avascular-vascular transition zones.Even though the central area of the rat 50/10 OIR retina is vascularized, the vascularized retina remains gradient hypoxic which could be detected using HYPOX-4.This study may clarify our understanding of levels of retinal hypoxia in the ROP patient at the peripheral avascular retina; and also, at the vascularized areas of the retina which is significant and needs future clinical investigations.

Imaging gradient levels of retinal hypoxia is an important indicator of severity of ROP pathogenesis including neovascularization. We used HYPOX-4 as a direct method for imaging gradient levels of retinal hypoxia in a model of ROP.

We observed minimal levels of retinal hypoxia near the ciliary bodies in this rat 50/10 OIR model and increased levels of hypoxia towards the avascular-vascular transition zones.

Even though the central area of the rat 50/10 OIR retina is vascularized, the vascularized retina remains gradient hypoxic which could be detected using HYPOX-4.

This study may clarify our understanding of levels of retinal hypoxia in the ROP patient at the peripheral avascular retina; and also, at the vascularized areas of the retina which is significant and needs future clinical investigations.