Abstract
Purpose :
To assess the severity of retinal hypoxia using pimonidazole in a rabbit model of graded hypoxic-ischemic injury
Methods :
New Zealand pigmented rabbits (2.5-3.5kg) were divided into 2 groups of hypoxia severity (group 1: 50-60% hypoxia, group 2: 80-90% hypoxia) based on intraocular pO2 recordings. All procedures were in accordance with a protocol approved by the local IACUC and ARVO guidelines for animal care and use. Pimonidazole was given intravenously (150mg) or intravitreally in both eyes (1, 5 or 10mg) before the procedure. An intraocular oxygen probe connected with OxyLite pO2 monitor was placed adjacent to a retinal vessel within 2mm of the optic disk edge to measure real-time pO2 readings. Graded hypoxia was induced by infusing balanced salt solution into the anterior chamber to elevate intraocular pressure (IOP) using a Stellaris Vitrectomy System. IOP elevation was maintained to induce predetermined hypoxia severity for group 1 or 2 for 90 minutes. After euthanasia and enucleation, eyes were fixed, embedded in paraffin and immunohistochemistry for pimonidazole adducts was performed using anti-pimonidazole antibody.
Results :
Rabbits that received intravenous pimonidazole showed significant increase in immunoreactivity throughout all layers of the central retina following the 80-90% hypoxic-ischemic retinal injury compared with untreated eyes. Staining was most notable within the nerve fiber layer. Intravitreal injection of pimonidazole at all concentrations showed increased immunoreactivity at all layers of the central retina at 1 and 4mm away from the optic disk edge, in both 50-60% hypoxic group and 80-90% hypoxic group, comparing with untreated control group. Compared with 50-60% hypoxia group, 80-90% hypoxia group showed qualitatively increased immunoreactivity at nerve fiber layer at 1mm away from the optic disk edge (Figure). In controls, the peripheral retina (outside the vascular streak) demonstrated increased immunostaining compared to the area within the vascular streak.
Conclusions :
Intravitreal injection of 1-5mg pimonidazole with immunohistochemistry staining is a feasible and reliable method of assessing hypoxia in the rabbit retina.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.