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Raghavi Sudharsan, Kristina M Simone, Nathan P Anderson, Gustavo D Aguirre, William A Beltran; Development of an optimized light damage paradigm to assess efficacy of gene therapy in a canine model of RHO-ADRP. Invest. Ophthalmol. Vis. Sci. 2017;58(8):282.
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© ARVO (1962-2015); The Authors (2016-present)
To develop an optimized light damage protocol to rapidly assess the efficacy of gene therapy for Rhodopsin autosomal dominant retinitis pigmentosa (RHO-ADRP) using the light sensitive canine Rhodopsin T4R (RHO-T4R) model of ADRP.
The retinas of 11 RHO-T4R mutant dogs were exposed for 1 minute with a monocular Ganzfeld dome to one of five intensities of white light ranging from 0.1 to 1.0 mW/cm2 (170 to 1590 lux) at the level of the cornea. The effect was assessed at 24 hours and 2 weeks post light exposure (LE) using TUNEL assay, histology and immunohistochemistry on retinal cryosections. A semi-quantitative grading system was used to report the incidence of photoreceptor cell death at 24 hours on biaxial topographical maps. Spidergraphs of outer nuclear layer (ONL) thickness and detailed biaxial maps of inner segments (IS), outer segments (OS) and retinal pigment epithelium (RPE) alterations at 2 weeks post LE were made to document structural changes in the outer retina.
At 24 hours, a dose-dependent effect of light on outer retina integrity was observed with abundant photoreceptor cell death, increased internuclear spacing in the ONL, rod IS/OS disruption, and damage to the RPE cells after exposure to the highest doses (0.5 and 1 mW/cm2). Two weeks post LE, severe retinal damage was seen centrally, particularly in the tapetal region where the ONL was reduced to a single row of cone nuclei after exposure to the highest (1mW/cm2) dose. A 0.3 mW/cm2 exposure still caused some cell death and ONL thinning in the central tapetal retina, but no photoreceptor loss was seen with a 0.1mW/cm2 dose. This sublethal dose did, however, cause disruption of OS and Rho mislocalization, thus highlighting the extreme sensitivity of the RHO-T4R retina to levels of light commonly used in animal housing and clinical settings.
We have established a light damage paradigm, identified light dosage thresholds for retinal damage and photoreceptor cell death, and characterized histological outcome measures of retinal degeneration that will now be used to rapidly assess in a large animal model the efficacy of gene therapy for RHO-ADRP.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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