Assessment of visual function and retinal structure following acute light exposure in the light sensitive T4R rhodopsin mutant dog

Abstract

The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4–5.5 months) were used in this study: 3 homozygous mutant RHOT4R/T4R, 8 heterozygous mutant RHOT4R/+, and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHOT4R/T4R) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm2, 170 lux; 0.5 mW/cm2, 820 lux; or 1 mW/cm2, 1590 lux) for 1min. All 8 heterozygous (RHOT4R/+) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHOT4R/T4R and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2–36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHOT4R/T4R retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHOT4R/T4R retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHOT4R/T4R retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.

Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector

“…Suppression of Mutant RHO with shRNA 820 . To verify the efficiency of shRNA 820 in heterozygous mutant retinas that express both WT and mutant RHO alleles, subretinal injections of AAV-shRNA 820 were performed over a range of titers (1 × 10 11 to 10 × 10 11 vg/mL) in 10 RHO-mutant eyes that were followed for 8-10 wk postinjection (SI Appendix, Table S1 groups D and E) Since the RHO-mutant dog retinas are highly sensitive to light (45)(46)(47)(48), the animals were housed under dim red light from birth until the end of the study, and the surgical intervention was performed under infrared illumination (49). Four eyes were used for quantification of RHO knockdown efficiency at the RNA and protein levels ( Fig.…”

“…Next, we evaluated whether knockdown alone could arrest photoreceptor degeneration. Another set of four RHO-mutant eyes (SI Appendix, Table S1 group E) were also injected with the same range of titers of AAV-shRNA 820 , but at 6-8 wk postinjection they were exposed for 1 min to moderate-intensity white light known to cause acute retinal degeneration in this canine model (46)(47)(48). Two weeks after light exposure, the eye injected with the titers of 10 × 10 11 and 5 × 10 11 vg/mL showed a distinct region of ONL retention corresponding to the treatment area ( Fig.…”

Response to Comment on: Sector retinitis pigmentosa caused by mutations of the RHO gene

“…In their recent study, Orlans et al found that retinal degeneration in RhoP23H/+ mice was significantly slower when the mice were housed in red-tinted cages than in untinted cages [2]. Some studies have also shown that retinal damage in rhodopsin-related RP animal models was more sensitive to light and that the progression of retinal degeneration was slower in dark environments [3,4].…”

Large Animal Models of Inherited Retinal Degenerations: A Review

“…A spontaneous dog model of RHO adRP has been identified [18]. The c.11C>G, p.Thr4Arg mutant dog (Rho T4R ) develops a retinal degeneration that is greatly exacerbated by light exposure [19]. The phenotype of this dog model closely resembles that of the human p.Thr4Lys RHO mutation which results in dominant RP [20].…”