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Raghavi Sudharsan, Natalia Dolgova, Michael H Elliott, William A Beltran, ; Optimization of Rod Photoreceptor Culture and Rod Outer Segment Isolation from a Single Canine Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):352.
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© ARVO (1962-2015); The Authors (2016-present)
Rapid rod outer segment (ROS) disorganization followed by acute cell death of both photoreceptors and retinal pigment epithelium (RPE) occurs after brief exposure to light in the canine model of autosomal dominant retinitis pigmentosa (ADRP) caused by a naturally-occurring T4R mutation in the rhodopsin (RHO) gene. Cell death is seen as early as 6 hours after light exposure and peaks by 24-48 hours. The molecular pathways that are activated immediately following light exposure and culminate in death of rods and RPE are not well characterized. The purpose of this study is to optimize rod photoreceptor culture and rod outer segment isolation from single canine retinas that could eventually be used to elucidate the mechanisms of acute photoreceptor and RPE loss in the T4R RHO model of ADRP.
Rod photoreceptors were isolated under dim red illumination from a single wild type (WT) canine retina and cultured in dark. Various substrates (poly-d-lysine, wheat germ agglutinin, laminin and matrigel) were evaluated for optimum cell attachment and survival. Live cell imaging was used to study phenotypic and viability changes in the photoreceptors on exposure to light. Previously published sucrose step gradient protocol was scaled down to prepare ROS from a single WT canine retina in dark. Cultured ARPE19 cells were exposed to fluorescently-labeled or unlabeled ROS and efficiency of ROS phagocytosis by ARPE19 cells was evaluated by confocal microscopy.
Canine rod photoreceptors were successfully cultured for a period of 3 to 5 days when maintained in dark. Cell viability was not compromised when observed by confocal microscopy using a 633 nm far-red laser. Approximately 10,000 intact ROS could be obtained from a single canine retina. Phagocytosis of both fluorescently-labeled and unlabeled ROS by ARPE19 cells was observed.
Optimization of the isolation of rods and ROS from a single retina circumvents the limited access to abundant material when working with rare and valuable retinal tissues from animal models and/or humans. These techniques will be applied to investigate the mechanism of extreme light sensitivity in the T4R RHO canine retina and the pathways responsible for rod and RPE cell death.
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