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S.E. Pearce–Kelling, A. Nickle, J. Jordan, G. Antonini, G. Aguirre, G. Acland; Immunocytochemical and Tunel Staining Changes Following Light Exposure in the T4R Rho/RPE65–/– Double–Mutant Dog . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5791.
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© ARVO (1962-2015); The Authors (2016-present)
Exposure to bright light greatly influences the progression of degeneration in the T4R RHO dog and is associated with intense apoptotic activity in the retina. In contrast, light exposure produces no degenerative effect in the RPE65–/– mutant retina. To better understand the degenerative processes at work, we compared the two single mutants to double mutants (T4Rho/RPE65–/–) by examining immunocytochemical and apoptotic markers to characterize changes that occur at specific time points following light exposure.
Age matched, dark adapted T4R+/–, RPE65–/–, single and double mutants and wt dogs were exposed to full field illumination using light levels comparable to those used in clinical fundus photography, and enucleated at selected time points following exposure. Eyes were fixed in paraformaldehyde, embedded in OCT, and sectioned for immunocytochemistry and TUNEL staining. Morphological changes associated with the degenerations were examined in 1micron plastic sections following glutaraldehyde/formaldehyde and osmium fixation.
In contrast to the T4Rho and RPE65–/– single mutants, the double T4R+/–/RPE65–/– mutants showed an accelerated retinal degeneration that was independent of light exposure. The pattern of apoptotic markers, including TUNEL and activated Caspase–3 staining, differed between the three degeneration models. Unlike the T4R+/– single mutant, where TUNEL positivity following light exposure was limited to the light exposed region of the retina and was found primarily in the nuclei of the outer nuclear layer, only occasional TUNEL positive staining was observed in the T4R+/–/RPE65–/– double mutants. This was observed primarily in cells of the inner retina in both light exposed and non–exposed regions.
A different time course of degeneration is operative in the light–susceptible T4Rho mutant compared to the light independent T4R+/–/RPE65–/– double mutants.
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