Immunocytochemical and TUNEL Staining Changes Following Light Exposure in the T4R RHO Mutant Dog

Abstract

Abstract: : Purpose: Exposure to light greatly influences the progression of degeneration in the T4R RHO dog. To better understand the role of light in the degenerative process, we used immunocytochemical and apoptotic markers to characterize changes that occur at specific time points following focal or full field light exposure. Methods: Age matched, dark adapted T4R+/– and wt dogs were exposed to either focal (3mm diameter) or 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. Results:Apoptotic changes, including positive TUNEL staining, were noted within 2 hours of light exposure. Immunocytochemical changes of constituent retinal and RPE proteins, including rhodopsin, cone opsins, arrestin and RPE65, were also found. At 2 and 24 hrs following exposure, there was shortening, fragmentation and disorganization of outer segments; RPE cells became reactive, proliferated and migrated into the subretinal space. These changes were found exclusively in light exposed areas of the retina. Conclusions: Exposure to light levels like those used in clinical retinal examinations modifies the progression of retinal degeneration in the RHO T4R mutant dog. Dogs with this mutation provide an important large animal model to examine the interactions of mutation and environment in determining the severity of the retinal disease phenotype.

Keywords: retinal degenerations: hereditary • immunohistochemistry • retinal degenerations: cell biology