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R.K. Crouch, S. Znoiko, M. Kono, B. Rohrer, P. Goletz, J. Gresh, T.M. Redmond, J. Ma; Can a Delivery of 11-cis Retinal to the RPE65 KO Mouse Restore Normal Rod and Cone Function? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3506.
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Purpose: The RPE65 knockout (Rpe65-/-) mouse is an excellent model for studying the vitamin A deprived retina. These studies were designed to assess the alterations to rod opsin resulting from 11-cis retinal deprivation in Rpe65-/- mice and to determine if cone function can be restored on the administration of 11-cis retinal. Methods: Rod outer segment preparations of wild type (wt) and Rpe65-/- mice were analyzed for bound arrestin by Western blots and for transducin activation by a filter binding assay. Rpe65-/- mice were injected intraperitoneally with 11-cis retinal. Cone function was analyzed using light-adapted single-flash ERGs in response to blue (430 nm) light. The light sensitivity of b-waves was compared to uninjected littermates. Immunohistochemistry of flat-mounted retina with antibodies to S-opsin and PNA-lectin was used to examine the M and S-cone populations. Results: Opsin from either light- or dark-adapted Rpe65-/- mice, known to be phosphorylated (Ablonczy et al., 2002), was found to have arrestin bound at about the same level as light-adapted wt mice, despite the lack of photoactivatible pigment. The Rpe65-/- opsin did not activate transducin in the dark, unlike opsin from light adapted wt animals. These results indicate that the opsin which has not been exposed to 11-cis retinal may have different properties than opsin that has undergone several regeneration and bleaching cycles. Previous ERG measurements at =P 30 have shown only minimal rod function forRrpe65-/- mice. Administration of 11-cis retinal at P30 to 18 months restores significant rod function but no cone function (Rohrer et al., 2003). Immunohistochemistry showed that cone outer segments in Rpe65-/- mice degenerate after P14. This cone degeneration was partially prevented by early administration starting at P14 of 11-cis retinal as shown by an increased responsiveness to blue light stimuli in the ERG (p=0.001), and a significant increase in PNA-lectin (p=0.001) or S-cone opsin positive (p=0.02) cells in the central retina. Conclusions: The opsin in rod photoreceptors of Rpe65-/- mice does not have the same properties of opsin from light exposed wt animals. Rod function can be restored in these animals, at least partially, by administration of 11-cis retinal regardless of age. Partial cone degeneration can be prevented and function restored if 11- cis retinal is supplied within the first two weeks. These experiments have great importance in the consideration of gene therapy approaches for cases where RPE65 is dysfunctional.
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