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C. Chen, E. Tsina, C. Cornwall, R.K. Crouch, S. Vijayaraghavan, Y. Koutalos; Reduction of Retinal to Retinol in Living Mouse Rod Photoreceptor Outer Segments . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2859.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the reduction of all-trans retinal to retinol in living mouse rod photoreceptors following visual pigment bleaching. Methods: Experiments were carried out with dark-adapted retinas from wild type (strains c57bl/6, sv129, cd1, fvb) and rpe65-/- mice. Isolated retinas were embedded in 3% low-temperature-gelling agarose and the agarose blocks were sliced with a vibratome under dim red light. The slice thickness was typically 250 µm. Agarose slices containing the retinal slices were placed in a chamber on the stage of an upright microscope equipped with a high-sensitivity digital CCD camera. The slice was continuously perfused with mammalian Ringer’s. The reduction of retinal to retinol was monitored from the fluorescence of retinol (excitation: 360 nm; emission: 540 nm broadband). The spatial distribution of slice fluorescence was measured before and at various times after bleaching with intense white light. All experiments were carried out at room temperature. Results: In retinal slices from wild type mice, the fluorescence increases in the rod outer segment layer after bleaching and reaches a steady state in about 60 min. In retinal slices from rpe65-/- mice, there are no significant changes in the rod outer segment layer fluorescence after bleaching. Addition of 100 µM all-trans retinal to retinal slices of either wild type or rpe65-/- mice led to significant fluorescence increases in the rod outer segment layer, consistent with the reduction of the exogenously supplied all-trans retinal to retinol. Conclusions: The results demonstrate the reduction of all-trans retinal to retinol in living mammalian rod photoreceptors. The rpe65-/- rod photoreceptors lack 11-cis retinal and cannot produce retinol upon bleaching, but they readily reduce exogenously supplied all-trans retinal, suggesting that the dehydrogenase step is not compromised in these animals. Support: NIH grants EY11351, EY04939, EY01157 and Foundation Fighting Blindness.
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