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Nicholas Boyer, Daniel Higbee, Lorie R. Blakeley, Zsolt Ablonczy, Rosalie K. Crouch, Yiannis Koutalos; A2E And Lipofuscin Are Present In The Retinal Pigment Epithelium Of Nrl-/- Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3645. doi: https://doi.org/.
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To examine the A2E and lipofuscin content of the retinal pigment epithelium (RPE) of Nrl-/- mice, the retinas of which contain only cone-like photoreceptors. A2E, a major component of lipofuscin, has been suggested to be formed as a byproduct of reactions involving the visual pigment chromophore. Nrl-/- photoreceptor cells have outer segments which are small, contain only cone pigments, and have cone-like responses when stimulated by light.
Nrl-/- mice of various ages were kept in cyclic light. Organic solvent extracts from Nrl-/- RPE-choroid samples were analyzed by HPLC and mass spectrometry (nano-LC-MS/MS). The results were compared against a synthetic A2E standard for confirmation of identity. Whole eyecups (with retina removed) were flattened and lipofuscin levels were measured at 10x magnification on a Leica Laser Scanning Confocal fluorescence microscope (ex= 488 nm, emission collected from 565-725 nm). Sample preparation was carried out under dim red light.
Comparison of Nrl-/- RPE-choroid extracts with a synthetic A2E standard by HPLC showed that the extracts contain a peak with the same retention time and absorbance spectra as A2E (dual absorbance max at ~335 and 440 nm). Mass spectrometry data showed a peak at 592 m/z, corresponding to A2E, with a matching MS/MS fragmentation pattern, confirming the presence of A2E in Nrl-/- RPE. The amount of A2E measured by HPLC was lower than that measured from wild type RPE. Data from fluorescence microscopy show total Nrl-/- RPE fluorescence to be less than that of wild type mice. The fluorescence emission spectrum of lipofuscin granules in Nrl-/- RPE has a broad peak with max~620 similar to that of granules in wild type.
Lipofuscin and A2E accumulate in the RPE of Nrl-/- mice as they do in mice with rod photoreceptors, but in lower total amounts. This is consistent with the lower amount of chromophore-containing pigment in Nrl-/- retinas. Our data demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E in the RPE. Support: NIH grants R01 EY014850 (YK), R01 EY004939 (RKC), R21 EY020661 (ZA/RKC), EY019065 (ZA), R24 EY14793 (MUSC vision core), Foundation Fighting Blindness, Inc. (Owings Mills, MD) (RKC); and unrestricted awards to the Departments of Ophthalmology at MUSC from Research to Prevent Blindness (RPB; New York); RKC is an RPB Senior Scientific Investigator.
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