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Stephanie G. Smith, Jindong Ding, Sina Farsiu, Melody Chan, Catherine Bowes Rickman; Morphometric Investigation of Human Retinal Pigment Epithelium (RPE) Flat Mounts: The Good, The Bad and The Ugly. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1761. doi: https://doi.org/.
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While the presence of a few small ‘hard’ drusen is a normal part of aging, the deposition of large, diffuse drusen in the macula is indicative of early age-related macular degeneration (AMD). Although the composition of drusen has been widely examined, flat mount analysis of their influence on the overlying RPE monolayer has been limited. In this study we analyzed morphometric and autofluorescence features of human RPE flat mounts on and around drusen by confocal microscopy.
Human eyes obtained from 46-77 year old donors, with and without a history of AMD, were supplied by the North Carolina Eye Bank and fixed in 4% paraformaldehyde. Six mm diameter punches were taken from the macula, and central and equatorial retina. The sclera and retina were removed, and the punch was placed RPE side down. The choroid and Bruch’s membrane were removed to expose the basal surface of the RPE. Some RPE flat mounts were stained with Hoeschst. RPE images were acquired using a Leica SP5 confocal microscope. Morphometric analysis and spectral measurements were performed with the Leica Application Suite, ImageJ and the Duke Ophthalmic Cell Analysis Program.
Typical morphology of the RPE included tightly joined hexagonal cells containing single nuclei and autofluorescent lipofuscin granules. These aggregates exhibited characteristic emission spectra with a peak around 530 nm under excitation by a 405 nm laser. In contrast, atypical changes found in association with drusen included cell enlargement, multi-nucleate cells, hyper- and hypo-pigmentation and distortions of the RPE sheet. Interestingly, the autofluorescent spectra of some abnormal cells were blue-shifted with a peak around 500 nm in comparison to healthy cells, and these resembled the measured spectra of sub-RPE deposits.
RPE flat mounts imaged from the basal side is an informative technique for assessing the negative impact of drusen on the RPE monolayer. This approach represents a novel perspective for imaging aging- and AMD-related changes to the RPE.
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