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Thomas Ach, Jeffrey D Messinger, Mark J Bentley, Francois C Delori, Theodore Smith, Kenneth R Sloan, Christine A Curcio; Impact of age-related macular degeneration (AMD) on human retinal pigment epithelium autofluorescence (AF), cell number, and packing geometry. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4532. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To describe the impact of different stages of AMD on cell number, AF, and packing geometry of human retinal pigment epithelium (RPE) flat-mounts, in comparison with age-matched normal eyes.
Internal fundus examination of human donor eyes revealed different AMD stages: early AMD, geographic atrophy, and late exudative AMD. Chorioretinal tissues were fixed in paraformaldehyde and cryo-preserved in glycerol/PBS, then RPE flat-mounts were prepared. Photo-documentation of RPE flatmount preparation steps (detaching retina; removing choroid) allowed preserving the foveal position throughout the preparation process. RPE cytoskeleton (labeled with AlexaFluor647-phalloidin) and AF (exc. 460-490nm, em. > 505 nm) were imaged on a spinning disk fluorescence microscope. AF was evaluated with the integrated software and normalized to a fluorescence reference (Delori et al., PMID 22016060). Custom Java software computed Voronoi diagrams and integrated AF intensity within each Voronoi region. RPE cell number, packing geometry, and normalized AF intensities were compared to corresponding points from age-matched normal RPE flatmounts (Ach et al., ARVO 2013, 54:5497).
Four eyes (three with early AMD, one with exudative AMD) have been analyzed to date. AMD impacts individual RPE cells by: 1) enlarging of RPE cell area (up to 10 times that of a normal cell), 2) frequent encapsulation and concentration of multiple lipofuscin granules within specific subcellular domains, 3) emptying and absence of lipofuscin granules from affected RPE cells, and 4) RPE cell displacement around drusen. Adjacent to areas of pathology are areas of normal RPE cells. Compared to normal eyes, AMD impacts mean AF intensity, RPE cell density and cell size, and packing geometry (for representative data for two retinal locations, see Table).
This study is the first analysis of RPE flat-mounts from AMD eyes to include histological autofluorescence descriptions and measurements in addition to packing geometry. While affected areas seem to be focal with surrounding normal RPE, AF and packing geometry data might indicate AMD affecting the entire RPE more diffusely. Further AMD tissues, including geographic atrophy, are currently under investigation.
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