Lipofuscin Redistribution and Loss Accompanied by Cytoskeletal Stress in Retinal Pigment Epithelium of Eyes With Age-Related Macular Degeneration

Purpose: Lipofuscin (LF) and melanolipofuscin (MLF) of the retinal pigment epithelium (RPE) are the principal source of autofluorescence (AF) signals in clinical fundus-AF imaging. Few details about how its subcellular distribution changes in age-related macular degeneration (AMD) are available. We describe the impact of aging and AMD on RPE morphology revealed by the distribution of autofluorescent LF/MLF granules and the actin cytoskeleton in human tissues. Methods: Thirty-five RPE-Bruch´s membrane flatmounts from 35 donors were prepared (post-mortem: ≤ 4 hours). Ex vivo fundus examination at the time of accession revealed either absence of chorioretinal pathologies (10 tissues; mean age; 83.0 ± 2.6 years) or stages of AMD (25 tissues; 85.0±5.8 years): incipient AMD, geographic atrophy, and late exudative AMD. RPE cytoskeleton was labeled with AlexaFluor647-Phalloidin. Tissues were imaged on a spinning-disk fluorescence microscope and a high resolution structured illumination microscope. Results: AMD impacts individual RPE cells by: 1) lipofuscin re-distribution by a) degranulation (granule-by-granule loss) and/or b) aggregation and shedding into extracellular space; 2) enlarging RPE cell area and converting cell bodies from convex to irregular and sometimes concave polygons; 3) cytoskeleton derangement including separations and breaks around subretinal deposits, thickening, and stress fibers. Conclusions: We report the first extensive and systematic en face analysis of LF/MLF-AF in AMD eyes. Re-distribution and loss of AF granules are among the earliest AMD changes, suggesting that cells with numerous and orderly-arranged granules are healthy. Data can enhance the interpretation of clinical fundus-AF and provide a basis for future quantitative studies.