Autofluorescent Organelles Within the Retinal Pigment Epithelium in Human Donor Eyes With and Without Age-Related Macular Degeneration

Abstract

Purpose Human retinal pigment epithelium (RPE) cells contain lipofuscin, melanolipofuscin, and melanosome organelles that impact clinical autofluorescence (AF) imaging. Here, we quantified the effect of age-related macular degeneration (AMD) on granule count and histologic AF of RPE cell bodies. Methods Seven AMD-affected human RPE-Bruch's membrane flatmounts (early and intermediate = 3, late dry = 1, and neovascular = 3) were imaged at fovea, perifovea, and near periphery using structured illumination and confocal AF microscopy (excitation 488 nm) and compared to RPE-flatmounts with unremarkable macula ( n = 7, >80 years). Subsequently, granules were marked with computer assistance, and classified by their AF properties. The AF/cell was calculated from confocal images. The total number of granules and AF/cell was analyzed implementing a mixed effect analysis of covariance (ANCOVA). Results A total of 152 AMD-affected RPE cells were analyzed (fovea = 22, perifovea = 60, and near-periphery = 70). AMD-affected RPE cells showed increased variability in size and a significantly increased granule load independent of the retinal location (fovea: P = 0.02, perifovea: P = 0.04, and near periphery: P < 0.01). The lipofuscin fraction of total organelles decreased and the melanolipofuscin fraction increased in AMD, at all locations (especially the fovea). AF was significantly lower in AMD-affected cells (fovea: <0.01, perifovea: <0.01, and near periphery: 0.02). Conclusions In AMD RPE, lipofuscin was proportionately lowest in the fovea, a location also known to be affected by accumulation of soft drusen and preservation of cone-mediated visual acuity. Enlarged RPE cell bodies displayed increased net granule count but diminished total AF. Future studies should also assess the impact on AF imaging of RPE apical processes containing melanosomes.

Histologic Cell Shape Descriptors for the Retinal Pigment Epithelium in Age-Related Macular Degeneration: A Comparison to Unaffected Eyes

“…A recent histologic study showed that in AMD RPE cells, intracellular organelle distribution (apical: melanosomes/melanolipofuscin; basal/basolateral: lipofuscin dominated) was comparable to aged RPE cells. 42 Further, this study showed that lipofuscin and melanolipofuscin, the predominant source of short-wavelength AF of the human RPE, were reduced in AMD RPE cells. 42 Histologic studies have documented RPE degranulation, shedding, and anterior migration to be at least partly culpable for the reduced AF of the monolayer.…”

“… 42 Further, this study showed that lipofuscin and melanolipofuscin, the predominant source of short-wavelength AF of the human RPE, were reduced in AMD RPE cells. 42 Histologic studies have documented RPE degranulation, shedding, and anterior migration to be at least partly culpable for the reduced AF of the monolayer. 1 Further, there is an increasing amount of evidence that AMD is associated with decreased rather than increased autofluorescence in clinical imaging.…”

Impact of lens autofluorescence and opacification on retinal imaging

“…On a cellular and subcellular level, FAF mainly originates from fluorophores in the outer retina and the retinal pigment epithelium (RPE). In RPE cells, these fluorophores are linked to intracellular granules, which show different autofluorescence (AF) properties: melanolipofuscin and lipofuscin granules with peak excitation in the short wavelength and melanin in melanosomes and melanolipofuscin granules with peak excitation in the near-infrared (IR) spectrum 3–5…”

Quantitative autofluorescence in central serous chorioretinopathy

“…Lipofuscin and melanolipofuscin arise as a byproduct of this phagocytosis and are accumulated from a family of autofluorescent bisretinoid compounds at the level of the RPE that can be measured using AF imaging devices. [15][16][17] Autofluorescence in patients with CSC shows multiple patterns, including mottled, hypofluorescent, and hyperfluorescent patterns. 18 In the acute phase, CSC shows a decreased AF signal, presumably caused by the presence of subretinal fluid underneath the retinal detachment.…”

“…Lipofuscin and melanolipofuscin arise as a byproduct of this phagocytosis and are accumulated from a family of autofluorescent bisretinoid compounds at the level of the RPE that can be measured using AF imaging devices. 15–17…”