January 2022
Volume 63, Issue 1
Open Access
Retinal Cell Biology  |   January 2022
Autofluorescent Organelles Within the Retinal Pigment Epithelium in Human Donor Eyes With and Without Age-Related Macular Degeneration
Author Affiliations & Notes
  • Katharina Bermond
    Department of Ophthalmology, Ludwigshafen Hospital, Ludwigshafen, Germany
  • Leon von der Emde
    Department of Ophthalmology, University Hospital Bonn, Bonn, Germany
  • Ioana-Sandra Tarau
    Department of Ophthalmology, University Hospital Würzburg, Würzburg, Germany
  • Leonie Bourauel
    Department of Ophthalmology, University Hospital Bonn, Bonn, Germany
  • Rainer Heintzmann
    Leibniz Institute of Photonic Technology, Jena, Germany
    Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Jena, Germany
  • Frank G. Holz
    Department of Ophthalmology, University Hospital Bonn, Bonn, Germany
  • Christine A. Curcio
    Department of Ophthalmology, University of Alabama at Birmingham, Alabama, AL, United States
  • Kenneth R. Sloan
    Department of Ophthalmology, University of Alabama at Birmingham, Alabama, AL, United States
  • Thomas Ach
    Department of Ophthalmology, University Hospital Bonn, Bonn, Germany
  • Correspondence: Thomas Ach, FEBO, University Hospital Bonn, Ernst Abbe Straße 2, Bonn 53127, Germany; thomas.ach@ukbonn.de
  • Footnotes
    *  KB and LE contributed equally and should be considered co-first authors.
Investigative Ophthalmology & Visual Science January 2022, Vol.63, 23. doi:https://doi.org/10.1167/iovs.63.1.23
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      Katharina Bermond, Leon von der Emde, Ioana-Sandra Tarau, Leonie Bourauel, Rainer Heintzmann, Frank G. Holz, Christine A. Curcio, Kenneth R. Sloan, Thomas Ach; Autofluorescent Organelles Within the Retinal Pigment Epithelium in Human Donor Eyes With and Without Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2022;63(1):23. doi: https://doi.org/10.1167/iovs.63.1.23.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

Retinal pigment epithelium (RPE) cells are essential for outer retinal health and play a key role in the pathogenesis of age-related macular degeneration (AMD). RPE cells absorb light, phagocytize photoreceptor outer segments, exchange metabolites with photoreceptors, and recycle retinoids of the visual cycle.1 A hallmark of RPE cells is the presence of hundreds of granules relevant to clinical imaging via autofluorescence (AF; lipofuscin and melanolipofuscin) and optical coherence tomography (OCT; lipofuscin and melanolipofuscin, plus mitochondria); the vast majority of these organelles in any one cell are lipofuscin and melanolipofuscin.24 
Lipofuscin and melanolipofuscin within the RPE are histologically detectable early in life.5,6 The accumulation continues for decades resulting in increased RPE AF, both in histology and clinical imaging.7,8 At advanced age and in AMD, AF granules demonstrate a characteristic histologic redistribution911 that could also impact clinical AF imaging. Recently, alteration and loss of clinical AF signal have been identified as precursors of geographic atrophy,12 consistent with this expectation. Quantitative autofluorescence (QAF), an in vivo imaging technique using a camera with an internal standard, revealed that AF levels in clinical fundus autofluorescence (FAF) of AMD eyes is reduced. In a cohort of intermediate AMD eyes with large drusen,13 QAF levels were reduced compared to healthy eyes and further decreased over time, confirming findings from other groups that AF intensity in AMD is generally decreased.14,15 
Using high-resolution structured illumination microscopy (SIM), we previously imaged individual RPE granules exhibiting AF and further subdivided the major granule classes (lipofuscin, melanolipofuscin, and melanosomes) into nine different phenotypes.4 In a recent survey of more than 195,000 RPE granules in young and aged human donor eyes with unremarkable maculae, lipofuscin was low at the fovea and increased with age and with eccentricity (highest at the perifovea).4 Melanolipofuscin on the other hand was the major granule type in foveal RPE cell bodies and remained stable across the above-mentioned locations and over time.4 However, it is valuable to the interpretation of clinical imaging to determine how the content of RPE cell granules and their intracellular distribution is impacted by AMD, as determined with the nine-phenotype classification system. 
The purpose of this study was to three-dimensionally image human RPE cell bodies of AMD-affected donor eyes using high resolution SIM, characterize granule content, distribution, and correlate granules with AF. These histologic results will help to further clarify whether RPE cells and its granules are altered in the presence of AMD. In addition, our data will benefit the interpretation of clinical AF imaging in patients with AMD. 
Methods
The protocols adhered to the Tenets of the Declaration of Helsinki and the study was approved by the Institutional Review Boards of the University of Alabama at Birmingham and the Ethics Committees of the University of Würzburg and the University of Bonn, Germany. 
Tissue
For this study, seven previously documented AMD-affected human donor eyes were utilized. 
Tissue preparation of human donor eyes has previously been described in detail.7 Briefly, the globes were collected from the Advancing Sight Network (Birmingham, AL, USA) within 4.2 hours of death, prepared and preserved by immersion in 4% paraformaldehyde/0.1-M phosphate buffered saline, and inspected under a dissection microscope equipped with trans- and epi-illumination. The neuroretina and choriocapillaris were removed in a multistep preparation and imaging process to ensure preservation of the exact foveal position (for details, see Supplementary Fig. S1 in Ach et al.)7 RPE-Bruch's membrane flat mounts were then imaged at three predefined locations using the regional definitions by Polyak16: fovea (0.4 ± 0.5 mm superior to the foveal center), perifovea (4.1 ± 0.2 mm superior to the foveal center), and near-periphery (8.6 ± 1.3 mm superior to the foveal center). These regions were chosen for the distinctive content of overlying photoreceptors (local rod:cone ratio 0, 17.5, approximately 25, respectively),17,18 because RPE lipofuscin originates as ingested outer segments. 
Imaging Protocol
For AMD RPE cell imaging, a commercially available SIM device (ELYRA-S.1; Carl Zeiss Microscopy, Jena, Germany) was used (excitation wavelength = 488 nm and emission = 510–750 nm). Images were captured using a 63X, numerical aperture 1.40, plan apochromat oil immersion objective and an iXon 885 EMCCD camera (Andor Technology Ltd., Belfast, Northern Ireland, UK), cooled to –63°C. Further SIM specifications (grid pattern and exposure times) have been published.9 The scanning area of 75.56 × 75.40 µm2 captured 20 to 25 RPE cells per location (for exceptions, see below). Z-stacks of SIM images were acquired from apical RPE (first granules in focus) to basal RPE (last granules out of focus) in 100-nm steps. SIM superimposes illumination grids on the tissues, and during imaging the grids are differently positioned and oriented. Each z-stack slab consisted of 15 raw images (5 grating positions × 3 rotations of the illumination grid). All slabs were postprocessed to extract and reconstruct the high-resolution images (device's internal software, ZEN 2010). 
At each location (fovea, perifovea, and near-periphery), adjacent RPE cells were selected from reconstructed SIM images based on visibility of cell boundaries (tiny gaps between two adjacent cells at the basolateral side, visible while scrolling through the z-stack). RPE cell borders were identified by a granule free void between RPE cells, as the RPE cell border and tight junctions do not display AF properties (see Supplementary Fig. S1). At locations severely affected by AMD, adjacent cells were not detectable in all cases due to loss of cell boundaries and the presence of giant and, in some cases, multinucleate RPE cells. Therefore, for the fovea, instead of using individual RPE cells, areas equal to the size of 10 foveal RPE cells were analyzed (169 µm2, equivalent to the area of an average foveal healthy cell) in some cases. Due to loss of cell boundaries at the perifovea in one tissue (late exudative AMD), we also analyzed a predefined area instead of individual cells (217 µm2, equivalent to the area of an average perifoveal healthy cell; 14% of perifoveal AMD cells were analyzed in this manner). 
Image Analysis and Granule Classification
For image analysis and granule subclassification, we used custom written FIJI plugins.19 A demarcation line was manually drawn with computer assistance around each cell within the reconstructed SIM images, and cell area and height (number of z-stack slabs × 100-nm step size) were reported. RPE cell borders were identified by a granule free void between RPE cells, as the RPE cell border and tight junctions do not display AF properties. A custom FIJI plugin was used for recording granule counts and classifications made by human observers (code for the plugin, TA_Pick_Particles, is available online, http://sites.imagej.net/CreativeComputation). To further highlight possible differences in apical and basal granule distribution, RPE cells were partitioned into four equally sized apical-to-basal quartiles (Q1–Q4), where Q1 to Q3 covered the cushion of granules visible on SIM images and Q4 was known to contain abundant non-AF mitochondria.3,4 
All identifiable granules within the selected RPE cell bodies were manually tagged with computer assistance and independently classified by one of two trained graders on the basis of granule morphology (see below). While the observer scrolled through the z-stacks, the center of each granule was marked with a dot in the slab that revealed the maximum granule dimension. Typically, granules expanded over 6 to 8 slabs corresponding to an average granule size of about 1 µm. Previously marked granules were automatically displayed in preceding and subsequent slabs to avoid duplicate assignments. For each tagged granule, the classification code (see below) and its x, y, z coordinates within the stack were registered and written to a text file for further statistical analysis. However, granule marking was done manually; the plugin served only to record the coordinates. 
Granule Types
Granule type characterization was carried out as previously described in healthy RPE cells.4 There, we defined a total of 9 different granule phenotypes: lipofuscin 1, uniform lipofuscin granules and lipofuscin 2, conglomerates (variable-shaped accumulation of hyper-AF material with irregular intragranular AF patterns, larger than lipofuscin 1); melanolipofuscin 1, melanolipofuscin with complete AF coating and minimal AF in the center; melanolipofuscin 2, melanolipofuscin with complete AF coating and hypo- to iso-AF in the center; melanolipofuscin 3, melanolipofuscin with incomplete AF coating; melanolipofuscin 4, bull's-eye-shaped melanolipofuscin with complete AF coating and AF core; and melanolipofuscin 5, large round melanolipofuscin granules with AF coating (greater than twice the volume of a typical lipofuscin granule). Due to a limited inter-reader agreement in the distinction among melanolipofuscins 1 to 3 in our previous work, results for these three subtypes were pooled (see below). Finally, in line with recent distinction of melanosomes from organelle mapping studies, round (melanosome 1) and spindle-shaped (melanosome 2) melanosomes could be detected, neither of which showed notable AF components on the surface nor the granule body.24 For analysis, results for melanolipofuscins 1 to 3, melanosomes 1 to 2, and lipofuscins 1 and 2 were each pooled. 
Determination of Total Autofluorescence Per Cell
In addition to SIM imaging, using a Zeiss LSM 780 laser scanning confocal fluorescence microscope (excitation = 488 nm, emission = 490–695 nm, and 24 channels, 8.9-nm spectral channel width), flat mounts were imaged at the identical locations documented in SIM imaging. Cells were imaged from the apical aspect (first granules in focus) to the basal aspect (last granules out of focus) in 390-nm steps and total AF/cell was determined as previously described.4 The scanning area covered 224.92 × 224.92 µm2. Laser scanning microscopy (LSM) was performed before SIM imaging, because of the possible bleaching effects due to longer and more intense laser power in SIM.4 The AF settings were identical between donors and retinal location, however, analyses of total AF have to be interpreted cautiously as no reference AF bar has been used. 
Statistical Analysis
Statistical analyses were achieved in the software environments R (R Foundation for Statistical Computing, IBM) and SPSS. A mixed effect analysis of covariance (ANCOVA; between effect: presence of AMD; within effect: cells nested in participant; and covariate: age) was implemented to analyze the effect of AMD on the total number of granules (log transformation to reach log-normal distribution), granule density and AF in the fovea, perifovea, and near periphery respectively. ANCOVA was followed up by Mauchly's sphericity test and, if necessary, a Greenhouse-Geisser test for validation. We correlated granule count with cell area, and AF with granule count and cell area using the Spearman correlation coefficient. 
Results
Seven RPE flat mounts from seven donor eyes (4 women and 3 men, mean age ± SD = 85 ± 3 years, and range = 81–90 years) with three early/intermediate, one late dry, and three neovascular AMD manifestation were included. A total of 152 RPE cells (or equivalent squares) at the 3 canonical locations were analyzed (fovea = 22 cells, perifovea = 60 cells, and near-periphery = 70 cells; Fig. 1). 
Figure 1.
 
SIM-images of AMD-affected and unremarkable tissue. Each image shows a representative slide at the apical surface of a SIM z-stack of the fovea (A–D) or perifovea (E–H) of AMD-affected (A–C, E–G) or unremarkable (D, H) tissues. The AMD-affected tissues show deterioration of cell structures like dissolving cell borders, partial loss of intracellular granule organization and cell loss. Areas of RPE cell loss probably associated with deposits (basal laminar deposits/basal linear deposits) are marked with a blue asterisk. These deposits affected the fovea and, in some tissues, extended to the perifovea (E, F). Where the perifovea was not (yet) affected by AMD (G), the tissue appeared comparable to healthy tissue (D, H) which shows uniform cell shapes and orderly intracellular granule distribution. Note: the granule free areas within the cell bodies in D and H represent the non-autofluorescent cell nucleus. In panels A, B, and E, we were unable to distinguish cell boundaries and used cell-size equivalent squares instead. A 3D-version of panel C is further depicted in the supplemental video. Early or intermediate AMD: 83-year-old woman (F), 84-year-old man B, and 86-year-old woman A. Late non-exudative AMD: 81-year-old man C, G. Late exudative AMD: 90-year-old woman E. Unremarkable tissue: 83-year-old woman D and 90-year-old woman H. Scale bar = 10 µm.
Figure 1.
 
SIM-images of AMD-affected and unremarkable tissue. Each image shows a representative slide at the apical surface of a SIM z-stack of the fovea (A–D) or perifovea (E–H) of AMD-affected (A–C, E–G) or unremarkable (D, H) tissues. The AMD-affected tissues show deterioration of cell structures like dissolving cell borders, partial loss of intracellular granule organization and cell loss. Areas of RPE cell loss probably associated with deposits (basal laminar deposits/basal linear deposits) are marked with a blue asterisk. These deposits affected the fovea and, in some tissues, extended to the perifovea (E, F). Where the perifovea was not (yet) affected by AMD (G), the tissue appeared comparable to healthy tissue (D, H) which shows uniform cell shapes and orderly intracellular granule distribution. Note: the granule free areas within the cell bodies in D and H represent the non-autofluorescent cell nucleus. In panels A, B, and E, we were unable to distinguish cell boundaries and used cell-size equivalent squares instead. A 3D-version of panel C is further depicted in the supplemental video. Early or intermediate AMD: 83-year-old woman (F), 84-year-old man B, and 86-year-old woman A. Late non-exudative AMD: 81-year-old man C, G. Late exudative AMD: 90-year-old woman E. Unremarkable tissue: 83-year-old woman D and 90-year-old woman H. Scale bar = 10 µm.
Fovea
Data from foveal RPE are available from four donors. One eye with early/intermediate AMD showed a giant RPE cell with densely packed granules and no apparent nuclei; one eye with late non-exudative AMD also showed a giant RPE cell at the fovea with 12 nuclei and/or vacuoles. In 2 donor eyes, no RPE cell boundaries could be distinguished, and analysis was based on 10 cell size equivalent squares each (see below, 90% of foveal AMD cells were analyzed in this manner). The three donor eyes with late exudative AMD with macular atrophy in addition to neovascularization showed no remaining RPE tissue at the fovea and, therefore, were excluded from analysis. 
The SIM scanned foveal areas (Fig. 2) from the 2 donors were analyzed within 20 squares, each equivalent to the area of a typical RPE cell in a healthy fovea. These squares displayed a total granule load per square of 613 ± 267, which was significantly increased compared to healthy aged foveal RPE cell of the same size (339 ± 137; P value/η2 value: 0.02/0.31; Tables 1, 2). Melanolipofuscin granules were the dominant population (lipofuscin = 111 ± 58 and melanolipofuscin = 497 ± 278 (no melanolipofuscin 4 and no melanolipofuscin 5; and melanosome, 5 ± 6 (see Table 1). Percentage-wise fractional distribution showed a reduced lipofuscin fraction compared to healthy aged cells (Table 3). Intracellular spatial granule distribution showed a slightly increased granule load in the central quartile (Q2) compared to healthy cells (AMD/healthy Q2: 49%/44% fractional granule load). 
Figure 2.
 
En face (A) and cross section (B) view of intracellular granule distribution. Each analyzed granule was color-coded and plotted (lipofuscin: yellow, melanolipofuscin 1–3: blue, melanolipofuscin 4: pink, melanolipofuscin 5: magenta, and M: orange). Only few melanosomes, melanolipofuscin 4, and melanolipofuscin 5 were detectable. Red lines represent cell borders or artificial squares, respectively. At four locations (three foveae and one perifovea), no granule analysis was possible due to large RPE-atrophy. Foveal tissue showed distinctive alterations in cell structure with either formation of enlarged cells (83-year-old woman and 81-year-old man) or primarily faded cell borders and a moth-eaten appearance (84-year-old man and 86-year-old woman [early/intermediate AMD]). The predominant granule type at the fovea remained melanolipofuscin, like in unremarkable tissues.4 At the perifovea and near periphery, all tissues that showed a regular cell structure in the en face view also displayed two bands in the cross-section view: an apical band with predominantly melanolipofuscin and lipofuscin and a basal band with predominantly lipofuscin. At locations where the cell structures were deteriorated (83-year-old perifovea and near periphery, 90-year-old perifovea) these two bands could not be distinguished. Scale bar = 10 µm.
Figure 2.
 
En face (A) and cross section (B) view of intracellular granule distribution. Each analyzed granule was color-coded and plotted (lipofuscin: yellow, melanolipofuscin 1–3: blue, melanolipofuscin 4: pink, melanolipofuscin 5: magenta, and M: orange). Only few melanosomes, melanolipofuscin 4, and melanolipofuscin 5 were detectable. Red lines represent cell borders or artificial squares, respectively. At four locations (three foveae and one perifovea), no granule analysis was possible due to large RPE-atrophy. Foveal tissue showed distinctive alterations in cell structure with either formation of enlarged cells (83-year-old woman and 81-year-old man) or primarily faded cell borders and a moth-eaten appearance (84-year-old man and 86-year-old woman [early/intermediate AMD]). The predominant granule type at the fovea remained melanolipofuscin, like in unremarkable tissues.4 At the perifovea and near periphery, all tissues that showed a regular cell structure in the en face view also displayed two bands in the cross-section view: an apical band with predominantly melanolipofuscin and lipofuscin and a basal band with predominantly lipofuscin. At locations where the cell structures were deteriorated (83-year-old perifovea and near periphery, 90-year-old perifovea) these two bands could not be distinguished. Scale bar = 10 µm.
Table 1.
 
Number of Granules and Mean Cell Area in AMD and Normal Aged RPE Cells4
Table 1.
 
Number of Granules and Mean Cell Area in AMD and Normal Aged RPE Cells4
Table 2.
 
Granule Composition in Different AMD Disease Manifestations. Reported are Number of Granules Per Cell ± Standard Deviation
Table 2.
 
Granule Composition in Different AMD Disease Manifestations. Reported are Number of Granules Per Cell ± Standard Deviation
Table 3.
 
Granule Distribution at the Fovea, Perifovea, and Near Periphery
Table 3.
 
Granule Distribution at the Fovea, Perifovea, and Near Periphery
Of note, two foveae of two donors revealed significantly enlarged cells (see one in the Supplementary Video). In a donor with early/intermediate AMD, one cell demonstrated a total granule load of 4624 granules (751 lipofuscin, 3820 melanolipofuscin, and 53 melanosomes), whereas no nucleus could be identified. In a donor with late non-exudative AMD, one cell displayed a total granule load of 3785 granules (1384 lipofuscin, 2386 melanolipofuscin, and 15 melanosomes). Twelve spherical non-autofluorescent areas were identified in this cell. These could be nuclei, vacuoles, or both. 
Due to the limitations of using cell-size equivalent squares at the fovea we further reported 2D granule density (granule/area = 100 µm2Table 4). Granule density was not significantly increased compared to healthy aged cells (P value/η2 value: 0.07/0.8). 
Table 4.
 
Granule Density at the Fovea and Perifovea
Table 4.
 
Granule Density at the Fovea and Perifovea
Perifovea
The perifovea revealed the highest total granule load per cell among all three locations. Cells had 1069 ± 507 granules within their cell body, significantly more than in healthy aged perifoveal cells (508 ± 194, P value/η2 value of 0.04/0.37). Granule density (see Table 4) was not significantly increased compared to healthy aged cells (P value/η2 value: 0.6/0.16). Cells contained 580 ± 270 lipofuscin, 486 ± 302 melanolipofuscin (1 ± 2 melanosomes 4 and 3 ± 3 melanolipofuscin 5), and 3 ± 3 melanosomes on average (see Table 1). 
Granule load varied among AMD manifestations with 1136 ± 513 (early/intermediate), 1017 ± 598 (late exudative), and 973 ± 225 (late non-exudative AMD; see Table 2). Percentage-wise granule distribution showed an increase in the melanolipofuscin fraction compared to the unaffected macula (see Table 3). Intracellular spatial granule distribution was in line with aged healthy cells with melanolipofuscin, melanolipofuscin 4, and melanosomes found mainly in Q1 (apically) and lipofuscin and melanolipofuscin 5 distributed mainly in the Q2 and Q3 (basolateral). 
Near Periphery
The total granule load per cell at the near periphery was 956 ± 380, which was significantly higher than healthy aged cells at the near periphery (522 ± 316; P value/η2 value <0.01/>0.37). Granule load varied among AMD manifestations (early/intermediate, late exudative, and late non-exudative AMD) with (mean ± SD) 969 ± 383, 976 ± 42, and 819 ± 213, respectively (see Table 2). Cells were composed of 418 ± 208 lipofuscin, 535 ± 229 melanolipofuscin (melanolipofuscin 4, 2 ± 3; melanolipofuscin 5, 3 ± 4), and 3 ± 4 melanosomes granules (see Table 1). Intracellular spatial granule distribution mimicked healthy aged RPE cells with melanolipofuscin, melanolipofuscin 4, and melanosomes found rather apically (Q1) and lipofuscin and melanolipofuscin 5 mainly spread in the basolateral quartiles (Q2 and Q3). 
RPE Morphology
Cell areas (Fig. 3) were markedly increased in AMD RPE cells with an average area of 265 ± 213 µm2 compared to 201 ± 73 µm2 for healthy aged cells. Cells of the near periphery (249 ± 98 µm2 in AMD versus 215 ± 90 µm2 in healthy aged eyes) were slightly larger than perifoveal cells (235 ± 116 µm2 in AMD versus 216 ± 66 µm2 in healthy aged eyes). Two giant RPE cells (1.3% of all RPE cells) found in each of two foveae were 1775 µm2 and 1742 µm2, respectively, and were about 10 times larger than the average normal aged foveal RPE cell (174 ± 5 µm2). Although these cells were grossly enlarged and densely packed with granules, these were the only two giant cells encountered. Total granule load was significantly associated with cell area, with a Spearman correlation coefficient 0.62 (P = 0.02). 
Figure 3.
 
Granule load per cell area. The graph shows the area (x-axis) and total granule load (y-axis) of RPE cells from donors with age-related macular degeneration (top row) and aged donors with unremarkable macula (bottom row). Whereas aged RPE cells displayed a clear trend between cell area and granule load, AMD RPE cells demonstrated a dissociation between the two. Using repeated measures correlation to adjust for the dependence of different cells from one tissue we found a strong correlation between cell area and granule load in AMD RPE cells as well (perifovea: r = 0.77 [95% confidence interval [0.61, 0.87], P value < 0.001/near periphery: r = 0.86 [95% confidence interval 0.78, 0.91] P value < 0.001). These results show that the relationship for granule load and cell area is stronger within-tissue than between-tissue. RPE cells in AMD presented an increased cell area and granule load. This trend was most pronounced at the fovea. The graph also illustrates an increased variability in cell area and granule load in AMD.
Figure 3.
 
Granule load per cell area. The graph shows the area (x-axis) and total granule load (y-axis) of RPE cells from donors with age-related macular degeneration (top row) and aged donors with unremarkable macula (bottom row). Whereas aged RPE cells displayed a clear trend between cell area and granule load, AMD RPE cells demonstrated a dissociation between the two. Using repeated measures correlation to adjust for the dependence of different cells from one tissue we found a strong correlation between cell area and granule load in AMD RPE cells as well (perifovea: r = 0.77 [95% confidence interval [0.61, 0.87], P value < 0.001/near periphery: r = 0.86 [95% confidence interval 0.78, 0.91] P value < 0.001). These results show that the relationship for granule load and cell area is stronger within-tissue than between-tissue. RPE cells in AMD presented an increased cell area and granule load. This trend was most pronounced at the fovea. The graph also illustrates an increased variability in cell area and granule load in AMD.
RPE Autofluorescence Per Cell
RPE cells (and cell equivalent squares) in AMD had a significantly lower total AF as compared to healthy eyes at all three locations, after correcting for age (P value/η2 value; fovea: <0.01/0.85; perifovea: <0.01/0.68; and near periphery: 0.02/0.25). These differences occur despite AMD cells being larger and having a higher overall granule load (see above). The two large cells at the fovea (not included in the analysis above) demonstrated increased AF/µm2 compared to cell area equivalent AMD squares. 
Discussion
This study compares RPE AF granule counts and AF intensity at predefined retinal locations from donor eyes with AMD to values in unaffected donor eyes. We found significantly decreased AF despite increased granule load in human RPE cells, as compared to RPE cells from healthy macula. Furthermore, granule load correlated with cell area. AMD cells demonstrated a tendency to be enlarged. We demonstrated an increase in total granule load in AMD independent of retinal location and AMD manifestation. At the same time, an increased variance of granule load in AMD RPE cells was reported, especially at the fovea, an area essential for cone mediated vision20,21 yet also prone to the accumulation of soft drusen.22,23 
Our published high-resolution imaging studies of RPE cells4 demonstrated an increase of lipofuscin and melanolipofuscin with age in adulthood, confirming Feeney's early data showing increase of granules during aging, despite methodological differences. It is important to mention that studies using QAF have found that AF might decrease in participants over the age of 60 years.12 However, our previous studies using histology as well as studies displaying marked differences of QAF dependent on lens opacification challenge this theory.7,24 A decrease in QAF at higher age (>80 years), however, is possible but remains to be tested. In AMD, however, the relationship is not as straightforward. RPE cells in AMD show granule aggregation and subsequent basolateral shedding of granule aggregates, a phenomenon uniquely attributed to AMD so far.9 
The major granule types (lipofuscin, melanolipofuscin, and melanosomes) revealed a tendency for proportionally reduced lipofuscin and increased melanolipofuscin in AMD. Granule subtypes were proportionally nearly equal, with lipofuscin 1 and melanolipofuscins 1 to 3 being the common determined granules. Our finding of proportionally decreased lipofuscin in AMD supports recent reports of lipofuscin redistribution and a probable loss in AMD rather than excessive accumulation.9 The histological loss of lipofuscin with subsequent reduced or loss of AF in AMD RPE cells was independent of retinal location and persisted after correcting for age, and comports with clinical imaging. Several groups have shown reduced AF levels in patients with AMD using quantitative FAF, a phenomenon not attributable to light-blocking subretinal drusenoid deposits because AF is also reduced in eyes with sub-RPE drusen.12,15,25 Therefore, it is most likely that the decrease in AF is due to changes at the RPE level. Photoreceptors are also impacted by AMD. Photoreceptor damage can result in increased AF signal, if light absorbing photopigment from photoreceptor outer segments is reduced or missing, as shown in AMD and other retinal diseases.2628 
We propose three different explanations for the increased granule load and the proportional change of lipofuscin and melanolipofuscin in AMD cells. First, AMD cells were shown to increase in size and to lose their typical shape, further underscored by our previous findings of vastly increased RPE cell areas in AMD without RPE cell loss.9 Thus, we could have misjudged granule density if the cells’ most apical and basal parts were out of focus and the cytoskeleton had not been labelled to define the actual cell dimensions. Future studies using alternative imaging techniques should focus on determining lipofuscin density in a 3D RPE cell depiction. 
Second, the AF signal could be derived mainly from certain molecular components or granule subtypes rather than all of lipofuscin and melanolipofuscin.29 Mass spectrometry studies revealed differing lipid signals in RPE cells dependent on retinal locations.30 Topography of AF and photoreceptors are congruent with increased AF in areas of high rod density.17,31 Further, it has been shown that rod mediated vision loss precedes and exceeds those of cones.3234 Therefore, it is possible that RPE cells demonstrate an overall increased granule load but with essential fluorophores for AF (possibly derived from rod outer segments) missing, changed, or not detectable in the short wavelength AF. Identifying the fluorophore(s) driving the FAF signal is pivotal for interpretation of AF in AMD. 
Third, melanosomes, preferentially localized in the apical processes of RPE cells,2,35 may shadow AF of more posteriorly localized lipofuscin and melanolipofuscin. At this point, however, no data are available on how AMD affects apical processes and melanosome load. It is believed that melanosomes are at least partly responsible for the near infrared (NIR)-AF signal.3638 So, future NIR-AF imaging and electron microscopy studies may help to elucidate differential melanosome compositions in the apical processes and cell bodies of AMD affected RPE cells, as 488 nm excitation does not display melanosome AF properties. 
The finding of a changed lipofuscin/melanolipofuscin ratio in AMD eyes also impacts the ongoing debate of lipofuscin's role in AMD progression. If the natural course of RPE cells in AMD is to re-organize the intracellular granule distribution with subsequent lipofuscin granule loss, to shift granule ratio toward melanolipofuscin, or both, then all these mechanisms give the impression of RPE cells trying to reduce lipofuscin load. An alternative hypothesis, however, would be that lipofuscin reduction and loss is not the cause but the consequence of other known (presence of basal laminar deposit and soft drusen material)3942 and unknown mechanisms that impact RPE cell integrity and function. Whether any intervention from outside is beneficial to therapeutically remove or reduce lipofuscin in AMD is under debate (Fang et al. ARVO abstract 2017, Investigative Ophthalmology & Visual Science, 58(8), 256–256).4346 In addition, as shown earlier, the increase of lipofuscin in normal aging does not impact the RPE cell morphology and supports lipofuscin's role as a pigment of aging and indicator of RPE cell health.7 
Melanolipofuscin's role in RPE cells remains unclear. By SIM, melanolipofuscin was shown to be the most abundant granule type in normal and AMD foveal RPE cells, whereas in RPE cells at the perifovea and near periphery lipofuscin is prominent.4 In addition, a sequence of granule conversion from melanosomes to melanolipofuscin to lipofuscin has been proposed by Feeney,6 and recently revised.4 Whether melanolipofuscin are an intermediary step in this sequence or an independent granule type needs further investigation. Spectral analysis of individual granules is currently in progress and will further contribute to the knowledge on individual granule types’ role in RPE physiology. Changes in RPE cell morphometry and cell area at the areas heavily affected by AMD are probably related to cell fusion and/or failed cytokinesis.47,48 This hypothesis is underscored by a body of evidence showing multinucleate RPE cells in AMD at the fovea, an area void of multinucleate RPE cells in healthy eyes.4850 
The granule increase in AMD affected RPE cells could be a response to local pathology, which also impacts RPE cell morphology (displacement and compression of RPE cells) especially in areas of drusen39,51 or macular neovascularization.52 Future studies combining in vivo clinical imaging like OCT with histology may help better explain the great variance in RPE cells at distinct AMD lesions. 
Strengths of this study include imaging with well-preserved RPE flat mounts with short post-mortem time,7 a large cohort of healthy RPE donor cells for comparison, and high-resolution SIM visualizing AF of individual RPE granules.9 Limitations of our study include AF excitation of only one wavelength (488 nm)36 with lack of information on other excitable fluorophores at different wavelengths, RPE cells restricted to 10 cells per location with analysis limited to three retinal locations, and imaging confined to the RPE cell body only due to possible loss of RPE's apical processes and indwelling organelles during processing. Nonetheless, the low numbers of melanosomes found within the cell body of this study is striking and further underscores the role of RPE's apical processes in housing melanosomes and contributing to clinical NIR-AF imaging. Further, analysis of cell area, 3D granule load and shape were hindered by missing RPE cells and the use of squares with average RPE cell size in some locations and donors. This could have possibly skewed reported total number of granules. We therefore consider data in Table 3 as the most indicative of changes from healthy to AMD pathology. In addition, no information on RPE cells responsible for intraretinal hyper-reflective foci (anteriorly migrating RPE cells) is available, which are an important OCT risk factors for AMD progression in multiple studies.5356 Finally, difficulties in delimiting RPE cells at the fovea do not allow proper comparison between disease manifestation, but also substantiate that RPE cells are altered most at this region. Comparison between disease manifestations is further hindered by the fact that eyes with exudative AMD also displayed macular atrophy impeding analyses of possible different disease pathways of these two late-stage AMD manifestations. Future histologic imaging studies should also include a reference for quantification of AF intensities across tissues and retinal locations. This would facilitate further clinical-histologic comparison to clinical QAF imaging. 
In summary, compared to healthy RPE cells, RPE cells in AMD display reduced AF (excitation with 488 nm), increased total granule load, and an increased variance in granule load. These seemingly paradoxical findings can be explained by a combination of cell enlargement, possible cell fusion, and different proportions of granule subtypes and/or fluorophores.51 This study helps to corroborate clinical (Q)AF studies in AMD and guide future interventional approaches in AMD affecting granule composition and new imaging biomarkers. 
Acknowledgments
Supported by Dr. Werner Jackstädt Foundation (TA) and NIH/NEI 1R01EY027948-01 (T.A. and C.A.C.); human eye tissues were supported by NIH/NEI R01EY06109 (C.A.C.). 
Disclosure: K. Bermond, None; L. von der Emde, None; I.-S. Tarau, None; L. Bourauel, None; R. Heintzmann, None; F.G. Holz, Acucela (C, F), Allergan (F), Apellis (C, F), Bayer (C, F), Boehringer-Ingelheim (C), Bioeq/Formycon (F, C), CenterVue (F), Ellex (F), Roche/Genentech (C, F), Geuder (C, F), Graybug (C), Gyroscope (C), Heidelberg Engineering (C, F), IvericBio (C, F), Kanghong (C, F), LinBioscience (C), NightStarX (F), Novartis (C, F), Optos (F), Oxurion (C), Pixium Vision (C, F), Oxurion (C), Stealth BioTherapeutics (C), Zeiss (F, C); C.A. Curcio, Genentech (F), Regeneron (F), MacRegen Inc. (I); K.R. Sloan, MacRegen Inc. (I); T. Ach, Roche (C), Novartis (C), Novartis (R), MacRegen Inc. (I) 
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Figure 1.
 
SIM-images of AMD-affected and unremarkable tissue. Each image shows a representative slide at the apical surface of a SIM z-stack of the fovea (A–D) or perifovea (E–H) of AMD-affected (A–C, E–G) or unremarkable (D, H) tissues. The AMD-affected tissues show deterioration of cell structures like dissolving cell borders, partial loss of intracellular granule organization and cell loss. Areas of RPE cell loss probably associated with deposits (basal laminar deposits/basal linear deposits) are marked with a blue asterisk. These deposits affected the fovea and, in some tissues, extended to the perifovea (E, F). Where the perifovea was not (yet) affected by AMD (G), the tissue appeared comparable to healthy tissue (D, H) which shows uniform cell shapes and orderly intracellular granule distribution. Note: the granule free areas within the cell bodies in D and H represent the non-autofluorescent cell nucleus. In panels A, B, and E, we were unable to distinguish cell boundaries and used cell-size equivalent squares instead. A 3D-version of panel C is further depicted in the supplemental video. Early or intermediate AMD: 83-year-old woman (F), 84-year-old man B, and 86-year-old woman A. Late non-exudative AMD: 81-year-old man C, G. Late exudative AMD: 90-year-old woman E. Unremarkable tissue: 83-year-old woman D and 90-year-old woman H. Scale bar = 10 µm.
Figure 1.
 
SIM-images of AMD-affected and unremarkable tissue. Each image shows a representative slide at the apical surface of a SIM z-stack of the fovea (A–D) or perifovea (E–H) of AMD-affected (A–C, E–G) or unremarkable (D, H) tissues. The AMD-affected tissues show deterioration of cell structures like dissolving cell borders, partial loss of intracellular granule organization and cell loss. Areas of RPE cell loss probably associated with deposits (basal laminar deposits/basal linear deposits) are marked with a blue asterisk. These deposits affected the fovea and, in some tissues, extended to the perifovea (E, F). Where the perifovea was not (yet) affected by AMD (G), the tissue appeared comparable to healthy tissue (D, H) which shows uniform cell shapes and orderly intracellular granule distribution. Note: the granule free areas within the cell bodies in D and H represent the non-autofluorescent cell nucleus. In panels A, B, and E, we were unable to distinguish cell boundaries and used cell-size equivalent squares instead. A 3D-version of panel C is further depicted in the supplemental video. Early or intermediate AMD: 83-year-old woman (F), 84-year-old man B, and 86-year-old woman A. Late non-exudative AMD: 81-year-old man C, G. Late exudative AMD: 90-year-old woman E. Unremarkable tissue: 83-year-old woman D and 90-year-old woman H. Scale bar = 10 µm.
Figure 2.
 
En face (A) and cross section (B) view of intracellular granule distribution. Each analyzed granule was color-coded and plotted (lipofuscin: yellow, melanolipofuscin 1–3: blue, melanolipofuscin 4: pink, melanolipofuscin 5: magenta, and M: orange). Only few melanosomes, melanolipofuscin 4, and melanolipofuscin 5 were detectable. Red lines represent cell borders or artificial squares, respectively. At four locations (three foveae and one perifovea), no granule analysis was possible due to large RPE-atrophy. Foveal tissue showed distinctive alterations in cell structure with either formation of enlarged cells (83-year-old woman and 81-year-old man) or primarily faded cell borders and a moth-eaten appearance (84-year-old man and 86-year-old woman [early/intermediate AMD]). The predominant granule type at the fovea remained melanolipofuscin, like in unremarkable tissues.4 At the perifovea and near periphery, all tissues that showed a regular cell structure in the en face view also displayed two bands in the cross-section view: an apical band with predominantly melanolipofuscin and lipofuscin and a basal band with predominantly lipofuscin. At locations where the cell structures were deteriorated (83-year-old perifovea and near periphery, 90-year-old perifovea) these two bands could not be distinguished. Scale bar = 10 µm.
Figure 2.
 
En face (A) and cross section (B) view of intracellular granule distribution. Each analyzed granule was color-coded and plotted (lipofuscin: yellow, melanolipofuscin 1–3: blue, melanolipofuscin 4: pink, melanolipofuscin 5: magenta, and M: orange). Only few melanosomes, melanolipofuscin 4, and melanolipofuscin 5 were detectable. Red lines represent cell borders or artificial squares, respectively. At four locations (three foveae and one perifovea), no granule analysis was possible due to large RPE-atrophy. Foveal tissue showed distinctive alterations in cell structure with either formation of enlarged cells (83-year-old woman and 81-year-old man) or primarily faded cell borders and a moth-eaten appearance (84-year-old man and 86-year-old woman [early/intermediate AMD]). The predominant granule type at the fovea remained melanolipofuscin, like in unremarkable tissues.4 At the perifovea and near periphery, all tissues that showed a regular cell structure in the en face view also displayed two bands in the cross-section view: an apical band with predominantly melanolipofuscin and lipofuscin and a basal band with predominantly lipofuscin. At locations where the cell structures were deteriorated (83-year-old perifovea and near periphery, 90-year-old perifovea) these two bands could not be distinguished. Scale bar = 10 µm.
Figure 3.
 
Granule load per cell area. The graph shows the area (x-axis) and total granule load (y-axis) of RPE cells from donors with age-related macular degeneration (top row) and aged donors with unremarkable macula (bottom row). Whereas aged RPE cells displayed a clear trend between cell area and granule load, AMD RPE cells demonstrated a dissociation between the two. Using repeated measures correlation to adjust for the dependence of different cells from one tissue we found a strong correlation between cell area and granule load in AMD RPE cells as well (perifovea: r = 0.77 [95% confidence interval [0.61, 0.87], P value < 0.001/near periphery: r = 0.86 [95% confidence interval 0.78, 0.91] P value < 0.001). These results show that the relationship for granule load and cell area is stronger within-tissue than between-tissue. RPE cells in AMD presented an increased cell area and granule load. This trend was most pronounced at the fovea. The graph also illustrates an increased variability in cell area and granule load in AMD.
Figure 3.
 
Granule load per cell area. The graph shows the area (x-axis) and total granule load (y-axis) of RPE cells from donors with age-related macular degeneration (top row) and aged donors with unremarkable macula (bottom row). Whereas aged RPE cells displayed a clear trend between cell area and granule load, AMD RPE cells demonstrated a dissociation between the two. Using repeated measures correlation to adjust for the dependence of different cells from one tissue we found a strong correlation between cell area and granule load in AMD RPE cells as well (perifovea: r = 0.77 [95% confidence interval [0.61, 0.87], P value < 0.001/near periphery: r = 0.86 [95% confidence interval 0.78, 0.91] P value < 0.001). These results show that the relationship for granule load and cell area is stronger within-tissue than between-tissue. RPE cells in AMD presented an increased cell area and granule load. This trend was most pronounced at the fovea. The graph also illustrates an increased variability in cell area and granule load in AMD.
Table 1.
 
Number of Granules and Mean Cell Area in AMD and Normal Aged RPE Cells4
Table 1.
 
Number of Granules and Mean Cell Area in AMD and Normal Aged RPE Cells4
Table 2.
 
Granule Composition in Different AMD Disease Manifestations. Reported are Number of Granules Per Cell ± Standard Deviation
Table 2.
 
Granule Composition in Different AMD Disease Manifestations. Reported are Number of Granules Per Cell ± Standard Deviation
Table 3.
 
Granule Distribution at the Fovea, Perifovea, and Near Periphery
Table 3.
 
Granule Distribution at the Fovea, Perifovea, and Near Periphery
Table 4.
 
Granule Density at the Fovea and Perifovea
Table 4.
 
Granule Density at the Fovea and Perifovea
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