Quantifying Retinal Pigment Epithelium Dysmorphia and Loss of Histologic Autofluorescence in Age-Related Macular Degeneration.

Purpose: Lipofuscin and melanolipofuscin organelles in retinal pigment epithelium (RPE) cells are signal sources for clinical fundus autofluorescence (AF). To elucidate the subcellular basis of AF imaging, we identified, characterized, and quantified the frequency of RPE morphology and AF phenotypes in donor eyes with age-related macular degeneration (AMD). Methods: In 25 RPE-Bruch's membrane flat mounts from 25 eyes, we analyzed 0.4-µm z-stack epifluorescence images of RPE stained with phalloidin for actin cytoskeleton. Using a custom ImageJ plugin, we classified cells selected in a systematic unbiased fashion in six phenotypes representing increasing degrees of pathology. For each cell, area, AF intensity, and number of Voronoi neighbors were compared with phenotype 1 (uniform AF, polygonal morphology) via generalized estimating equations. We also analyzed each cell's neighborhood. Results: In 29,323 cells, compared with phenotype 1, all other phenotypes, in order of increasing pathology, had significantly larger area, reduced AF, and more variable number of neighbors. Neighborhood area and AF showed similar, but subtler, trends. Cells with highly autofluorescent granule aggregates are no more autofluorescent than others and are in fact lower overall in AF. Pre-aggregates were found in phenotype 1. Phenotype 2, which exhibited degranulation despite normal cytoskeleton, was the most numerous nonhealthy phenotype (16.23%). Conclusions: Despite aggregation of granules that created hyperAF aggregates within cells, overall AF on a per cell basis decreased with increasing severity of dysmorphia (abnormal shape). Data motivate further development of subcellular resolution in clinical fundus AF imaging and inform an ongoing reexamination of the role of lipofuscin in AMD.

Rod-Mediated Dark Adaptation and Macular Pigment Optical Density in Older Adults with Normal Maculas.

PURPOSE: To examine the association between macular pigment optical density (MPOD) and rod-mediated dark adaptation (RMDA) in persons ≥60 years old with normal maculas as determined by an accepted color fundus photography grading system. METHODS: This cross-sectional analysis used baseline data from eyes in the Alabama Study on Early Age-Related Macular Degeneration. Eyes at step 1 in the AREDS 9-step grading system were considered normal. Eyes were additionally assessed by spectral domain optical coherence tomography (SD-OCT). Foveal MPOD was estimated via heterochromatic flicker photometry, and RMDA was assessed with a computerized dark adaptometer. The association between RMDA and MPOD was examined via Spearman correlation coefficients adjusted for age. RESULTS: In 306 eyes from 306 persons (mean age 68.2 years) in normal macular health, MPOD was not associated with RMDA (age-adjusted rank correlation = 0.043, p = 0.45). After 81 eyes with incidental macular findings by SD-OCT evaluation were excluded, the association between MPOD and RMDA remained null (N = 225, age-adjusted r = 0.015, p = 0.82). CONCLUSION: In a large sample of normal aged eyes, RMDA, a visual function that is rate limited by retinoid availability to photoreceptors across the complex of retinal pigment epithelium, Bruch's membrane, and choriocapillaris, is not related to MPOD in the neurosensory retina.

Macular Atrophy Development and Subretinal Drusenoid Deposits in Anti-Vascular Endothelial Growth Factor Treated Age-Related Macular Degeneration.

Purpose: To explore the association between presence of subretinal drusenoid deposits (SDD) at baseline in eyes with neovascular age-related macular degeneration (nAMD) with the development of macular atrophy (MA) during anti-vascular endothelial growth factor (VEGF) therapy. Methods: There were 74 eyes without pre-existing MA receiving anti-VEGF therapy for nAMD for 2 years or longer analyzed. At least two image modalities that included spectral-domain optical coherence tomography, near-infrared reflectance, fluorescein angiography, and color fundus photos were used to assess for SDD presence, phenotype (dot and ribbon), and location, neovascularization type, and MA. Logistic regression models using generalized estimating equations assessed the association between SDD and the development of MA adjusting for age, neovascularization type, and choroidal thickness. Results: SDD were present in 46 eyes (63%) at baseline. MA developed in 38 eyes (51%) during the mean of 4.7 ± 1.2 years of follow-up. Compared with eyes without SDD, those with SDD at baseline were 3.0 times (95% confidence interval [CI] 1.1-8.5, P = 0.0343) more likely to develop MA. Eyes with SDD present in the inferior macula and inferior extramacular fields at baseline were 3.0 times and 6.5 times more likely to develop MA at follow-up than eyes without SDD in these locations (95% CI 1.0-8.9, P = 0.0461 and 95% CI 1.3-32.4, P = 0.0218, respectively). MA development was not associated with a specific SDD phenotype. Conclusions: MA frequently developed in eyes during anti-VEGF treatment. SDD were independently associated with MA development. The extension of SDD into the inferior fundus, particularly in the inferior extramacular field, conferred higher odds of subsequent MA development.

ASSOCIATION BETWEEN VISUAL FUNCTION AND SUBRETINAL DRUSENOID DEPOSITS IN NORMAL AND EARLY AGE-RELATED MACULAR DEGENERATION EYES.

PURPOSE: To examine the association between subretinal drusenoid deposits (SDDs) identified by multimodal retinal imaging and visual function in older eyes with normal macular health or in the earliest phases of age-related macular degeneration (AMD). METHODS: Age-related macular degeneration status for each eye was defined according to the Age-Related Eye Disease Study (AREDS) 9-step classification system (normal = Step 1, early AMD = Steps 2-4) based on color fundus photographs. Visual functions measured were best-corrected photopic visual acuity, contrast and light sensitivity, mesopic visual acuity, low-luminance deficit, and rod-mediated dark adaptation. Subretinal drusenoid deposits were identified through multimodal imaging (color fundus photographs, infrared reflectance and fundus autofluorescence images, and spectral domain optical coherence tomography). RESULTS: The sample included 1,202 eyes (958 eyes with normal health and 244 eyes with early AMD). In normal eyes, SDDs were not associated with any visual function evaluated. In eyes with early AMD, dark adaptation was markedly delayed in eyes with SDDs versus no SDD (a 4-minute delay on average), P = 0.0213. However, this association diminished after age adjustment, P = 0.2645. Other visual functions in early AMD eyes were not associated with SDDs. CONCLUSION: In a study specifically focused on eyes in normal macular health and in the earliest phases of AMD, early AMD eyes with SDDs have slower dark adaptation, largely attributable to the older ages of eyes with SDD; they did not exhibit deficits in other visual functions. Subretinal drusenoid deposits in older eyes in normal macular health are not associated with any visual functions evaluated.

Prevalence of Subretinal Drusenoid Deposits in Older Persons with and without Age-Related Macular Degeneration, by Multimodal Imaging.

PURPOSE: To assess the prevalence of subretinal drusenoid deposits (SDD) in older adults with healthy maculas and early and intermediate age-related macular degeneration (AMD) using multimodal imaging. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 651 subjects aged ≥60 years enrolled in the Alabama Study of Early Age-Related Macular Degeneration from primary care ophthalmology clinics. METHODS: Subjects were imaged using spectral domain optical coherence tomography (SD OCT) of the macula and optic nerve head (ONH), infrared reflectance, fundus autofluorescence, and color fundus photographs (CFP). Eyes were assessed for AMD presence and severity using the Age-Related Eye Disease Study (AREDS) 9-step scale. Criteria for SDD presence were identification on ≥1 en face modality plus SD OCT or on ≥2 en face modalities if absent on SD OCT. Subretinal drusenoid deposits were considered present at the person level if present in 1 or both eyes. MAIN OUTCOME MEASURES: Prevalence of SDD in participants with and without AMD. RESULTS: Overall prevalence of SDD was 32% (197/611), with 62% (122/197) affected in both eyes. Persons with SDD were older than those without SDD (70.6 vs. 68.7 years, P = 0.0002). Prevalence of SDD was 23% in subjects without AMD and 52% in subjects with AMD (P < 0.0001). Among those with early and intermediate AMD, SDD prevalence was 49% and 79%, respectively. After age adjustment, those with SDD were 3.4 times more likely to have AMD than those without SDD (95% confidence interval, 2.3-4.9). By using CFP only for SDD detection per the AREDS protocol, prevalence of SDD was 2% (12/610). Of persons with SDD detected by SD OCT and confirmed by at least 1 en face modality, 47% (89/190) were detected exclusively on the ONH SD OCT volume. CONCLUSIONS: Subretinal drusenoid deposits are present in approximately one quarter of older adults with healthy maculae and in more than half of persons with early to intermediate AMD, even by stringent criteria. The prevalence of SDD is strongly associated with AMD presence and severity and increases with age, and its retinal topography including peripapillary involvement resembles that of rod photoreceptors. Consensus on SDD detection methods is recommended to advance our knowledge of this lesion and its clinical and biologic significance.

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 sources of autofluorescence (AF) signals in clinical fundus-AF imaging. Few details about the subcellular distribution of AF organelles in AMD are available. We describe the impact of aging and AMD on RPE morphology revealed by the distribution of AF LF/MLF granules and actin cytoskeleton in human tissues. METHODS: Thirty-five RPE-Bruch's membrane flatmounts from 35 donors were prepared (postmortem: ≤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): early AMD, geographic atrophy, and late exudative AMD. Retinal pigment epithelium cytoskeleton was labeled with AlexaFluor647-Phalloidin. Tissues were imaged on a spinning-disk fluorescence microscope and a high-resolution structured illumination microscope. RESULTS: Age-related macular degeneration impacts individual RPE cells by (1) lipofuscin redistribution by (i) degranulation (granule-by-granule loss) and/or (ii) aggregation and apparent shedding into the extracellular space; (2) enlarged RPE cell area and conversion from convex to irregular and sometimes concave polygons; and (3) cytoskeleton derangement including separations and breaks around subretinal deposits, thickening, and stress fibers. CONCLUSIONS: We report an extensive and systematic en face analysis of LF/MLF-AF in AMD eyes. Redistribution and loss of AF granules are among the earliest AMD changes and could reduce fundus AF signal attributable to RPE at these locations. Data can enhance the interpretation of clinical fundus-AF and provide a basis for future quantitative studies.