Outer Retinal Thickness Is Associated With Cognitive Function in Normal Aging to Intermediate Age-Related Macular Degeneration.

Purpose: Research on Alzheimer's disease (AD) and precursor states demonstrates a thinner retinal nerve fiber layer (NFL) compared to age-similar controls. Because AD and age-related macular degeneration (AMD) both impact older adults and share risk factors, we asked if retinal layer thicknesses, including NFL, are associated with cognition in AMD. Methods: Adults ≥ 70 years with normal retinal aging, early AMD, or intermediate AMD per Age-Related Eye Disease Study (AREDS) nine-step grading of color fundus photography were enrolled in a cross-sectional study. Optical coherence tomography (OCT) volumes underwent 11-line segmentation and adjustments by a trained operator. Evaluated thicknesses reflect the vertical organization of retinal neurons and two vascular watersheds: NFL, ganglion cell layer-inner plexiform layer complex (GCL-IPL), inner retina, outer retina (including retinal pigment epithelium-Bruch's membrane), and total retina. Thicknesses were area weighted to achieve mean thickness across the 6-mm-diameter Early Treatment of Diabetic Retinopathy Study (ETDRS) grid. Cognitive status was assessed by the National Institutes of Health Toolbox cognitive battery for fluid and crystallized cognition. Correlations estimated associations between cognition and thicknesses, adjusting for age. Results: Based on 63 subjects (21 per group), thinning of the outer retina was significantly correlated with lower cognition scores (P < 0.05). No other retinal thickness variables were associated with cognition. Conclusions: Only the outer retina (photoreceptors, supporting glia, retinal pigment epithelium, Bruch's membrane) is associated with cognition in aging to intermediate AMD; NFL was not associated with cognition, contrary to AD-associated condition reports. Early and intermediate AMD constitute a retinal disease whose earliest, primary impact is in the outer retina. Our findings hint at a unique impact on the brain from the outer retina in persons with AMD.

Quantitative autofluorescence at AMD's beginnings highlights retinal topography and grading system differences: ALSTAR2 baseline.

PURPOSE: To describe baseline quantitative (short-wavelength) autofluorescence (qAF) findings in a large pseudophakic cohort at age-related macular degeneration (AMD)'s beginnings; to assess qAF8 as an outcome measure and evaluate AREDS and Beckman grading systems. METHODS: In the ALSTAR2 baseline cohort (NCT04112667), 346 pseudophakic eyes of 188 persons (74.0 ± 5.5 years) were classified as normal (N=160 by AREDS, 158 by Beckman), early (e)AMD (N=104, 66), and intermediate (i)AMD (N=82, 122). Groups were compared via mean qAF intensities in a 6°- 8° annulus (qAF8) and maps of differences between observations and overall mean, divided by standard deviation (Z-score). RESULTS: qAF8 did not differ significantly among diagnostic groups by either stratification (p = 0.0869 AREDS; p = 0.0569 by Beckman). Notably, 45 eyes considered eAMD by AREDS became iAMD by Beckman. For AREDS-stratified eyes, Z-score maps showed higher centrally located qAF for normal, near the mean in eAMD, and lower values for iAMD. Maps deviated from this pattern for Beckman-stratified eyes. CONCLUSIONS: In a large sample of pseudophakic eyes, qAF8 does not differ overall from normal aging to iAMD but also does not capture the earliest AMD activity in the macula lutea. AREDS classification gives results more consistent with a slow decline in histologic autofluorescence than Beckman classification.

Spatially Resolved Association of Structural Biomarkers on Retinal Function in Non-Exudative Age-Related Macular Degeneration Over 4 Years.

Purpose: To longitudinally assess the impact of high-risk structural biomarkers for natural disease progression in non-exudative age-related macular degeneration (AMD) on spatially resolved mesopic and scotopic fundus-controlled perimetry testing. Methods: Multimodal retinal imaging data and fundus-controlled perimetry stimuli points were semiautomatically registered according to landmark correspondences at each annual visit over a period of up to 4 years. The presence of sub-RPE drusen, subretinal drusenoid deposits, pigment epithelium detachments (PEDs), hyper-reflective foci (HRF), vitelliform lesions, refractile deposits, and incomplete RPE and outer retinal atrophy (iRORA) and complete RPE and outer retinal atrophy (cRORA) were graded at each stimulus position and visit. Localized retinal layer thicknesses were extracted. Mixed-effect models were used for structure-function correlation. Results: Fifty-four eyes of 49 patients with non-exudative AMD (mean age, 70.7 ± 9.1 years) and 27 eyes of 27 healthy controls (mean age, 63.4 ± 8.9 years) were included. During study course, presence of PED had the highest functional impact with a mean estimated loss of -1.30 dB (P < 0.001) for mesopic and -1.23 dB (P < 0.001) for scotopic testing, followed by HRF with -0.89 dB (mesopic, P = 0.001) and -0.87 dB (scotopic, P = 0.005). Subretinal drusenoid deposits were associated with a stronger visual impairment (mesopic, -0.38 dB; P = 0.128; scotopic, -0.37 dB; P = 0.172) compared with sub-RPE drusen (-0.22 dB, P = 0.0004; -0.18 dB, P = 0.006). With development of c-RORA, scotopic retinal sensitivity further significantly decreased (-2.15 dB; P = 0.02). Thickening of the RPE-drusen-complex and thinning of the outer nuclear layer negatively impacted spatially resolved retinal sensitivity. Conclusions: The presence of PED and HRF had the greatest prognostic impact on progressive point-wise sensitivity losses. Higher predominant rod than cone-mediated localized retinal sensitivity losses with early signs of retinal atrophy development indicate photoreceptor preservation as a potential therapeutic target for future interventional AMD trials.

Age-Related Macular Degeneration, a Mathematically Tractable Disease.

A progression sequence for age-related macular degeneration onset may be determinable with consensus neuroanatomical nomenclature augmented by drusen biology and eye-tracked clinical imaging. This narrative review proposes to supplement the Early Treatment of Diabetic Retinopathy Study (sETDRS) grid with a ring to capture high rod densities. Published photoreceptor and retinal pigment epithelium (RPE) densities in flat mounted aged-normal donor eyes were recomputed for sETDRS rings including near-periphery rich in rods and cumulatively for circular fovea-centered regions. Literature was reviewed for tissue-level studies of aging outer retina, population-level epidemiology studies regionally assessing risk, vision studies regionally assessing rod-mediated dark adaptation (RMDA), and impact of atrophy on photopic visual acuity. The 3 mm-diameter xanthophyll-rich macula lutea is rod-dominant and loses rods in aging whereas cone and RPE numbers are relatively stable. Across layers, the largest aging effects are accumulation of lipids prominent in drusen, loss of choriocapillary coverage of Bruch's membrane, and loss of rods. Epidemiology shows maximal risk for drusen-related progression in the central subfield with only one third of this risk level in the inner ring. RMDA studies report greatest slowing at the perimeter of this high-risk area. Vision declines precipitously when the cone-rich central subfield is invaded by geographic atrophy. Lifelong sustenance of foveal cone vision within the macula lutea leads to vulnerability in late adulthood that especially impacts rods at its perimeter. Adherence to an sETDRS grid and outer retinal cell populations within it will help dissect mechanisms, prioritize research, and assist in selecting patients for emerging treatments.

Volumetric Reconstruction of a Human Retinal Pigment Epithelial Cell Reveals Specialized Membranes and Polarized Distribution of Organelles.

Purpose: Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles. Methods: A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods. Results: In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin. Conclusions: Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.

Retest variability and patient reliability indices of quantitative fundus autofluorescence in age-related macular degeneration: a MACUSTAR study report.

This study aimed to determine the retest variability of quantitative fundus autofluorescence (QAF) in patients with and without age-related macular degeneration (AMD) and evaluate the predictive value of patient reliability indices on retest reliability. A total of 132 eyes from 68 patients were examined, including healthy individuals and those with various stages of AMD. Duplicate QAF imaging was conducted at baseline and 2 weeks later across six study sites. Intraclass correlation (ICC) analysis was used to evaluate the consistency of imaging, and mean opinion scores (MOS) of image quality were generated by two researchers. The contribution of MOS and other factors to retest variation was assessed using mixed-effect linear models. Additionally, a Random Forest Regressor was trained to evaluate the extent to which manual image grading of image quality could be replaced by automated assessment (inferred MOS). The results showed that ICC values were high for all QAF images, with slightly lower values in AMD-affected eyes. The average inter-day ICC was found to be 0.77 for QAF segments within the QAF8 ring and 0.74 for peripheral segments. Image quality was predicted with a mean absolute error of 0.27 on a 5-point scale, and of all evaluated reliability indices, MOS/inferred MOS proved most important. The findings suggest that QAF allows for reliable testing of autofluorescence levels at the posterior pole in patients with AMD in a multicenter, multioperator setting. Patient reliability indices could serve as eligibility criteria for clinical trials, helping identify patients with adequate retest reliability.

Discernibility of the Interdigitation Zone (IZ), a Potential Optical Coherence Tomography (OCT) Biomarker for Visual Dysfunction in Aging.

PURPOSE: Photoreceptor (PR) outer segments, retinal pigment epithelium apical processes, and inter-PR matrix contribute to the interdigitation zone (IZ) of optical coherence tomography (OCT). We hypothesize that this interface degrades over adulthood, in concert with a delay of rod mediated dark adaptation (RMDA). To explore this idea, we determined IZ discernibility and RMDA in younger and older adults. METHODS: For this cross-sectional study, eyes of 20 young (20-30 years) and 40 older (≥60 years) participants with normal maculas according to the AREDS 9-step grading system underwent OCT imaging and RMDA testing at 5° superior to the fovea. Custom FIJI plugins enabled analysis for IZ discernibility at 9 eccentricities in 0.5 mm steps on one single horizontal B-scan through the fovea. Locations with discernible IZ met two criteria: visibility on B-scans and a distinct peak on a longitudinal reflectivity profile. The frequency of sites meeting both criteria was compared between both age groups and correlated with rod intercept time (RIT). RESULTS: The median number of locations with discernible IZ was significantly higher (foveal, 4 vs. 0, p = 0.0099; extra-foveal 6 vs. 0, p < 0.001) in eyes of young (26 ± 3 years) compared to older (73 ± 5 years) participants. For the combined young and older sample, the higher frequency of discernible IZ was correlated with shorter RIT (faster dark adaptation) (rs = -0.56, p < 0.0001). This association was significant within young eyes (rs = -0.54; p = 0.0134) and not within older eyes (rs = -0.29, p = 0.706). CONCLUSIONS: Results suggest that the interface between outer segments and apical processes degrades in normal aging, potentially contributing to delayed rod-mediated dark adaptation. More research is needed to verify an age-related association between IZ discernibility and rod-mediated dark adaptation. If confirmed in a large sample, IZ discernibility might prove to be a valuable biomarker and predictor for visual function in aging.

Quantitative Fundus Autofluorescence in Systemic Chloroquine/Hydroxychloroquine Therapy: One Year Follow-Up.

Purpose: Systemic chloroquine/hydroxychloroquine (CQ/HCQ) can cause severe ocular side effects including bull's eye maculopathy (BEM). Recently, we reported higher quantitative autofluorescence (QAF) levels in patients with CQ/HCQ intake. Here, QAF in patients taking CQ/HCQ in a 1-year follow-up is reported. Methods: Fifty-eight patients currently or previously treated with CQ/HCQ (cumulative doses 94-2435 g) and 32 age- and sex-matched healthy subjects underwent multimodal retinal imaging (infrared, red free, fundus autofluorescence [FAF], QAF [488 nm], and spectral-domain optical coherence tomography (SD-OCT). For analysis, custom written FIJI plugins were used for image processing, multimodal image stacks assembling, and QAF calculation. Results: Thirty patients (28 without BEM and 2 with BEM, age range = 25-69 years) were followed up (370 ± 63 days). QAF values in patients taking CQ/HCQ showed a significant increase between baseline and follow-up examination: 282.0 ± 67.9 to 297.7 ± 70.0 (QAF a.u.), P = 0.002. An increase up to 10% was observed in the superior macular hemisphere. Eight individuals (including 1 patient with BEM) had a pronounced QAF increase of up to 25%. Compared to healthy controls, QAF levels in patients taking CQ/HCQ were significantly increased (P = 0.04). Conclusions: Our study confirms our previous finding of increased QAF in patients taking CQ/HCQ with a further significant QAF increase from baseline to follow-up. Whether pronounced QAF increase might predispose for rapid progression toward structural changes and BEM development is currently investigated in ongoing studies. Translational Relevance: In addition to standard screening tools during systemic CQ/HCQ treatment, QAF imaging might be useful in CQ/HCQ monitoring and could serve as a screening tool in the future.

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence.

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear. This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids. AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions. This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Macular and Plasma Xanthophylls Are Higher in Age-related Macular Degeneration than in Normal Aging: Alabama Study on Early Age-related Macular Degeneration 2 Baseline.

Purpose: Quantification of retinal xanthophyll carotenoids in eyes with and without age-related macular degeneration (AMD) via macular pigment optical volume (MPOV), a metric for xanthophyll abundance from dual wavelength autofluorescence, plus correlations to plasma levels, could clarify the role of lutein (L) and zeaxanthin (Z) in health, AMD progression, and supplementation strategies. Design: Cross-sectional observational study (NCT04112667). Participants: Adults ≥ 60 years from a comprehensive ophthalmology clinic, with healthy maculas or maculas meeting fundus criteria for early or intermediate AMD. Methods: Macular health and supplement use was assessed by the Age-related Eye Disease Study (AREDS) 9-step scale and self-report, respectively. Macular pigment optical volume was measured from dual wavelength autofluorescence emissions (Spectralis, Heidelberg Engineering). Non-fasting blood draws were assayed for L and Z using high-performance liquid chromatography. Associations among plasma xanthophylls and MPOV were assessed adjusting for age. Main Outcome Measures: Age-related macular degeneration presence and severity, MPOV in fovea-centered regions of radius 2.0° and 9.0°; plasma L and Z (µM/ml). Results: Of 809 eyes from 434 persons (89% aged 60-79, 61% female), 53.3% eyes were normal, 28.2% early AMD, and 18.5% intermediate AMD. Macular pigment optical volume 2° and 9° were similar in phakic and pseudophakic eyes, which were combined for analysis. Macular pigment optical volume 2° and 9° and plasma L and Z were higher in early AMD than normal and higher still in intermediate AMD (P < 0.0001). For all participants, higher plasma L was correlated with higher MPOV 2° (Spearman correlation coefficient [Rs] = 0.49; P < 0.0001). These correlations were significant (P < 0.0001) but lower in normal (Rs = 0.37) than early and intermediate AMD (Rs = 0.52 and 0.51, respectively). Results were similar for MPOV 9°. Plasma Z, MPOV 2°, and MPOV 9° followed this same pattern of associations. Associations were not affected by supplement use or smoking status. Conclusions: A moderate positive correlation of MPOV with plasma L and Z comports with regulated xanthophyll bioavailability and a hypothesized role for xanthophyll transfer in soft drusen biology. An assumption that xanthophylls are low in AMD retina underlies supplementation strategies to reduce progression risk, which our data do not support. Whether higher xanthophyll levels in AMD are due to supplement use cannot be determined in this study.

Hyper-Reflective Foci in Intermediate Age-Related Macular Degeneration: Spatial Abundance and Impact on Retinal Morphology.

Purpose: The purpose of this study was to analyze spatially resolved structural changes at retinal locations in presence (+) or absence (-) of hyper-reflective foci (HRF) in eyes with subretinal pigment epithelium (RPE) drusen in intermediate age-related macular degeneration (iAMD). Methods: Patients with IAMD (n = 40; mean age = 69.7 ± 9.2 [SD] years) and healthy controls (n = 27; 64.2 ± 9.0) underwent spectral-domain optical-coherence-tomography imaging and fundus-controlled perimetry testing. After reviewing retinal layer segmentation, presence of HRF was annotated and retinal layer thicknesses (RLTs) extracted using ImageJ. Localized RLTs were compared between +HRF and -HRF positions. Univariate mixed linear models were used to investigate associations among RLT, HRF presence, and HRF size. Results: In iAMD eyes, a mean of 11.1 ± 12.5 HRF were detected with a peak abundance at 0.5 to 1.5 mm eccentricity to the fovea. At +HRF positions, outer nuclear layer (ONL; P = 0.0013, average difference = -12.4 µm) and retinal pigment epithelium drusen complex (RPEDC; P < 0.0001, +45.6 µm) thicknesses differed significantly compared to -HRF positions, even after correcting for accompanying drusen-related RPEDC layer thickening (P = 0.01). Mixed linear models revealed a significant association between increasing HRF area and decreasing ONL (association score = -0.17, P < 0.0001; 95% confidence interval [CI] = -0.22 to -0.11), and inner photoreceptor segments (IS) layer thicknesses (-0.08, P = 0.005; 95% CI = -0.14 to -0.03). Spearman rank correlation analysis yielded a significant correlation between total HRF area and mesopic (P = 0.015), but not scotopic (P = 0.305) retinal sensitivity losses. Conclusions: Descriptive analysis of this study demonstrated a predominant distribution of HRF at a foveal eccentricity of 0.5 to 1.5 mm, whereas further refined topographic analysis revealed a significant ONL layer thinning in presence of HRF even after correction for sub-RPE drusen presence compared to lesions in absence of HRF. Longitudinal studies are further needed to analyze the prognostic impact as well as the role of HRF presence in the context of iAMD.

Impact of the Aging Lens and Posterior Capsular Opacification on Quantitative Autofluorescence Imaging in Age-Related Macular Degeneration.

Purpose: The purpose of this study was to investigate quantitative autofluorescence (qAF8) in patients with and without early or intermediate age-related macular degeneration (AMD); to determine the impact of the aged crystalline lens and posterior capsular opacification (PCO). Methods: In phakic and pseudophakic eyes ≥60 years, AMD status was determined by the Beckman system. PCO presence and severity was extracted from clinical records. qAF8 was calculated using custom FIJI plugins. Differences in qAF8, stratified by lens status, PCO severity, and AMD status, were analyzed using generalized estimating equations. Results: In 210 eyes of 115 individuals (mean age = 75.7 ± 6.6 years), qAF8 was lower in intermediate AMD compared to early AMD (P = 0.05). qAF8 did not differ between phakic and pseudophakic eyes (P = 0.8909). In phakic (n = 83) and pseudophakic (n = 127) eyes considered separately, qAF8 did not differ by AMD status (P = 0.0936 and 0.3494, respectively). Qualitative review of qAF images in phakic eyes illustrated high variability. In pseudophakic eyes, qAF8 did not differ with PCO present versus absent (54.5% vs. 45.5%). Review of implanted intraocular lenses (IOLs) revealed that 43.9% were blue-filter IOLs. Conclusions: qAF8 was not associated with AMD status, up to intermediate AMD, considering only pseudophakic eyes to avoid noisy images in phakic eyes. In pseudophakic eyes, qAF8 was not affected by PCO. Because blue-filter IOLs may reduce levels of exciting light for qAF8, future studies investigating qAF in eyes with different IOL types are needed. Translational Relevance: To reduce variability in observational studies and clinical trials requiring qAF8, pseudophakic participants without blue-filter IOLs or advanced PCO should be preferentially enrolled.

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

Purpose: Phenotype alterations of the retinal pigment epithelium (RPE) are a main characteristic of age-related macular degeneration (AMD). Individual RPE cell shape descriptors may help to delineate healthy from AMD-affected cells in early disease stages. Methods: Twenty-two human RPE flatmounts (7 eyes with AMD [early, 3; geographic atrophy, 1; neovascular, 3); 15 unaffected eyes [8 aged ≤51 years; 7 aged >80 years)] were imaged at the fovea, perifovea, and near periphery (predefined sample locations) using a laser-scanning confocal fluorescence microscope. RPE cell boundaries were manually marked with computer assistance. For each cell, 11 shape descriptors were calculated and correlated with donor age, cell autofluorescence (AF) intensity, and retinal location. Statistical analysis was performed using an ensemble classifier based on logistic regression. Results: In AMD, RPE was altered at all locations (most pronounced at the fovea), with area, solidity, and form factor being the most discriminatory descriptors. In the unaffected macula, aging had no significant effect on cell shape factors; however, with increasing distance to the fovea, area, solidity, and convexity increased while form factor decreased. Reduced AF in AMD was significantly associated with decreased roundness and solidity. Conclusions: AMD results in an altered RPE with enlarged and deformed cells that could precede clinically visible lesions and thus serve as early biomarkers for AMD onset. Our data may also help guide the interpretation of RPE morphology in in vivo studies utilizing high-resolution single-cell imaging. Translational Relevance: Our histologic RPE cell shape data have the ability to identify robust biomarkers for the early detection of AMD-affected cells, which also could serve as a basis for automated segmentation of RPE sheets.

Distribution of macular pigments in macular telangiectasia type 2 and correlation with optical coherence tomography characteristics and visual acuity.

BACKGROUND: To estimate macular pigment values in macular telangiectasia (MacTel) Type 2 in comparison with healthy subjects in the South Indian population across different spatial profiles and to quantify the regional differences of macular pigment optical density (MPOD) in MacTel Type 2. METHODS: In this prospective cross-sectional study, healthy controls and patients diagnosed with various stages of MacTel Type 2 underwent MPOD measurement using dual-wavelength autofluorescence technique with Spectralis HRA + OCT. RESULTS: Sixty eyes of 31 healthy subjects and 41 eyes of 22 MacTel type 2 patients were included. We found an overall decrease in MPOD values in MacTel type 2 patients (-0.109, -0.11, -0.001) in comparison with healthy subjects (0.38, 0.23, 0.06) at 1°, 2° & 6° foveal eccentricities (P < 0.001). In various stages of MacTel type 2, the mean MPOD was found to be higher in the peripheral region compared to the central region. We found a significantly lower mean MPOD in the central region in association with specific optical coherence tomography (OCT) parameters like inner retinal cavities (P = 0.035) and ellipsoid zone disruption (P = 0.034). CONCLUSIONS: In MacTel type 2, MPOD distribution varies in different spatial profiles with higher MPOD levels in the peripheral region compared to the central region. The macular pigment levels are associated with inner retinal cavities and ellipsoid zone disruption seen on OCT.

Spatial Dissociation of Subretinal Drusenoid Deposits and Impaired Scotopic and Mesopic Sensitivity in AMD.

Purpose: Subretinal drusenoid deposits (SDD) first appear in the rod-rich perifovea and can extend to the cone-rich fovea. To refine the spatial relationship of visual dysfunction with SDD burden, we determined the topography of mesopic and scotopic light sensitivity in participants with non-neovascular AMD with and without SDD. Methods: Thirty-three subjects were classified into three groups: normal (n = 9), AMD-Drusen (with drusen and without SDD; n = 12), and AMD-SDD (predominantly SDD; n = 12). Mesopic and scotopic microperimetry were performed using 68 targets within the Early Treatment Diabetic Retinopathy Study grid, including points at 1.7° from the foveal center (rod:cone ratio, 0.35). Age-adjusted linear regression was used to compare mesopic and scotopic light sensitivities across groups. Results: Across the entire Early Treatment Diabetic Retinopathy Study grid and within individual subfields, the three groups differed significantly for mesopic and scotopic light sensitivities (all P < 0.05). The AMD-SDD group exhibited significantly decreased mesopic and scotopic sensitivity versus both the normal and the AMD-Drusen groups (all P < 0.05), while AMD-Drusen and normal eyes did not significantly differ (all P > 0.05). The lowest relative sensitivities were recorded for scotopic light levels, especially in the central subfield, in the AMD-SDD group. Conclusions: SDD-associated decrements in rod-mediated vision can be detected close to the foveola, and these deficits are proportionately worse than functional loss in the rod-rich perifovea. This finding suggests that factors other than the previously hypothesized direct cytotoxicity to photoreceptors and local transport barrier limitations may negatively impact vision. Larger prospective studies are required to confirm these observations.

Histology and Clinical Lifecycle of Acquired Vitelliform Lesion, a Pathway to Advanced Age-Related Macular Degeneration.

PURPOSE: To evaluate hypotheses about the role of acquired vitelliform lesion (AVL) in age-related macular degeneration pathophysiology. DESIGN: Laboratory histology study; retrospective, observational case series. METHODS: Two donor eyes in a research archive with AVL and age-related macular degeneration were analyzed with light and electron microscopy for AVL content at locations matched to ex vivo B-scans. A retrospective, observational clinical cohort study of 42 eyes of 30 patients at 2 referral clinics determined the frequency of optical coherence tomography features stratified by AVL fate. RESULTS: Histologic and clinical cases showed subretinal drusenoid deposit and drusen. Ultrastructural AVL components in 2 donor eyes included retinal pigment epithelium (RPE) organelles (3%-22% of volume), outer segments (2%-10%), lipid droplets (0.2%-12%), and a flocculent material (57%-59%). Of 48 AVLs (mean follow-up 46 ± 39 months), 50% collapsed to complete RPE and outer retinal atrophy, 38% were stable, 10% resorbed, and 2% developed neovascularization. The Early Treatment Diabetic Retinopathy Study grid central subfield contained 77% of AVLs. Hyperreflective foci, ellipsoid zone disruption, and hyperreflective thickening of the RPE-basal lamina-Bruch membrane band were common at maximum AVL expansion. Collapsing and noncollapsing AVLs had different growth rates (rapid vs slow, respectively). CONCLUSIONS: AVL deposits contain unexpectedly low levels of RPE organelles and outer segments. Subfoveal predilection, reflectivity on optical coherence tomography, hyperautofluorescence, yellow color, and growth-regression phases suggest dysregulation of lipid transfer pathways specific to cone photoreceptors and supporting cells in formation of AVL deposit, analogous to drusen and subretinal drusenoid deposit. Prediction of AVL outcomes via growth rates should be confirmed in larger clinical studies.

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

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.

Characteristics of normal human retinal pigment epithelium cells with extremes of autofluorescence or intracellular granule count.

BACKGROUND: Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities. METHODS: RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5th or above the 95th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation. RESULTS: Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell. CONCLUSIONS: In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscins role in aging and AMD further into perspective.

Quantitative Fundus Autofluorescence in the Developing and Maturing Healthy Eye.

Purpose: Quantitative fundus autofluorescence (QAF) enables comparisons of autofluorescence intensities among participants. While clinical QAF reports mostly focused on the healthy and diseased adult retina, only very limited data of QAF in the maturing eye are available. Here, we report QAF in a large cohort of healthy children. Methods: In this prospective monocentric cross-sectional study, 70 healthy Caucasian children (5-18 years) were multimodal imaged, including QAF and spectral domain optical coherence tomography. QAF and retinal thicknesses were measured at predefined locations (along horizontal meridian; Early Treatment Diabetic Retinopathy Study [ETDRS] grid) and correlated using custom written Fiji plugins. Standard retinae for different age groups were generated. Results: Fifty-three participants were included. QAF was low in childhood but increased steadily (P < 0.001), also at the fovea (P < 0.001), with no gender differences (P = 0.61). The QAF distribution was similar to adults showing highest values superior-temporally. At individual points, retinal thickness remained stable, while using the ETDRS pattern, the retinal pigment epithelium (RPE) thickness increased significantly with aging. Standard QAF retinae of age groups also showed an increase with aging. Conclusions: QAF can be reliably performed in young children. Function-structure correlation showed a thickening of the RPE and an increasing QAF with aging, probably related to the histologic low number of RPE autofluorescent granules at a younger age but further deposition of these granules during maturation. Standard retinae help to distinguish abnormal QAF in the diseased retina of age-matched patients. Translational Relevance: Our data bridge the gap between preclinical QAF and clinical data application and structural OCT correlation in children.

Topographic Distribution and Progression of Soft Drusen Volume in Age-Related Macular Degeneration Implicate Neurobiology of Fovea.

Purpose: To refine estimates of macular soft drusen abundance in eyes with age-related macular degeneration (AMD) and evaluate hypotheses about drusen biogenesis, we investigated topographic distribution and growth rates of drusen by optical coherence tomography (OCT). We compared results to retinal features with similar topographies (cone density and macular pigment) in healthy eyes. Methods: In a prospective study, distribution and growth rates of soft drusen in eyes with AMD were identified by human observers in OCT volumes and analyzed with computer-assistance. Published histologic data for macular cone densities (n = 12 eyes) and in vivo macular pigment optical density (MPOD) measurements in older adults with unremarkable maculae (n = 31; 62 paired eyes, averaged) were revisited. All values were normalized to Early Treatment Diabetic Retinopathy Study (ETDRS) subfield areas. Results: Sixty-two eyes of 44 patients were imaged for periods up to 78 months. Soft drusen volume per unit volume at baseline is 24.6-fold and 2.3-fold higher in the central ETDRS subfield than in outer and inner rings, respectively, and grows most prominently there. Corresponding ratios (central versus inner and central versus outer) for cone density in donor eyes is 13.3-fold and 5.1-fold and for MPOD, 24.6 and 23.9-fold, and 3.6 and 3.6-fold. Conclusions: Normalized soft drusen volume in AMD eyes as assessed by OCT is ≥ 20-fold higher in central ETDRS subfields than in outer rings, paralleling MPOD distribution in healthy eyes. Data on drusen volume support this metric for AMD risk assessment and clinical trial outcome measure. Alignment of different data modalities support the ETDRS grid for standardizing retinal topography in mechanistic studies of drusen biogenesis.