Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function.

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (APOB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic depletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor-associated cholesterol deposits, and photoreceptor cell death, and loss of rod but not cone function. RPE-specific reduction in Mttp had no significant effect on plasma lipids and lipoproteins. While APOB was decreased in the RPE, most ocular retinoids remained unchanged, with the exception of the storage form of retinoid, retinyl ester. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but is not directly involved in plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

Shotgun lipidomics of human subretinal fluids under rod-dominant retina reveals cone-dominated lipids.

Subretinal fluid (SRF) accumulates between photoreceptor outer segments and retinal pigment epithelium during rhegmatogenous retinal detachment. Biomolecular components such as lipids originate from cells surrounding the SRF. Knowledge of the composition of these molecules in SRF potentially provides mechanistic insight into the physiologic transfer of lipids between retinal tissue compartments. Using mass spectrometry and tandem mass spectrometry analysis on an electrospray ionization quadrupole-time-of-flight mass spectrometer, we identified a total of 115 lipid molecular species of 11 subclasses and 9 classes in two samples from two patients with rhegmatogenous retinal detachment. These included 47 glycerophosphocholines, 6 glycerophosphoethanolamines, 1 glycerophosphoinositol, 18 sphingomyelins, 9 cholesteryl esters, free cholesterol, 3 ceramides, 22 triacylglycerols and 8 free fatty acids. Glycerophosphocholines were of the highest intensity. By minimizing the formation of different adduct forms or clustering ions of different adducts, we determined the relative intensity of lipid molecular species within the same subclasses. The profiles were compared with those of retinal cells available in the published literature. The glycerophosphocholine profile of SRF was similar to that of cone outer segments, suggesting that outer segment degradation products are constitutively released into the interphotoreceptor matrix, appearing in SRF during detachment. This hypothesis was supported by the retinal distributions of corresponding lipid synthases' mRNA expression obtained from an online resource based on publicly available single-cell sequencing data. In contrast, based on lipid profiles and relevant gene expression in this study, the sources of free cholesterol and cholesteryl esters in SRF appeared more ambiguous, possibly reflecting that outer retina takes up plasma lipoproteins. Further studies to identify and quantify lipids in SRF will help better understand etiology of diseases relevant to outer retina.

Quantitative Autofluorescence at AMD's Beginnings Highlights Retinal Topography and Grading System Differences: ALSTAR2 Baseline.

INTRODUCTION: The aims of the study were to describe baseline quantitative (short-wavelength) autofluorescence (qAF) findings in a large pseudophakic cohort at age-related macular degeneration (AMD)'s beginnings and to assess qAF8 as an outcome measure and evaluate Age-Related Eye Disease Study (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 AMD (eAMD) (N = 104, 66), and intermediate AMD (iAMD) (N = 82, 122). Groups were compared via mean qAF intensities in a 6°-8° annulus (qAF8) and maps of differences between observations and the 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.

HISTOLOGY, DIMENSIONS, AND FLUORESCEIN STAINING CHARACTERISTICS OF NODULAR AND CUTICULAR DRUSEN IN AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To enable in vivo analysis of drusen composition and lifecycle, the macular nodular and cuticular drusen were assessed using histology. METHODS: Median and interquartile range of base widths of single (nonconfluent) nodular drusen in three sources were determined histologically: 43 eyes of 43 clinically undocumented donors, in an online resource; one eye with punctate hyperfluorescence in fluorescein angiography; and two eyes of one patient with bilateral "starry sky" cuticular drusen. All tissues were processed for high-resolution epoxy-resin histology and for cuticular drusen, transmission electron microscopy. RESULTS: All drusen localized between the retinal pigment epithelium basal lamina and inner collagenous layer of the Bruch membrane. They were solid, globular, homogeneously stained with toluidine blue, and uncovered by basal laminar deposit and basal mounds. Median base widths were 13.0 µ m (Source 1, N = 128 drusen, interquartile range 7.7, 20.0 µ m), 15.3 µ m (Source 2, N = 87, interquartile range 10.6, 20.5 µ m), and 7.3 µ m (Source 3, N = 78, interquartile range 3.9, 14.1 µ m). CONCLUSION: In three samples, >90% of solitary nodular drusen were <30 µ m, the visibility threshold in color fundus photography; these drusen are hyperfluorescent in fluorescein angiography. Whether these progress to soft drusen, known as high-risk from epidemiology studies and hypofluorescent, may be determinable from multimodal imaging datasets that include fluorescein angiography.

INFLAMMATORY CELL ACTIVITY IN TREATED NEOVASCULAR AGE-RELATED MACULAR DEGENERATION: A Histologic Case Study.

BACKGROUND: Imaging indicators of macular neovascularization risk can help determine patient eligibility for new treatments for geographic atrophy secondary to age-related macular degeneration. Because type 1 macular neovascularization includes inflammation, we assessed by histology the distribution of cells with inflammatory potential in two fellow eyes with age-related macular degeneration. METHODS: Two eyes of a White woman in her 90's with type 3 macular neovascularization treated with antivascular endothelial growth factor were prepared for high-resolution histology. Eye-tracked spectral domain optical coherence tomography applied to the preserved donor eyes linked in vivo imaging to histology. Cells were enumerated in the intraretinal, subretinal, and subretinal retinal pigment epithelium (RPE)-basal lamina compartments on 199 glass slides. Cells with numerous organelles were considered to RPE-derived; cells with sparse RPE organelles were considered non-RPE phagocytes. RESULTS: Both eyes had soft drusen and abundant subretinal drusenoid deposit. In the retina and subretinal space, RPE-derived cells, including hyperreflective foci, were common (n = 125 and 73, respectively). Non-RPE phagocytes were infrequent (n = 5 in both). Over drusen, RPE morphology transitioned smoothly from the age-normal layer toward the top, suggesting transdifferentiation. The sub-RPE-basal lamina space had RPE-derived cells (n = 87) and non-RPE phagocytes (n = 49), including macrophages and giant cells. CONCLUSION: Numerous sub-RPE-basal lamina cells of several types are consistent with the documented presence of proinflammatory lipids in drusen and aged Bruch's membrane. The relatively compartmentalized abundance of infiltrating cells suggests that drusen contents are more inflammatory than subretinal drusenoid deposit, perhaps reflecting their environments. Ectopic RPE occurs frequently. Some manifest as hyperreflective foci. More cells may be visible as optical coherence tomography technologies evolve.

Untargeted Lipidomic Profiling of Aged Human Retina With and Without Age-Related Macular Degeneration (AMD).

The molecular characterization of extracellular deposits is crucial to understanding the clinical progression of AMD. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is a powerful analytical discovery tool capable of identifying lipids in an untargeted manner. NanoLC-MS/MS is an analytical tool capable of identifying lipids with high sensitivity and minimum sample usage. Hence, the purpose of this study was to compare retina lipid identification from RPE-choroid samples using high flow LC-MS/MS and nanoLC-MS/MS. Manually dissected paraformaldehyde-fixed human donor tissues sections were used for LC-MS/MS and nanoLC-MS/MS analysis. Lipids were extracted with MeOH/MTBE/CHCl3 (MMC) and were analyzed by LC-MS/MS and nanoLC-MS/MS using negative and positive ionization modes. Untargeted lipidomics using LC-MS/MS identified 215 lipids from 4 lipid classes and 15 subclasses. We observed a 78% increase in lipid identifications using nanoLC-MS/MS with lipid numbers totaling 384. The nanoLC-MS/MS method is expected to provide extensive lipid identifications from small retina samples, e.g., from drusen and drusenoid deposits in aged and AMD eyes, and could help elucidate how lipids are involved in extracellular deposit formation in AMD.

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal Laminar Deposit of Age-Related Macular Degeneration.

Pathologies of the retina are clinically visualized in vivo with OCT and ex vivo with immunohistochemistry. Although both techniques provide valuable information on prognosis and disease state, a comprehensive method for fully elucidating molecular constituents present in locations of interest is desirable. The purpose of this work was to use multimodal imaging technologies to localize the vast number of molecular species observed with matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) in aged and diseased retinal tissues. Herein, MALDI IMS was utilized to observe molecular species that reside in photoreceptor cells and also a basal laminar deposit from two human donor eyes. The molecular species observed to accumulate in these discrete regions can be further identified and studied to attempt to gain a greater understanding of biological processes occurring in debilitating eye diseases such as age-related macular degeneration (AMD).

Transthyretin proteoforms of intraocular origin in human subretinal fluid.

Understanding the molecular composition of ocular tissues and fluids could inform new approaches to prevalent causes of blindness. Subretinal fluid accumulating between the photoreceptor outer segments and retinal pigment epithelium (RPE) is potentially a rich source of proteins and lipids normally cycling among outer retinal cells and choroid. Herein, intact post-translationally modified proteins (proteoforms) were extracted from subretinal fluids of five patients with rhegmatogenous retinal detachment (RRD), analyzed by tandem mass spectrometry, and compared to published data on these same proteins as synthesized by other organs. Single-nuclei transcriptomic data from non-diseased human retina/RPE were used to identify whether proteins in subretinal fluid were of potential ocular origin. Two human donor eyes with normal maculas were immunoprobed for transthyretin (TTR) with appropriate controls. The three most abundant proteins detected in subretinal fluid were albumin, TTR, and apolipoprotein A-I. Remarkably, TTR relative to the other proteins was more abundant than its serum counterpart, suggestive of TTR being synthesized predominantly locally. Six proteoforms of TTR were detected, with the relative amount of glutathionylated TTR being much higher in the subretinal fluid (12-43%) than values reported for serum (<5%) and cerebrospinal fluid (0.4-13%). Moreover, a putative glycosylated TTR dimer of 32,428 Da was detected as the fourth most abundant protein. The high abundance of TTR and putative TTR dimer in subretinal fluid was supported by analysis of available single-nuclei transcriptomic data, which showed strong and specific signal for TTR in RPE. Immunohistochemistry further showed strong diffuse TTR immunoreactivity in choroidal stroma that contrasted with vertically aligned signal in the outer segment zone of the subretinal space and negligible signal in RPE cell bodies. These results suggest that TTR in the retina is synthesized intraocularly, and glutathionylation is crucial for its normal function. Further studies on the composition, function, and quantities of TTR and other proteoforms in subretinal fluid could inform mechanisms, diagnostic methods, and treatment strategies for age-related macular degeneration, familial amyloidosis, and other retinal diseases involving dysregulation of physiologic lipid transfer and oxidative stress.

Histology and clinical imaging lifecycle of black pigment in fibrosis secondary to neovascular age-related macular degeneration.

PURPOSE: Melanotic cells with large spherical melanosomes, thought to originate from retinal pigment epithelium (RPE), are found in eyes with neovascular age-related macular degeneration (nvAMD). To generate hypotheses about RPE participation in fibrosis, we correlate histology to clinical imaging in an eye with prominent black pigment in fibrotic scar secondary to nvAMD. METHODS: Macular findings in a white woman with untreated inactive subretinal fibrosis due to nvAMD in her right eye were documented over 9 years with color fundus photography (CFP), fundus autofluorescence (FAF) imaging, and optical coherence tomography (OCT). After death (age 90 years), this index eye was prepared for light and electron microscopy to analyze 7 discrete zones of pigmentation in the fibrotic scar. In additional donor eyes with nvAMD, we determined the frequency of black pigment (n = 36 eyes) and immuno-labeled for retinoid, immunologic, and microglial markers (RPE65, CD68, Iba1, TMEM119; n = 3 eyes). RESULTS: During follow-up of the index eye, black pigment appeared and expanded within a hypoautofluorescent fibrotic scar. The blackest areas correlated to melanotic cells (containing large spherical melanosomes), some in multiple layers. Pale areas had sparse pigmented cells. Gray areas correlated to cells with RPE organelles entombed in the scar and multinucleate cells containing sparse large spherical melanosomes. In 94% of nvAMD donor eyes, hyperpigmentation was visible. Certain melanotic cells expressed some RPE65 and mostly CD68. Iba1 and TMEM119 immunoreactivity, found both in retina and scar, did not co-localize with melanotic cells. CONCLUSION: Hyperpigmentation in CFP results from both organelle content and optical superimposition effects. Black fundus pigment in nvAMD is common and corresponds to cells containing numerous large spherical melanosomes and superimposition of cells containing sparse large melanosomes, respectively. Melanotic cells are molecularly distinct from RPE, consistent with a process of transdifferentiation. The subcellular source of spherical melanosomes remains to be determined. Detailed histology of nvAMD eyes will inform future studies using technologies for spatially resolved molecular discovery to generate new therapies for fibrosis. The potential of black pigment as a biomarker for fibrosis can be investigated in clinical multimodal imaging datasets.

Inside out: Relations between the microbiome, nutrition, and eye health.

Age-related macular degeneration (AMD) is a complex disease with increasing numbers of individuals being afflicted and treatment modalities limited. There are strong interactions between diet, age, the metabolome, and gut microbiota, and all of these have roles in the pathogenesis of AMD. Communication axes exist between the gut microbiota and the eye, therefore, knowing how the microbiota influences the host metabolism during aging could guide a better understanding of AMD pathogenesis. While considerable experimental evidence exists for a diet-gut-eye axis from murine models of human ocular diseases, human diet-microbiome-metabolome studies are needed to elucidate changes in the gut microbiome at the taxonomic and functional levels that are functionally related to ocular pathology. Such studies will reveal new ways to diminish risk for progression of- or incidence of- AMD. Current data suggest that consuming diets rich in dark fish, fruits, vegetables, and low in glycemic index are most retina-healthful during aging.

Visual outcomes of macula-involving rhegmatogenous retinal detachment in patients with and without age-related macular degeneration.

BACKGROUND: The prognosis for patients with macula-off rhegmatogenous retinal detachment (RRD) and concomitant age-related macular degeneration (AMD) is not well known. The purpose of this study is to compare visual outcomes in macula-off RRD in eyes with AMD versus a group of comparison eyes without AMD. METHODS: This was a retrospective chart review of 1149 patients. A total of 191 eyes met study criteria, 162 non-AMD eyes (controls), and 29 AMD eyes. The main outcome measure was postoperative visual acuity following pars plana vitrectomy (PPV), scleral buckle (SB), or combined PPV/SB in control eyes versus AMD eyes. This was compared using Fisher's exact test. RESULTS: There was a statistically significant difference in postoperative visual acuity by AMD status, with those without AMD having a worse visual outcome overall (p = 0.0048). A similar percentage of AMD versus non-AMD eyes achieved vision better than 20/40. More patients in the non-AMD group achieved a final visual acuity between 20/40 and 20/200. Of patients with AMD, more had vision worse than 20/200 though 58% maintained functional vision (better than 20/200). Those without AMD had a higher frequency of Count Fingers (CF), Hand Motion (HM), Light Perception (LP), or No Light Perception (NLP) vision (p = 0.023). CONCLUSIONS: Though postoperative visual acuity was worse overall in the non-AMD group with a higher frequency of patients having final vision of CF, HM, LP, or NLP, this is likely a function of the difference in sample size and composition between the two groups. Importantly, this study suggests AMD patients can expect similar outcomes to non-AMD patients after RRD repair. We conclude that AMD patients can achieve functional vision after RRD surgery, similar to those without AMD.

BACILLARY LAYER DETACHMENT: MULTIMODAL IMAGING AND HISTOLOGIC EVIDENCE OF A NOVEL OPTICAL COHERENCE TOMOGRAPHY TERMINOLOGY: Literature Review and Proposed Theory.

PURPOSE: To clarify the histologic basis of bacillary layer detachment (BALAD) through a review of the current literature and an analysis of retinal imaging. METHODS: The literature for previous reports of BALAD were reviewed. An analysis of retinal images was performed to support anatomical conclusions. RESULTS: A total of 164 unique patients with BALAD on optical coherence tomography (OCT) were identified from the published literature. Twenty-two underlying etiologies, all associated with subretinal exudation, were identified. Forty-one different OCT terminologies were found. The defining OCT feature of BALAD was a split at the level of the photoreceptor inner segment myoid creating a distinctive intraretinal cavity. Resolution of BALAD was followed by a rapid restoration of the ellipsoid zone. Histology of age-related macular degeneration eyes suggests that individual photoreceptors can shed inner segments. Furthermore, detachment of the entire layer of inner segments is a common postmortem artifact. It is proposed that BALAD occurs when outwardly directed forces promoting attachment of photoreceptor outer segments to the retinal pigment epithelium exceed the tensile strength of the photoreceptor inner segment myoid. CONCLUSION: This review serves to strengthen the OCT nomenclature "bacillary layer detachment," based on specific reflectance information obtained by OCT and previously published histologic observations.

Erythropsia and Chromatopsia: Case Study and Brief Review.

A 65-year-old woman presented with erythropsia (red-tinged vision) in the right eye from a subfoveal macula dehaemoglobinised intraretinal haemorrhage. Erythropsia is a type of chromatopsia, a condition in which objects appear to be abnormally coloured or tinged with colour. This manuscript provides a brief review of colour vision abnormalities including chromatopsia, and additionally we discuss dyschromatopsia and achromatopsia defined as deficiency and absence of colour vision respectively, both of which may be congenital or acquired. We theorise that the mechanism of the chromatopsia may be selective damage of ganglion cells involved in colour opponency.

Nonexudative Macular Neovascularization Supporting Outer Retina in Age-Related Macular Degeneration: A Clinicopathologic Correlation.

PURPOSE: Type 1 macular neovascularization (MNV) secondary to age-related macular degeneration (AMD) may sustain hypoxic and micronutrient-insufficient outer retinal cells compensatorily. We explored this hypothesis via histologic analysis of an eye with a shallow irregular retinal pigment epithelial elevation (SIRE) on OCT and good vision. DESIGN: Case study and clinicopathologic correlation. PARTICIPANT: A white woman with untreated nonexudative neovascular AMD and 20/30 visual acuity (left eye) and neovascular AMD (right eye), with 9 years' multimodal imaging before dying at 90 years of age. METHODS: The left eye was preserved 6.25 hours after death and prepared for submicrometer epoxy resin sections and transmission electron microscopy aligned to clinical OCT B-scans. Inside and outside the MNV area, layer thicknesses, phenotypes, and vascular density of native choriocapillaris and neovessels were measured. Lengths of choriocapillaries and intervening gaps in the index eye and in early AMD eyes and healthy eyes with similar age (n = 19 each) from the Project MACULA (Maculopathy Unveiled by Laminar Analysis) online histopathologic resource (http://projectmacula.cis.uab.edu/) were measured with custom software (Caps and Gaps). MAIN OUTCOME MEASURES: Descriptive features, vascular density, histologic and OCT layer thicknesses, and distribution of choriocapillaries and intervening gaps. RESULTS: The SIRE correlated to a type 1 MNV that expanded slowly without evidence of exudation and with numerous choroidal vessels traversing Bruch's membrane defects, some visible on OCT. Tissue layers in and adjacent to the MNV area showed continuous RPE and characteristic AMD deposits. Capillary-like neovessels with fenestrations and caveolae resembling native choriocapillaris lined the retinal pigment epithelium (RPE) with a vascular density comparable with surrounding non-MNV areas. Relative to early AMD and healthy aged eyes, the index eye showed similar capillary lengths but larger gaps between vessels, indicating dropout. Outer nuclear layer thickness was preserved and showed less photoreceptor degeneration over areas of relative choriocapillaris health, including the type 1 MNV. CONCLUSIONS: Eyes with nonexudative type 1 MNV in AMD may progress to exudation, yet this stable MNV complex supported outer retinal structure for 9 years. Distinguishing features were numerous connecting vessels, high density of neovessels, continuous RPE, and slow growth. Maintaining beneficial type 1 MNV may be a therapeutic strategy.

Incomplete Retinal Pigment Epithelial and Outer Retinal Atrophy in Age-Related Macular Degeneration: Classification of Atrophy Meeting Report 4.

PURPOSE: To describe the defining features of incomplete retinal pigment epithelium (RPE) and outer retinal atrophy (iRORA), a consensus term referring to the OCT-based anatomic changes often identified before the development of complete RPE and outer retinal atrophy (cRORA) in age-related macular degeneration (AMD). We provide descriptive OCT and histologic examples of disease progression. DESIGN: Consensus meeting. PARTICIPANTS: Panel of retina specialists, including retinal imaging experts, reading center leaders, and retinal histologists. METHODS: As part of the Classification of Atrophy Meeting (CAM) program, an international group of experts analyzed and discussed longitudinal multimodal imaging of eyes with AMD. Consensus was reached on a classification system for OCT-based structural alterations that occurred before the development of atrophy secondary to AMD. New terms of iRORA and cRORA were defined. This report describes in detail the CAM consensus on iRORA. MAIN OUTCOME MEASURES: Defining the term iRORA through OCT imaging and longitudinal cases showing progression of atrophy, with histologic correlates. RESULTS: OCT was used in cases of early and intermediate AMD as the base imaging method to identify cases of iRORA. In the context of drusen, iRORA is defined on OCT as (1) a region of signal hypertransmission into the choroid, (2) a corresponding zone of attenuation or disruption of the RPE, and (3) evidence of overlying photoreceptor degeneration. The term iRORA should not be used when there is an RPE tear. Longitudinal studies confirmed the concept of progression from iRORA to cRORA. CONCLUSIONS: An international consensus classification for OCT-defined anatomic features of iRORA are described and examples of longitudinal progression to cRORA are provided. The ability to identify these OCT changes reproducibly is essential to understand better the natural history of the disease, to identify high-risk signs of progression, and to study early interventions. Longitudinal data are required to quantify the implied risk of vision loss associated with these terms. The CAM classification provides initial definitions to enable these future endeavors, acknowledging that the classification will be refined as new data are generated.

Linear and planar reflection artifacts on swept-source and spectral-domain optical coherence tomography due to hyperreflective crystalline deposits.

PURPOSE: To describe novel spectral-domain (SD) and swept-source (SS) optical coherence tomography (OCT) linear and planar reflection artifacts produced by hyperreflective crystalline deposits (HCD). METHODS: Imaging from 10 eyes with HCD producing linear and planar artifacts on OCT was retrospectively analyzed. All eyes had SD-OCT (Spectralis HRA + OCT, Heidelberg Engineering, Germany) and SS-OCT angiography (PLEX Elite 9000, Carl Zeiss Meditec, Inc., Dublin, CA) acquired on the same day. The horizontal extent of planar artifacts and the corresponding HCD on B-scans was measured using a digital caliper. Artifact features from HCD in eyes with non-neovascular age-related macular degeneration (AMD) were analyzed and compared to those seen in two eyes with the "onion sign," an OCT signature previously shown to represent cholesterol crystals (CC) in the sub-retinal pigment epithelium-basal laminar space of eyes with neovascular AMD. A third eye with the "onion sign" was imaged with dense B-scan (DB)-OCTA. RESULTS: Ten eyes of ten patients (77.4 ± 8.7 years) with HCD were analyzed. On SS-OCTA, HCD produced linear artifacts of high signal intensity passing through the HCD and spanning the entire scan depth. On SD-OCT, HCD produced planar artifacts located anterior to both the retina and a hyporeflective space representing normal vitreous signal. The horizontal extent of the artifact did not differ significantly from the corresponding HCD on OCT B-scans (P = 0.62). The OCT artifacts produced by the "onion sign" appeared similar to those of HCD. The additional eye with neovascular AMD imaged with DB-OCTA was characterized by a single, vertical, linear false-flow signal crossing retinal layers. CONCLUSIONS: To the authors' knowledge, this is the first description of SD- and SS-OCT/OCTA artifacts corresponding to both HCD and the "onion sign." These artifacts are likely due to highly reflective CC previously shown on histology to correspond to both of these OCT signatures.

SUBRETINAL DRUSENOID DEPOSIT IN AGE-RELATED MACULAR DEGENERATION: Histologic Insights Into Initiation, Progression to Atrophy, and Imaging.

PURPOSE: To clarify the role of subretinal drusenoid deposits (SDD; pseudodrusen) in the progression of age-related macular degeneration through high-resolution histology. METHODS: In 33 eyes of 32 donors (early age-related macular degeneration, n = 15; geographic atrophy, n = 9; neovascular age-related macular degeneration, n = 7; unremarkable, n = 2), and 2 eyes of 2 donors with in vivo multimodal imaging including optical coherence tomography, examples of SDD contacting photoreceptors were assessed. RESULTS: Subretinal drusenoid deposits were granular extracellular deposits at the apical retinal pigment epithelium (RPE); the smallest were 4-µm wide. Outer segment (OS) fragments and RPE organelles appeared in some larger deposits. A continuum of photoreceptor degeneration included OS disruption, intrusion into inner segments, and disturbance of neurosensory retina. In a transition to outer retinal atrophy, SDD appeared to shrink, OS disappeared, inner segment shortened, and the outer nuclear layer thinned and became gliotic. Stage 1 SDD on optical coherence tomography correlated with displaced OS. Confluent and disintegrating Stage 2 to 3 SDD on optical coherence tomography and dot pseudodrusen by color fundus photography correlated with confluent deposits and ectopic RPE. CONCLUSION: Subretinal drusenoid deposits may start at the RPE as granular, extracellular deposits. Photoreceptor OS, RPE organelles, and cell bodies may appear in some advanced deposits. A progression to atrophy associated with deposit diminution was confirmed. Findings support a biogenesis hypothesis of outer retinal lipid cycling.

ABUNDANCE AND MULTIMODAL VISIBILITY OF SOFT DRUSEN IN EARLY AGE-RELATED MACULAR DEGENERATION: A Clinicopathologic Correlation.

PURPOSE: To determine the abundance and multimodal visibility of drusen and basal linear deposit (BLinD) in early age-related macular degeneration. METHODS: A 69-year-old white man was imaged by color fundus photography and red free photography, fundus autofluorescence, and optical coherence tomography. From en face images, we determined the drusen field, drusen area, and equivalent diameters of individual drusen. From high-resolution light-microscopic histology (6 months after the last clinic visit), we determined the area of drusen, BLinD, and pre-BLinD in a subretinal pigment epithelium-basal lamina lipid field. RESULTS: In right and left eyes, respectively, BLinD covered 40% and 46% of the lipid field, versus 21% and 14% covered by drusen. The lipid field was covered 60% to 61% by Drusen + BLinD and 65% to 72% by BLinD + pre-BLinD. In the left eye, the drusen area on color fundus photography (0.18 mm) and red free (0.28 mm) was smaller than the drusen area on histology (1.16 mm). Among drusen confirmed by optical coherence tomography, 55.1% and 56.6% were observed on red free and fundus autofluorescence, respectively. CONCLUSION: Basal linear deposit covered 1.9 and 3.4-fold more fundus area than soft drusen, silently increasing progression risk. Improved visualization of BLinD and readouts of the retinal pigment epithelium health over lipid will assist population surveillance, early detection, and trial outcome measures.

Functionally validated imaging endpoints in the Alabama study on early age-related macular degeneration 2 (ALSTAR2): design and methods.

BACKGROUND: Age-related macular degeneration (AMD), a leading cause of irreversible vision impairment in the United States and globally, is a disease of the photoreceptor support system involving the retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris in the setting of characteristic extracellular deposits between outer retinal cells and their blood supply. Research has clearly documented the selective vulnerability of rod photoreceptors and rod-mediated (scotopic) vision in early AMD, including delayed rod-mediated dark adaptation (RMDA) and impaired rod-mediated light and pattern sensitivity. The unifying hypothesis of the Alabama Study on Early Macular Degeneration (ALSTAR2) is that early AMD is a disease of micronutrient deficiency and vascular insufficiency, due to detectable structural changes in the retinoid re-supply route from the choriocapillaris to the photoreceptors. Functionally this is manifest as delayed rod-mediated dark adaptation and eventually as rod-mediated visual dysfunction in general. METHODS: A cohort of 480 older adults either in normal macular health or with early AMD will be enrolled and followed for 3 years to examine cross-sectional and longitudinal associations between structural and functional characteristics of AMD. Using spectral domain optical coherence tomography, the association between (1) subretinal drusenoid deposits and drusen, (2) RPE cell bodies, and (3) the choriocapillaris' vascular density and rod- and cone-mediated vision will be examined. An accurate map and timeline of structure-function relationships in aging and early AMD gained from ALSTAR2, especially the critical transition from aging to disease, will identify major characteristics relevant to future treatments and preventative measures. DISCUSSION: A major barrier to developing treatments and prevention strategies for early AMD is a limited understanding of the temporal interrelationships among structural and functional characteristics while transitioning from aging to early AMD. ALSTAR2 will enable the development of functionally valid, structural biomarkers for early AMD, suitable for use in forthcoming clinical trials as endpoint/outcome measures. The comprehensive dataset will also allow hypothesis-testing for mechanisms that underlie the transition from aging to AMD, one of which is a newly developed Center-Surround model of cone resilience and rod vulnerability. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT04112667, October 7, 2019.

RNA expression in human retina.

Recent Genome-wide Association Studies (GWASs) for eye diseases/traits have delivered a number of novel findings across a diverse range of diseases, including age-related macular degeneration (AMD), glaucoma and refractive error. However, despite this astonishing rate of success, the major challenge still remains to not only confirm that the genes implicated in these studies are truly the genes conferring protection from or risk of disease but also to define the functional roles these genes play in disease. Ongoing evidence is accumulating that the single nucleotide polymorphisms (SNPs) used in GWAS and fine mapping studies have causal effects through their influence on gene expression rather than affecting protein function. The biological interpretation of SNP regulatory effects for a tissue requires knowledge of the transcriptome for that tissue. We summarize the reasons to characterize the complete retinal transcriptome as well as the evidence to include an assessment of differences in regional retinal expression.