Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged ≥80 years old, with a death-to-preservation time (DtoP) of ≤6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30° macula cube, 30 µm spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 ± 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Implication of specific retinal cell-type involvement and gene expression changes in AMD progression using integrative analysis of single-cell and bulk RNA-seq profiling.

Age-related macular degeneration (AMD) is a blinding eye disease with no unifying theme for its etiology. We used single-cell RNA sequencing to analyze the transcriptomes of ~ 93,000 cells from the macula and peripheral retina from two adult human donors and bulk RNA sequencing from fifteen adult human donors with and without AMD. Analysis of our single-cell data identified 267 cell-type-specific genes. Comparison of macula and peripheral retinal regions found no cell-type differences but did identify 50 differentially expressed genes (DEGs) with about 1/3 expressed in cones. Integration of our single-cell data with bulk RNA sequencing data from normal and AMD donors showed compositional changes more pronounced in macula in rods, microglia, endothelium, Müller glia, and astrocytes in the transition from normal to advanced AMD. KEGG pathway analysis of our normal vs. advanced AMD eyes identified enrichment in complement and coagulation pathways, antigen presentation, tissue remodeling, and signaling pathways including PI3K-Akt, NOD-like, Toll-like, and Rap1. These results showcase the use of single-cell RNA sequencing to infer cell-type compositional and cell-type-specific gene expression changes in intact bulk tissue and provide a foundation for investigating molecular mechanisms of retinal disease that lead to new therapeutic targets.

Comprehensive analysis of gene expression in human retina and supporting tissues.

Understanding the influence of gene expression on the molecular mechanisms underpinning human phenotypic diversity is fundamental to being able to predict health outcomes and treat disease. We have carried out whole transcriptome expression analysis on a series of eight normal human postmortem eyes by RNA sequencing. Here we present data showing that ∼80% of the transcriptome is expressed in the posterior layers of the eye and that there is significant differential expression not only between the layers of the posterior part of the eye but also between locations of a tissue layer. These differences in expression also extend to alternative splicing and splicing factors. Differentially expressed genes are enriched for genes associated with psychiatric, immune and cardiovascular disorders. Enrichment categories for gene ontology included ion transport, synaptic transmission and visual and sensory perception. Lastly, allele-specific expression was found to be significant for CFH, C3 and CFB, which are known risk genes for age-related macular degeneration. These expression differences should be useful in determining the underlying biology of associations with common diseases of the human retina, retinal pigment epithelium and choroid and in guiding the analysis of the genomic regions involved in the control of normal gene expression.

Quantification of fluorescein-stained drusen associated with age-related macular degeneration.

BACKGROUND: Previous studies of age-related macular degeneration have not quantified the number of drusen that accumulate fluorescein. Histopathologic studies have demonstrated druse subregions with different degrees of hydrophobicity, and these subregions might potentially exhibit different degrees of fluorescein uptake. METHODS: We evaluated macular drusen from 35 age-related macular degeneration patients by measuring druse area in color digital images and fluorescein angiograms, using 2 morphometric methods. RESULTS: Of 828 drusen evaluated, 405 had a corresponding fluorescein angiogram signal. About half of all drusen per eye (49.57%) stained in each participant. Among fluorescein-stained drusen, druse size measured in color images did not differ significantly from the sizes measured in corresponding fluorescein images (P = 0.8105), across the range of druse sizes. CONCLUSION: These findings indicate that our understanding of drusen subregion staining may not directly correlate to in vivo observations of macular drusen in age-related macular degeneration.

Retinal pigment epithelial expression of complement regulator CD46 is altered early in the course of geographic atrophy.

In geographic atrophy (GA), the non-neovascular end stage of age-related macular degeneration (AMD), the macular retinal pigment epithelium (RPE) progressively degenerates. Membrane cofactor protein (MCP, CD46) is the only membrane-bound regulator of complement expressed on the human RPE basolateral surface. Based on evidence of the role of complement in AMD, we hypothesized that altered CD46 expression on the RPE would be associated with GA development and/or progression. Here we report the timeline of CD46 protein expression changes across the GA transition zone, relative to control eyes, and relative to events in other chorioretinal layers. Eleven donor eyes (mean age 87.0 ± 4.1 yr) with GA and 5 control eyes (mean age 84.0 ± 8.9 yr) without GA were evaluated. Macular cryosections were stained with PASH for basal deposits, von Kossa for calcium, and for CD46 immunoreactivity. Internal controls for protein expression were provided by an independent basolateral protein, monocarboxylate transporter 3 (MCT3) and an apical protein, ezrin. Within zones defined by 8 different semi-quantitative grades of RPE morphology, we determined the location and intensity of immunoreactivity, outer segment length, and Bruch's membrane calcification. Differences between GA and control eyes and between milder and more severe RPE stages in GA eyes were assessed statistically. Increasing grades of RPE degeneration were associated with progressive loss of polarity and loss of intensity of staining of CD46, beginning with the stages that are considered normal aging (grades 0-1). Those GA stages with affected CD46 immunoreactivity exhibited basal laminar deposit, still-normal photoreceptors, and concomitant changes in control protein expression. Activated or anteriorly migrated RPE (grades 2-3) exhibited greatly diminished CD46. Changes in RPE CD46 expression thus occur early in GA, before there is evidence of morphological RPE change. At later stages of degeneration, CD46 alterations occur within a context of altered RPE polarity. These changes precede degeneration of the overlying retina and suggest that therapeutic interventions be targeted to the RPE.

Imaging of Dalen-Fuchs Nodules in a Likely Case of Sympathetic Ophthalmia by Fluorescein Angiography and OCT.

Sympathetic ophthalmia (SO) is a well-known but rare autoimmune disease in which the sympathizing eye suffers granulomatous panuveitis after trauma to the fellow eye. An unusual case of SO occurring 32 years after trauma to the fellow eye, and 1 year after unsuccessful vitrectomy/scleral buckle repair of an acute retinal detachment in the inciting eye was presented. An optical coherence tomography imagery of Dalen-Fuchs nodules, not previously reported, and rare angiographic imaging of SO in its acute phase was demonstrated.

Branch retinal artery occlusion: visual prognosis.

PURPOSE: To evaluate the visual prognosis in eyes with branch retinal artery occlusion (BRAO). DESIGN: Retrospective, observational, consecutive case series. METHODS: Retrospective medical record review of 52 consecutive eyes of 52 patients with BRAO seen at two vitreoretinal practices in Birmingham, Alabama. Only eyes with decreased central macular perfusion on fluorescein angiography (FA) were included. Eyes with anterior segment or optic nerve disease, lack of retinal whitening or a delay in arterial filling on FA, central retinal artery occlusion, and cilioretinal artery occlusion were excluded. The main outcome measure was presenting best-corrected visual acuity (BCVA) and its relationship to final BCVA. RESULTS: On presentation, 54% of eyes with BRAO had BCVA of 20/40 or better. At the mean 14-month visit, 60% of all eyes had visual acuity (VA) of 20/40 or better. VAs of 20/40 or better were retained by 89% of eyes with baseline BCVA of 20/40 or better. Only 14% of eyes with 20/100 or worse BCVA improved to 20/40 or better. Neither visible emboli (P > or = .244) nor the region of macular involvement (P = .142) were significant with respect to visual improvement. CONCLUSIONS: Visual prognosis after BRAO seems to be correlated to presenting VA. Eyes with initial VA of 20/40 or better usually remained at 20/40 or better. Individuals with poor VA of 20/100 or worse generally did not show the significant improvement reported in previous studies.

Clinical examination accurately locates capillary nonperfusion in diabetic retinopathy.

PURPOSE: To help the ophthalmologist who wishes to know the perfusion status of the retina outside the macula, we determined whether clinical examination can predict capillary nonperfusion in diabetic retinopathy (DR) as accurately as fluorescein angiography (FA). DESIGN: Prospective observational case series. SETTING: Clinical practice. STUDY POPULATION: Both eyes of 20 patients with clinically significant diabetic macular edema (CSME). OBSERVATION PROCEDURE: The location of capillary nonperfusion was recorded following 90-diopter lens slit-lamp biomicroscopy, and the same measurements made on FA. MAIN OUTCOME MEASURE: In the nasal and temporal quadrants of each eye, the clinical results were compared with the "gold standard" FA. RESULTS: Most patients (84%) had definite peripheral capillary nonperfusion. Biomicroscopy results were highly correlated with angiography, with only a few differences of clinical importance. CONCLUSIONS: Clinical examination can determine the location of capillary nonperfusion in DR with a high degree of accuracy.