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).
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.
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%).
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.
The retinal pigment epithelium (RPE) is a unique epithelium, with major roles which are essential in the visual cycle and homeostasis of the outer retina. The RPE is a monolayer of polygonal and ...pigmented cells strategically placed between the neuroretina and Bruch membrane, adjacent to the fenestrated capillaries of the choriocapillaris. It shows strong apical (towards photoreceptors) to basal/basolateral (towards Bruch membrane) polarization. Multiple functions are bound to a complex structure of highly organized and polarized intracellular components: the cytoskeleton. A strong connection between the intracellular cytoskeleton and extracellular matrix is indispensable to maintaining the function of the RPE and thus, the photoreceptors. Impairments of these intracellular structures and the regular architecture they maintain often result in a disrupted cytoskeleton, which can be found in many retinal diseases, including age-related macular degeneration (AMD). This review article will give an overview of current knowledge on the molecules and proteins involved in cytoskeleton formation in cells, including RPE and how the cytoskeleton is affected under stress conditions-especially in AMD.
Antibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) have been reported in patients with aquaporin-4 antibody (AQP4-IgG)-negative neuromyelitis optica spectrum disorders (NMOSD). The ...objective of this study was to describe optic neuritis (ON)-induced neuro-axonal damage in the retina of MOG-IgG-positive patients in comparison with AQP4-IgG-positive NMOSD patients.
Afferent visual system damage following ON was bilaterally assessed in 16 MOG-IgG-positive patients with a history of ON and compared with that in 16 AQP4-IgG-positive NMOSD patients. In addition, 16 healthy controls matched for age, sex, and disease duration were analyzed. Study data included ON history, retinal optical coherence tomography, visual acuity, and visual evoked potentials.
Eight MOG-IgG-positive patients had a previous diagnosis of AQP4-IgG-negative NMOSD with ON and myelitis, and eight of (mainly recurrent) ON. Twenty-nine of the 32 eyes of the MOG-IgG-positive patients had been affected by at least one episode of ON. Peripapillary retinal nerve fiber layer thickness (pRNFL) and ganglion cell and inner plexiform layer volume (GCIP) were significantly reduced in ON eyes of MOG-IgG-positive patients (pRNFL = 59 ± 23 μm; GCIP = 1.50 ± 0.34 mm
) compared with healthy controls (pRNFL = 99 ± 6 μm, p < 0.001; GCIP = 1.97 ± 0.11 mm
, p < 0.001). Visual acuity was impaired in eyes after ON in MOG-IgG-positive patients (0.35 ± 0.88 logMAR). There were no significant differences in any structural or functional visual parameters between MOG-IgG-positive and AQP4-IgG-positive patients (pRNFL: 59 ± 21 μm; GCIP: 1.41 ± 0.27 mm
; Visual acuity = 0.72 ± 1.09 logMAR). Importantly, MOG-IgG-positive patients had a significantly higher annual ON relapse rate than AQP4-IgG-positive patients (median 0.69 vs. 0.29 attacks/year, p = 0.004), meaning that on average a single ON episode caused less damage in MOG-IgG-positive than in AQP4-IgG-positive patients. pRNFL and GCIP loss correlated with the number of ON episodes in MOG-IgG-positive patients (p < 0.001), but not in AQP4-IgG-positive patients.
Retinal neuro-axonal damage and visual impairment after ON in MOG-IgG-positive patients are as severe as in AQP4-IgG-positive NMOSD patients. In MOG-IgG-positive patients, damage accrual may be driven by higher relapse rates, whereas AQP4-IgG-positive patients showed fewer but more severe episodes of ON. Given the marked damage in some of our MOG-IgG-positive patients, early diagnosis and timely initiation and close monitoring of immunosuppressive therapy are important.
To seek pathways of retinal pigment epithelium (RPE) fate in age-related macular degeneration via a morphology grading system; provide nomenclature, visualization targets, and metrics for clinical ...imaging and model systems.
Donor eyes with geographic atrophy (GA) or choroidal neovascularization (CNV) and one GA eye with previous clinical spectral-domain optical coherence tomography (SDOCT) imaging were processed for histology, photodocumented, and annotated at predefined locations. Retinal pigment epithelial cells contained spindle-shaped melanosomes, apposed a basal lamina or basal laminar deposit (BLamD), and exhibited recognizable morphologies. Thicknesses and unbiased estimates of frequencies were obtained.
In 13 GA eyes (449 locations), 'Shedding,' 'Sloughed,' and 'Dissociated' morphologies were abundant; 22.2% of atrophic locations had 'Dissociated' RPE. In 39 CNV eyes (1363 locations), 37.3% of locations with fibrovascular/fibrocellular scar had 'Entombed' RPE; 'Sloughed,' 'Dissociated,' and 'Bilaminar' morphologies were abundant. Of abnormal RPE, CNV and GA both had ~35% 'Sloughed'/'Intraretinal,' with more Intraretinal in CNV (9.5% vs. 1.8%). 'Shedding' cells associated with granule aggregations in BLamD. The RPE layer did not thin, and BLamD remained thick, with progression. Granule-containing material consistent with three morphologies correlated to SDOCT hyperreflective foci in the previously examined GA patient.
Retinal pigment epithelium morphology indicates multiple pathways in GA and CNV. Atrophic/scarred areas have numerous cells capable of transcribing genes and generating imaging signals. Shed granule aggregates, possibly apoptotic, are visible in SDOCT, as are 'Dissociated' and 'Sloughed' cells. The significance of RPE phenotypes is addressable in longitudinal, high-resolution imaging in clinic populations. Data can motivate future molecular phenotyping studies.
To describe, illustrate, and account for two cell types plausibly derived from RPE in geographic atrophy (GA) and choroidal neovascularization (CNV) of AMD, using melanosomes, lipofuscin, and basal ...laminar deposit (BLamD) as anatomical markers.
Human donor eyes with GA (n = 13) or CNV (n = 39) were histologically processed, photodocumented, and analyzed for frequencies of occurrence. We defined RPE as cells containing spindle-shaped melanosomes and RPE lipofuscin, internal to basal lamina or BLamD, if present, or Bruch's membrane if not, and RPE-derived cells as those plausibly derived from RPE and not attached to basal lamina or BLamD.
'Subducted' cells contain RPE melanosomes and localize to the sub-RPE space, on Bruch's membrane. Credible transitional forms from RPE cells were seen. Grades of RPE overlying 'Subducted' cells were 'Atrophic with BLamD' (32.2% vs. 37.0% of 'Subducted,' for GA and CNV eyes, respectively), 'Dissociated' (22.0% vs. 21.7%), 'Nonuniform' (22.0% vs. 23.9%), and 'Sloughed' RPE (10.2% vs. 4.3%). Found exclusively in CNV scars, 'Melanotic' cells containing spherical melanosomes were adjacent to 'Entombed' RPE with spindle-shaped and spherical melanosomes. Of subretinal 'Melanotic' cells, 40.0% associated with 'Atrophy with BLamD,' 36.8% with 'Atrophy without BLamD,' and 20.6% with 'Entombed.'
'Dissociated' RPE within atrophic areas may be the source of 'Subducted' cells. 'Entombed' RPE within fibrovascular and fibrocellular scars may be the source of 'Melanotic' cells. An imaging correlate for 'Subducted' cells awaits discovery; 'Melanotic' cells appear gray-black in the CNV fundus. Results provide a basis for future molecular phenotyping studies.
To enable future studies of retinal pigment epithelium (RPE) fate in the macular atrophy occurring in eyes with neovascular age-related macular degeneration (nvAMD), the authors determined how RPE ...morphology changes across the transition from health to atrophy in donor eyes with nvAMD.
In RPE-Bruch membrane flat mounts of 5 nvAMD eyes, the terminations of organized RPE cytoskeleton and autofluorescent material were compared. In high-resolution histologic sections of 27 nvAMD eyes, RPE phenotypes were assessed at ±500 μm and ±100 μm from the descent of the external limiting membrane (ELM) toward the Bruch membrane. Thicknesses of RPE, basal laminar deposit (BLamD), and RPE + BLamD were determined. Shapes of the ELM descent were recorded.
Approaching the ELM descent, the percentage of different RPE phenotypes and the thickness of RPE, BLamD, and RPE + BLamD each stayed roughly constant. Compared with a separately described cohort of eyes with geographic atrophy, eyes with nvAMD were more likely to have RPE dysmorphia that did not worsen toward the atrophy border, thinner BLamD overall (3.25 ± 3.46 μm vs. 7.99 ± 7.49 μm for geographic atrophy), and a higher proportion of oblique ELM descents (47.9 vs. 31.9%).
The distribution of RPE phenotypes at the transition to macular atrophy in eyes with nvAMD differs from that in primary geographic atrophy, likely reflecting greater photoreceptor loss and the effects of exudation in nvAMD. This distribution, the shape of ELM descents, and thickness profiles may be useful metrics in clinical studies of macular atrophy using optical coherence tomography and fundus autofluorescence.
To assess outer retinal tubulation (ORT) morphology from spectral-domain optical coherence tomography (SD-OCT) volumes and donor eye histology, analyze ORT reflectivity, and estimate the number of ...cones surviving in ORT.
In SD-OCT volumes from nine patients with advanced AMD, ORT was analyzed en face and in B-scans. The hyperreflective ORT border in cross-section was delineated and surface area calculated. Reflectivity was compared between ORT types (Closed, Open, Forming, and Branching). A flatmount retina from a donor with neovascular AMD was labeled to visualize the external limiting membrane that delimits ORT and allow measurements of cross-sectional cone area, center-to-center cone spacing, and cone density. The number of cones surviving in ORT was estimated.
By en face SD-OCT, ORT varies in complexity and shape. Outer retinal tubulation networks almost always contain Closed cross-sections. Spectral-domain OCT volumes containing almost exclusively Closed ORTs showed no significant direction-dependent differences in hyperreflective ORT border intensity. The surface areas of partial ORT assessed by SD-OCT volumes ranged from 0.16 to 1.76 mm2. From the flatmount retina, the average cross-sectional area of cone inner segments was 49.1 ± 7.9 μm2. The average cone spacing was 7.5 ± 0.6 μm. Outer retinal tubulation cone density was 20,351 cones/mm2. The estimated number of cones in ORT in a macula ranged from 26,399 to 186,833 cones, which is 6% to 44% of the cones present in a healthy macula.
These first estimates for cone density and number of cones surviving in ORT suggest that ORT formation considerably distorts the photoreceptor mosaic. Results provide additional insight into the reflectivity characteristics and number of ORT cones observable in living patients by SD-OCT, as cones persist and disease progresses.
Choroidal melanocytic lesions require reliable and precise clinical examination and diagnosis to differentiate benign choroidal nevi from choroidal melanoma, as the latter may become life-threatening ...through metastatic disease. To come to an accurate diagnosis, as well as for monitoring, and to assess the efficacy of therapy, various imaging modalities may be used, one of which is non-invasive fundus autofluorescence (FAF) imaging using novel high-resolution digital imaging technology. FAF imaging is based on the visualization of intrinsic fluorophores in the ocular fundus. Lipofuscin and melanolipofuscin within the postmitotic retinal pigment epithelium (RPE) cells represent the major fluorophores that contribute to the FAF signal. In addition, the presence or loss of absorbing molecular constituents may have an impact on the FAF signal. A choroidal melanoma can cause secondary retinal and RPE alterations that affect the FAF signal (e.g., occurrence of orange pigment). Therefore, FAF imaging supports multimodal imaging and gives additional information over and above conventional imaging modalities regarding retinal metabolism and RPE health status. This article summarises the features of FAF imaging and the role of FAF imaging in the context of choroidal melanoma, both before and following therapeutic intervention.
Abstract
Clinical discrimination of posterior uveitis entities remains a challenge. This exploratory, cross-sectional study investigated the green (GEFC) and red emission fluorescent components ...(REFC) of retinal and choroidal lesions in posterior uveitis to facilitate discrimination of the different entities. Eyes were imaged by color fundus photography, spectrally resolved fundus autofluorescence (Color-FAF) and optical coherence tomography. Retinal/choroidal lesions’ intensities of GEFC (500–560 nm) and REFC (560–700 nm) were determined, and intensity-normalized Color-FAF images were compared for birdshot chorioretinopathy, ocular sarcoidosis, acute posterior multifocal placoid pigment epitheliopathy (APMPPE), and punctate inner choroidopathy (PIC). Multivariable regression analyses were performed to reveal possible confounders. 76 eyes of 45 patients were included with a total of 845 lesions. Mean GEFC/REFC ratios were 0.82 ± 0.10, 0.92 ± 0.11, 0.86 ± 0.10, and 1.09 ± 0.19 for birdshot chorioretinopathy, sarcoidosis, APMPPE, and PIC lesions, respectively, and were significantly different in repeated measures ANOVA (
p
< 0.0001). Non-pigmented retinal/choroidal lesions, macular neovascularizations, and fundus areas of choroidal thinning featured predominantly GEFC, and pigmented retinal lesions predominantly REFC. Color-FAF imaging revealed involvement of both, short- and long-wavelength emission fluorophores in posterior uveitis. The GEFC/REFC ratio of retinal and choroidal lesions was significantly different between distinct subgroups. Hence, this novel imaging biomarker could aid diagnosis and differentiation of posterior uveitis entities.
PDF
