Dataset Information

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

ABSTRACT:

Purpose

Lipofuscin and melanolipofuscin organelles in retinal pigment epithelium (RPE) cells are signal sources for clinical fundus autofluorescence (AF). To elucidate the subcellular basis of AF imaging, we identified, characterized, and quantified the frequency of RPE morphology and AF phenotypes in donor eyes with age-related macular degeneration (AMD).

Methods

In 25 RPE-Bruch's membrane flat mounts from 25 eyes, we analyzed 0.4-?m z-stack epifluorescence images of RPE stained with phalloidin for actin cytoskeleton. Using a custom ImageJ plugin, we classified cells selected in a systematic unbiased fashion in six phenotypes representing increasing degrees of pathology. For each cell, area, AF intensity, and number of Voronoi neighbors were compared with phenotype 1 (uniform AF, polygonal morphology) via generalized estimating equations. We also analyzed each cell's neighborhood.

Results

In 29,323 cells, compared with phenotype 1, all other phenotypes, in order of increasing pathology, had significantly larger area, reduced AF, and more variable number of neighbors. Neighborhood area and AF showed similar, but subtler, trends. Cells with highly autofluorescent granule aggregates are no more autofluorescent than others and are in fact lower overall in AF. Pre-aggregates were found in phenotype 1. Phenotype 2, which exhibited degranulation despite normal cytoskeleton, was the most numerous nonhealthy phenotype (16.23%).

Conclusions

Despite aggregation of granules that created hyperAF aggregates within cells, overall AF on a per cell basis decreased with increasing severity of dysmorphia (abnormal shape). Data motivate further development of subcellular resolution in clinical fundus AF imaging and inform an ongoing reexamination of the role of lipofuscin in AMD.

SUBMITTER: Gambril JA

PROVIDER: S-EPMC6557619 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Gambril J Alan JA Sloan Kenneth R KR Swain Thomas A TA Huisingh Carrie C Zarubina Anna V AV Messinger Jeffrey D JD Ach Thomas T Curcio Christine A CA

Investigative ophthalmology & visual science 20190601 7

<h4>Purpose</h4>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).<h4>Methods</h4>In 25 RPE-Bruch's membrane flat mounts from 25 eyes, we analyzed 0.4-μm z-stack epifluorescence images of RPE stained ...[more]

PMID: 31173079

Similar Datasets

Project description:PurposeLipofuscin contained in the retinal pigment epithelium (RPE) is the main source of fundus autofluorescence (FAF), the target of an imaging method useful for estimating the progression of geographic atrophy (GA) in clinical trials. To establish a cellular basis for hyperfluorescent GA border zones, histologic autofluorescence (HAF) was measured at defined stages of RPE pathologic progression.DesignExperimental study.Participants and controlsTen GA donor eyes (mean age ± standard deviation, 87.1 ± 4.0 years) and 3 age-matched control eyes (mean age ± standard deviation, 84.0 ± 7.2 years) without GA.MethodsThe 10-micrometer-thick sections were divided into zones of RPE morphologic features according to an 8-point scale. Any HAF excited by 488 nm light was imaged by laser confocal microscopy. The HAF intensity summed along vertical lines perpendicular to Bruch's membrane at 0.2-?m intervals served as a surrogate for FAF. Intensity profiles in 151 zones were normalized to grade 0 at a standard reference location in each eye. Cross-sectional area, mean, and sum autofluorescence for individual RPE cells were measured (cellular autofluorescence [CAF]).Main outcome measuresStatistically significant differences in intensity and localization of HAF and CAF at defined stages of RPE morphologic progression for GA and control eyes.ResultsThe RPE morphologic features were most abnormal (cell rounding, sloughing, and layering; grade 2) and HAF intensity profiles were highest and most variable immediately adjacent to atrophic areas. Peaks in HAF intensity frequently were associated with vertically superimposed cells. The HAF value that optimally separated reactive RPE was 0.66 standard deviations more than the mean for uninvolved RPE and was associated with a sensitivity of 75.8% and a specificity of 76.3%. When variable cell area was accounted for, neither mean nor sum CAF differed significantly among the RPE pathologic grades.ConclusionsAreas with advanced RPE alterations are most likely to exhibit clinically recognizable patterns of elevated FAF around GA, but may not predict cells about to die, because of vertically superimposed cells and cellular fragments. These data do not support a role for lipofuscin-related cell death and call into question the rationale of treatments targeting lipofuscin.

Dataset Information

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

Purpose

Methods

Results

Conclusions

Publications

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

Similar Datasets

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