Thomas Ach

Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium

PURPOSE Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. METHODS Retinal pigment epithelium-Bruchs membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. RESULTS Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. CONCLUSIONS Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LFs role in AMD.

Lipofuscin Redistribution and Loss Accompanied by Cytoskeletal Stress in Retinal Pigment Epithelium of Eyes With Age-Related Macular Degeneration

PURPOSE Lipofuscin (LF) and melanolipofuscin (MLF) of the retinal pigment epithelium (RPE) are the principal sources of autofluorescence (AF) signals in clinical fundus-AF imaging. Few details about the subcellular distribution of AF organelles in AMD are available. We describe the impact of aging and AMD on RPE morphology revealed by the distribution of AF LF/MLF granules and actin cytoskeleton in human tissues. METHODS Thirty-five RPE-Bruchs membrane flatmounts from 35 donors were prepared (postmortem: ≤4 hours). Ex vivo fundus examination at the time of accession revealed either absence of chorioretinal pathologies (10 tissues; mean age: 83.0 ± 2.6 years) or stages of AMD (25 tissues; 85.0 ± 5.8 years): early AMD, geographic atrophy, and late exudative AMD. Retinal pigment epithelium cytoskeleton was labeled with AlexaFluor647-Phalloidin. Tissues were imaged on a spinning-disk fluorescence microscope and a high-resolution structured illumination microscope. RESULTS Age-related macular degeneration impacts individual RPE cells by (1) lipofuscin redistribution by (i) degranulation (granule-by-granule loss) and/or (ii) aggregation and apparent shedding into the extracellular space; (2) enlarged RPE cell area and conversion from convex to irregular and sometimes concave polygons; and (3) cytoskeleton derangement including separations and breaks around subretinal deposits, thickening, and stress fibers. CONCLUSIONS We report an extensive and systematic en face analysis of LF/MLF-AF in AMD eyes. Redistribution and loss of AF granules are among the earliest AMD changes and could reduce fundus AF signal attributable to RPE at these locations. Data can enhance the interpretation of clinical fundus-AF and provide a basis for future quantitative studies.

VISUALIZING RETINAL PIGMENT EPITHELIUM PHENOTYPES IN THE TRANSITION TO GEOGRAPHIC ATROPHY IN AGE-RELATED MACULAR DEGENERATION.

Purpose: To inform the interpretation of clinical optical coherence tomography and fundus autofluorescence imaging in geographic atrophy (GA) of age-related macular degeneration by determining the distribution of retinal pigment epithelium (RPE) phenotypes in the transition from health to atrophy in donor eyes. Methods: In RPE–Bruch membrane flat mounts of two GA eyes, the terminations of organized RPE cytoskeleton and autofluorescent material were compared. In high-resolution histological sections of 13 GA eyes, RPE phenotypes were assessed at ±500 and ±100 &mgr;m from the descent of the external limiting membrane (ELM) toward Bruch membrane. The ELM descent was defined as curved, reflected, or oblique in shape. Thicknesses of RPE, basal laminar deposit (BLamD), and RPE plus BLamD were measured. Results: A border of atrophy that can be precisely delimited is the ELM descent, as opposed to the termination of the RPE layer itself, because of dissociated RPE in the atrophic area. Approaching the ELM descent, the percentage of abnormal RPE morphologies increases, the percentage of age-normal cells decreases, overall RPE thickens, and BLamD does not thin. The combination of RPE plus BLamD is 19.7% thicker at −100 &mgr;m from the ELM descent than that at −500 &mgr;m (23.1 ± 10.7 &mgr;m vs. 19.3 ± 8.2 &mgr;m; P = 0.05). Conclusion: The distribution of RPE phenotypes at the GA transition supports the idea that these morphologies represent defined stages of a degeneration sequence. The idea that RPE dysmorphia including rounding and stacking helps explain variable autofluorescence patterns in GA is supported. The ELM descent and RPE plus BLamD thickness profile may have utility as spectral domain optical coherence tomography metrics in clinical trials.

Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution

BACKGROUND The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. METHODS Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. RESULTS A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. CONCLUSION This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

Simultaneous decomposition of multiple hyperspectral data sets: signal recovery of unknown fluorophores in the retinal pigment epithelium

Upon excitation with different wavelengths of light, biological tissues emit distinct but related autofluorescence signals. We used non-negative matrix factorization (NMF) to simultaneously decompose co-registered hyperspectral emission data from human retinal pigment epithelium/Bruchs membrane specimens illuminated with 436 and 480 nm light. NMF analysis was initialized with Gaussian mixture model fits and constrained to provide identical abundance images for the two excitation wavelengths. Spectra recovered this way were smoother than those obtained separately; fluorophore abundances more clearly localized within tissue compartments. These studies provide evidence that leveraging multiple co-registered hyperspectral emission data sets is preferential for identifying biologically relevant fluorophore information.

Ex Vivo Hyperspectral Autofluorescence Imaging and Localization of Fluorophores in Human Eyes with Age-Related Macular Degeneration

To characterize fluorophore signals from drusen and retinal pigment epithelium (RPE) and their changes in age related macular degeneration (AMD), the authors describe advances in ex vivo hyperspectral autofluorescence (AF) imaging of human eye tissue. Ten RPE flatmounts from eyes with AMD and 10 from eyes without AMD underwent 40× hyperspectral AF microscopic imaging. The number of excitation wavelengths tested was initially two (436 nm and 480 nm), then increased to three (436 nm, 480 nm, and 505 nm). Emission spectra were collected at 10 nm intervals from 420 nm to 720 nm. Non-negative matrix factorization (NMF) algorithms decomposed the hyperspectral images into individual emission spectra and their spatial abundances. These include three distinguishable spectra for RPE fluorophores (S1, S2, and S3) in both AMD and non-AMD eyes, a spectrum for drusen (SDr) only in AMD eyes, and a Bruch’s membrane spectrum that was detectable in normal eyes. Simultaneous analysis of datacubes excited atthree excitation wavelengths revealed more detailed spatial localization of the RPE spectra and SDr within drusen than exciting only at two wavelengths. Within AMD and non-AMD groups, two different NMF initialization methods were tested on each group and converged to qualitatively similar spectra. In AMD, the peaks of the SDr at ~510 nm (436 nm excitation) were particularly consistent. Between AMD and non-AMD groups, corresponding spectra in common, S1, S2, and S3, also had similar peak locations and shapes, but with some differences and further characterization warranted.