Elsevier

Ophthalmology

Volume 120, Issue 4, April 2013, Pages 821-828
Ophthalmology

Original article
Histologic Basis of Variations in Retinal Pigment Epithelium Autofluorescence in Eyes with Geographic Atrophy

https://doi.org/10.1016/j.ophtha.2012.10.007Get rights and content

Purpose

Lipofuscin 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.

Design

Experimental study.

Participants and Controls

Ten 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.

Methods

The 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-μm 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 Measures

Statistically significant differences in intensity and localization of HAF and CAF at defined stages of RPE morphologic progression for GA and control eyes.

Results

The 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.

Conclusions

Areas 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.

Financial Disclosure(s)

The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Section snippets

Eyes and Tissue Preparation

All research adhered to the tenets of the Declaration of Helsinki and received institutional review board approval. Ten donor eyes (donor age range, 73–93 years) with evidence of GA were received from the Alabama Eye Bank within 6 hours after death. Globes were fixed with 4% paraformaldehyde overnight and were stored in 1% paraformaldehyde at 4°C. All eyes were examined and photographed with epi-illumination and transillumination under a preparation microscope (SMZ-U; Nikon Instruments, Inc,

Results

Results are reported from 10 donors (7 women and 3 men) with GA (mean age, 87.1±4.0 years) and 3 control donors (2 women and 1 man; mean age, 84.0±7.2 years), all white. Available eye health history for 4 GA eyes indicated a clinical presentation of AMD at 1.8 to 87.8 months before death.8 Gross examination revealed appearances typical of GA (Fig 2, available at http://aaojournal.org), including central RPE atrophy and a mottled junctional zone. Some eyes had lobulated borders formed by the

Discussion

Because FAF is being advocated for imaging RPE metabolism, a more complete understanding of the sources of FAF variability and their clinical significance is essential. This study provides the first systematic and quantitative analysis of RPE HAF in AMD eyes. The principal finding is that a variability in RPE cellular morphologic features underlies the increased FAF at the transition between normal RPE and GA. Areas with advanced RPE alterations (grades 2 or 3) are most likely to cause

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    Manuscript no. 2011-1772.

    Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

    Supported by the International Retinal Research Foundation, Birmingham, Alabama; the National Eye Institute, National Institutes of Health, Bethesda, Maryland (grant no.: EY06109); Eye Sight Foundation of Alabama, Birmingham, Alabama; Research to Prevent Blindness, New York, New York; Deutsche Forschungsgemeinschaft (13942); Themenbezogene Forschungsförderung 2008 der Deutschen Ophthalmologischen Gesellschaft; and the AMD-Förderpreis 2009 der Deutschen Ophthalmologischen Gesellschaft. The funding organizations had no role in the design or conduct of this research. Dr. Read is a Research to Prevent Blindness Physician Scientist.

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