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M. Rudolf, S. D. Vogt, I. Rudolf, C. A. Curcio, R. W. Read; Autofluorescence in Histological Sections of Eyes With Geographic Atrophy (GA) Due to Age-Related Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2008;49(13):2237. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) is the main source of fundus autofluorescence (FAF). Patterns of increased FAF may be useful for estimating the progression of GA and evaluating future therapies for dry AMD. We measured RPE autofluorescence (AF) in different, histologically-defined stages of GA to determine the morphology of hyperfluorescent cells.
Ten µm-thick macular sections of 7 GA donor eyes (73-93 years) fixed with 4% paraformaldehyde were evaluated by differential interference contrast microscopy. In 3 sections/eye, zones of RPE alterations were graded (0 = normal; 1 = irregular cells, intact layer; 2 = rounded, enlarged, or heaping cells; 3 = migrating cells/ cell debris in retina; 4 = RPE absent). We then imaged AF of different RPE zones by confocal scanning laser (cSL) microscopy with settings (excitation 488 nm, emission 500+ nm, energy ~1 mW) close to parameters of cSL ophthalmoscopes. To obtain a surrogate measure for FAF we measured the sum of AF intensity along vertical lines at 0.2 µm intervals perpendicular to Bruch’s membrane across all RPE zones. AF sum intensity of all 101 graded zones was normalized by dividing by mean AF for zone 0 in the same eye. From 3 eyes, 43 zones were pooled for a preliminary determination of whether differences in AF among RPE grades were statistically significant.
From the outer macula towards the central atrophic area we defined zones in which the RPE grades passed almost steadily upward, indicating increased pathology. A) Average AF sum intensity was similar in zones 0 and 1 (p=0.12). B) Of particular interest clinically, grade 2 RPE alterations predominated within the area immediately adjacent to atrophy. AF sum intensity was highest in these areas (up to 2.4-fold; p=0.01) with 64% of grade 2 zones higher than grade 0. Conversely, AF sum intensity in 36% of grade 2 zones was identical to grade 0 zones. This variability in hyperfluorescence was also found in individual eyes, where not every grade 2 zone contained hyperfluorescence. C) If present, zone 3 was close to the atrophy margin. These zones often included AF from both intra-retinal and directly underlying in situ RPE cells, causing a sharp increase in the AF sum intensity at these locations.
Our results contribute to a cellular basis for FAF variation in GA and are useful for interpreting clinical data. Areas with advanced RPE alterations (grades 2/3) are most likely to cause clinically recognizable patterns of elevated fundus autofluorescence around RPE atrophy zones. However, data so far also indicate that many dysmorphic cells are not hyperfluorescent.
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