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Martin Rudolf, Anna Wagner, Christine A. Curcio, Russell W. Read; Autofluorescence of Individual Cells in Histological Sections of Eyes with Geographic Atrophy (GA). Invest. Ophthalmol. Vis. Sci. 2011;52(14):1763.
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© ARVO (1962-2015); The Authors (2016-present)
Previous histological studies demonstrated that areas with advanced RPE alterations are most likely to cause clinically recognizable patterns of elevated fundus autofluorescence (AF) which are useful for estimating the progression of GA. But many dysmorphic cells were not hyperfluorescent (36%, Rudolf, ARVO 2008). In histological sections we measured the AF of individual RPE cells at different, histologically-defined stages of GA to determine a relation between AF, grad of RPE alteration, and cell size.
Ten µm-thick macular sections of 10 GA donor eyes fixed with 4% paraformaldehyde were evaluated by light microscopy with differential interference contrast. 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 in retina; 4 = RPE absent). We then imaged the AF of different RPE zones by confocal scanning laser (cSL) microscope with settings (excitation 488 nm, emission 500+ nm, energy 1 mW) close to clinical parameters of cSL ophthalmoscopes. For each graded zone we measured in representative indiviual cells the cross-sectional area, average AF intensity, and total AF intensity.
From the outer macula towards the central RPE atrophic area we defined zones in which the RPE passed almost steadily upward through the grades of increased pathology. Concordantly with previous morphometric measurements the size of cells (cross-sectional area) increased from grad 0 to 2 significantly (p<0.05). No differences were found between cells graded 3 or 2 even though the latter showed the highest variability. After data normalisation no correlation between average AF intensity and cross-sectional area was detected (r=0.24) but a strong association of cell size and total AF intensity was found in all grades (r=0.78).
Hypertrophic RPE cells as signs of advanced cellular degeneration are directly correlated with increased total AF intensity which is most likely derived from cytotoxic lipofuscin. However, this increased accumulation of lipofuscin gets diluted in many large cells so that there average AF intensity is not increased compared to normal RPE. Our results explain why not all advanced RPE changes are detectable with increased fundus AF exclusively. A combination of fundus AF and high resolution OCT might be favorable to detect more advanced RPE changes in GA.
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