Abstract
Purpose :
In histology and clinical optical coherence tomography (OCT), HRF in AMD correlate to anteriorly migrating RPE; in neovascular (nv)AMD, HRF also include lipid-filled cells. HRF confer high risk for progression to advanced AMD. To assess HRF functional repertoire, we probed 15 RPE phenotypes (PMID 28785769) for immune cell markers (CD68, CD163) and retinoid pathway proteins (RPE65, CRALBP).
Methods :
Eyes of white donors (>80 years) were preserved by immersion in 4% paraformaldehyde (mean death-to-preservation, 3.9 hours). Ex vivoOCT scans (Spectralis, Heidelberg Engineering) were indexed to 12 µm-thick sections of posterior pole, which were stained with periodic acid Schiff hematoxylin for diagnosis, immuno-stained using enzymatic detection and a red substrate, and scanned with a 40X objective. RPE was recognized by abundant spindle-shape melanosomes and lipofuscin/ melanolipofuscin. Staining was assessed by one observer as 0 (none), 1 (some cells of a phenotype), 2 (all cells) and converted to percentage by dividing by the maximal allowable score for each phenotype. Positive controls were cells of inner retina and choroid (CD68, CD163) and Müller glia (CRALPB).
Results :
In 4 normal and 16 AMD eyes (early-intermediate AMD, n=7; geographic atrophy, n=6; nvAMD, n=3), 13 RPE phenotypes were stained by 4 markers each, in at least 3 different eyes. Vitelliform and vacuolated cells were not seen. RPE65 and CRALBP immunoreactivity was intense in uniform and non-uniform RPE (age-normal, in a continuous layer) and rare (<5%) in any other phenotype at any AMD stage. In atrophic areas Müller glia were strongly labeled by CRALBP and dissociated RPE was unlabeled. CD68 and CD163 immunoreactivity appeared in some non-uniform RPE and in all abnormal phenotypes, including intraretinal RPE, with CD68 much more prominent than CD163.
Conclusions :
This is the first attempt to probe functions of migrating RPE cells in AMD retinas. Immunoreactivity for CD68, a macrophage lysosome protein, in age-normal RPE layer, intraretinal RPE, and other phenotypes suggests that RPE cells transdifferentiate in situ then migrate. Loss of retinoid processing proteins in RPE and preservation in Müller glia supports a model of vulnerable rod vision and resilient cone vision due to glial sustenance. Markers for HRF will facilitate high-throughput discovery of their full repertoire.
This is a 2020 ARVO Annual Meeting abstract.