Abstract
Purpose :
Clinical trials for RPE transplantation for AMD have shown black pigment in the post-operative fundus. Black pigment in neovascular AMD is ascribed to melanotic cells, i.e., RPE-originated and packed with large spherical melanosomes (PMID 26024109). To inform interpretation of trial imaging outcomes, we provided histology of a clinically imaged eye with neovascular AMD and black pigment; in donor eyes, we performed immunohistochemistry to test whether melanotic cells are transdifferentiated RPE.
Methods :
A white woman with inactive subretinal fibrosis OD was followed for 9 years using fundus color and autofluorescence imaging, and optical coherence tomography (OCT). After patient death at age 90 years, OD was preserved 6.25 hours later and prepared for epoxy resin sections aligned to OCT and light and electron microscopy. Regions of distinct gray-black pigmentation in the fibrotic scar were identified in histology. To assess cellular functional repertoire, we used enzyme-linked colorimetric immunohistochemistry to detect retinoid (RPE65) and immune (CD68) proteins, in bleached sections of donor eyes with nvAMD and lacking clinical history.
Results :
Black and gray non-autofluorescent pigment appeared within the fibrotic scar and expanded during follow-up. The black pigment corresponded to cells with densely packed spherical black melanosomes. These contrasted both with spindle-shaped melanosomes of normal RPE, which were found infrequently, and tightly packed small spherical melanosomes in choroidal melanocytes. Some moderately pigmented melanotic cells expressed RPE65 and CD68. Only CD68 immunoreactivity, a marker for abnormal RPE (Cao ARVO2020), was seen in heavily pigmented cells.
Conclusions :
Black fundus pigment corresponds to melanotic cells, shown previously to arise from RPE entombed in fibrotic scars or enveloping neovascular membranes (PMID 26024109, 32855855). Large spherical melanosomes likely arise from organelles such as lipofuscin or newly described spherical melanosomes (PMID 32433758) rather than from native melanosomes, which are sparse in advanced disease. Cell based therapies should anticipate possible dynamism of lysosome-related organelles including melanosomes. Trial outcome measures should include multimodal imaging with autofluorescence to assess health of implanted cells.
This is a 2021 ARVO Annual Meeting abstract.