Histology and clinical imaging lifecycle of black pigment in fibrosis secondary to neovascular age-related macular degeneration

Purpose: Melanotic cells with large spherical melanosomes, thought to originate from retinal pigment epithelium (RPE), are found in eyes with neovascular age-related macular degeneration (nvAMD). To generate hypotheses about RPE participation in fibrosis, we correlate histology to clinical imaging in an eye with prominent black pigment in fibrotic scar secondary to nvAMD. Methods: Macular findings in a white woman with untreated inactive subretinal fibrosis due to nvAMD in her right eye were documented over 9 years with color fundus photography (CFP), fundus autofluorescence (FAF) imaging, and optical coherence tomography (OCT). After death (age 90 years), this index eye was prepared for light and electron microscopy to analyze 7 discrete zones of pigmentation in the fibrotic scar. In additional donor eyes with nvAMD, we determined the frequency of black pigment (n = 36 eyes) and immuno-labeled for retinoid, immunologic, and microglial markers (RPE65, CD68, Iba1, TMEM119; n = 3 eyes). Results: During follow-up of the index eye, black pigment appeared and expanded within a hypoautofluorescent fibrotic scar. The blackest areas correlated to melanotic cells (containing large spherical melanosomes), some in multiple layers. Pale areas had sparse pigmented cells. Gray areas correlated to cells with RPE organelles entombed in the scar and multinucleate cells containing sparse large spherical melanosomes. In 94% of nvAMD donor eyes, hyperpigmentation was visible. Certain melanotic cells expressed some RPE65 and mostly CD68. Iba1 and TMEM119 immunoreactivity, found both in retina and scar, did not co-localize with melanotic cells. Conclusion: Hyperpigmentation in CFP results from both organelle content and optical superimposition effects. Black fundus pigment in nvAMD is common and corresponds to cells containing numerous large spherical melanosomes and superimposition of cells containing sparse large melanosomes, respectively. Melanotic cells are molecularly distinct from RPE, consistent with a process of transdifferentiation. The subcellular source of spherical melanosomes remains to be determined. Detailed histology of nvAMD eyes will inform future studies using technologies for spatially resolved molecular discovery to generate new therapies for fibrosis. The potential of black pigment as a biomarker for fibrosis can be investigated in clinical multimodal imaging datasets.