Hyperspectral Autofluorescence (AF) and Mechanisms of Retinal Pigment Epithelium (RPE) Lipofuscin Loss in Age-Related Macular Degeneration (AMD)

Abstract

Purpose : RPE lipofuscin (LF) loss occurs in advanced aging and in AMD progression by at least 2 mechanisms, de-granulation and aggregation/shedding (Ach, PMID 25758814; Zanzottera, PMID 25813989). We examined the spectra of these subcellular mechanisms in AMD.

Methods : 12 locations from RPE flat mounts of 5 donors with AMD were examined with hyperspectral AF imaging, spectral recovery by matrix factorization, and quantification for ex vivo spectral signatures (Ben Ami, PMID 27226929). These include, for LF, S1, S2 (excitation 436 nm, emission peaks S1 535 ± 17 nm, S2 576 ± 20 nm, respectively) and for melanolipofuscin (MLF), S3, (trimodal, median peak 650 nm). Also extracted were S0, a signature of Bruch’s membrane, and SDr, a recently discovered non-LF signature that is sensitive and specific for drusen and subRPE deposits (peak 510 nm, Tong, PMID: 277496960).

Results : De-granulation was observed in 5 locations as large (2-4X normal diameter) attenuated cells emptying most of their contents, including individual LF granules, with notably decreased remaining S3 relative to S1 and S2 (Fig). Aggregation was observed in 8 locations, as multiple well-defined spherical groups of organelles 5-15 µm in diameter. 20 studied aggregates had higher proportions of the MLF spectrum S3 than organelles within cells (mean increase of MLF fraction, 0 to 44%).

Conclusions : Ex vivo histology is a snapshot in time that reveals spectra of organelles retained in degenerating cells. RPE LF loss in AMD is not random but proceeds by at least 2 stereotyped subcellular mechanisms. These 2 mechanisms may exhibit differential fates of specific organelles (MLF vulnerable in degranulation, LF in aggregation). Defining the spectrum of aggregates shed into underlying basal laminar deposits, and more samples overall, will complete this picture and thus help elucidate RPE damage and survival.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Figure. Large 8-sided emptying cell (rectangle); another 8-sided cell, beginning to empty (arrow). RGB: 40X AF micrograph. Magnified spectral abundances from inset are shown in false colors: S0, gray, Bruch’s membrane; SDr, azure, drusen/subRPE deposits, visible through attenuated cell; S1, green, 1 of 2 LF spectra; S3, red, MLF. S1/S3 overlay: Green and red for S1 and S3 are mixed (yellow) and roughly equal, in unaffected RPE. Granules in the emptying cell are mostly LF, with little MLF.