Abstract
Purpose :
The role of zinc in pathogenesis and prevention of age-related macular degeneration (AMD) is poorly understood and appears paradoxical. Use of an early fluorescein-based probe (Zinpyr-1) showed zinc as a major component of sub-retinal pigment epithelial (RPE) deposits (drusen), an early biomarker of AMD. However, Zinpyr-1 had limited sensitivity in ex-vivo analysis nor was it useful for in-vivo analysis. To assess the suitability of murine AMD models for assessing the role of zinc in AMD, and to develop in-vivo assays for detecting human and murine drusen, we assay the ability of a new fluorescein-based probe (ZPP1) and a novel, chemically-distinct probe (ZQuinT) to detect drusen.
Methods :
Flat mounts of post-mortem, human sub-RPE tissue were labeled with the probes and analyzed by fluorescence and confocal microscopy. Probe specificity for zinc was confirmed in parallel samples by addition of zinc-specific chelating agents prior to labeling. Flat mounts of murine sub-RPE tissue from wild-type and mice deficient in superoxide dismutase isoform 1 (CuZn-SOD) or isoform 2 (Mn-SOD) were similarly analyzed. The ability of intense light exposure to enhance drusen formation in mice was assessed. Murine samples were also analyzed by retinal imaging microscopy, optical coherence tomography, and histology.
Results :
In human sub-RPE tissue, drusen was detected in greater numbers and intensity with ZPP1 compared to Zinpyr-1. Drusen was detected in tissue from a 46-year old donor with no ocular history suggesting that ZPP1 might be sensitive enough to detect drusen at an early stage. Analysis of the same samples with ZQuinT suggested it was more sensitive for drusen than either fluorescein-based probe. Similar results about the sensitivity of the probes were obtained with murine tissue. Drusen formation in the mice was enhanced by both an SOD deficient genotype and light exposure. ZQuinT analysis revealed the presence of RPE cells with high levels of cytoplasmic zinc in light exposed mice.
Conclusions :
Through fluorescence induced by zinc binding, detection of sub-RPE deposits in human and murine samples was enhanced by the use of ZPP1, and even more so by ZQuinT. Enhanced zinc sensitivity makes possible an analysis of zinc in AMD using murine models, and could result in development of sensitive in-vivo probes for drusen to aid in research and clinical detection of AMD.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.