Imaging of Retinal Hydroxyapatite as an Early Screen for AMD

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Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly in developed countries, affecting over ten million Americans. The disease onset is gradual, with few symptoms and most patients unaware until irreversible vision loss is detected by eye examination. It is accepted that the buildup of deposits of fats and protein in the retina, the best known of which are called drusen, causes the death of the light-sensitive cells there. While the less common, “wet” form of AMD (CNV) can be arrested by drugs called VEGF inhibitors, the more common, “dry” form (GA) is currently untreatable, although several treatments are in development. Recently, we discovered that drusen contain microscopic spherules of hydroxyapatite (HAP), a form of calcium phosphate abundant in bones and teeth, and developed evidence indicating that the spherules nucleate the growth of drusen in the retina. We found that fluorescent stains developed to study bone growth would also stain the spherules, permitting them to be studied by fluorescence imaging of the retina. We inferred that detecting the HAP spherules early by such an imaging approach used in vivo might predict the appearance of the drusen and thus AMD development; this was confirmed in at least some cases. Thus, the thrust of this Bioengineering Research Grant is to develop and validate a retinal imaging approach for screening and early detection of AMD, both to cue treatment, and to follow the course of the disease to assess treatment. Some of the stains synthesized for bone studies in animal models perform well in vitro, but their behavior and safety is little known in animals, and there are no human studies at all. However, some tetracycline antibiotics also give bright fluorescence when bound to bone mineral, and we found that they have a distinct fluorescence lifetime under these conditions, which can be resolved from the background fluorescence of the retina by fluorescence lifetime imaging. The tetracyclines have well known behavior, can probably be administered orally, and are very safe in humans. To image the spherules with tetracycline staining we must construct a fluorescence lifetime imaging ophthalmoscope (FLIO), usable on humans or the only good animal model for AMD, macaques (monkeys). We will construct a FLIO based on an existing instrument, refine our procedures on donor cadavers, then test the FLIO and staining procedures on aged macaques (which develop spherules) and a Japanese macaque which develops drusen early and rapidly as a result of a high fat diet. Key questions to be answered are how reliably the appearance of spherules predicts the development of drusen and progression to AMD, and how safe the imaging procedure is for the monkeys; satisfactory results will likely lead to trials in humans.