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Richard Thompson, Kavita Rajeev Hegde, Henryk Szmacinski, Kristina Pugh, Adam Puche, Imre Lengyel; Infusion labeling and FLIM imaging of hydroxyapatite spherules in human sub-RPE deposits. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3466.
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Recently, we found that hydroxyapatite (HAP, Ca5(PO4)3OH), the hard mineral form of calcium phosphate in bone and teeth, is a principal constituent both of microscopic spherules, precursors for drusen formation (PMID 25605911), and nodules, a progression marker for geographic atrophy (PMID 30404862) in AMD. We proposed that HAP deposition may precede the development of or be associated with progression to AMD, and thus HAP could serve as a robust biomarker for AMD screening. HAP can be labeled and imaged by microscopy with fluorescent probes designed for bone growth studies (LiCor BoneTag and Perkin Elmer OsteoSense) and compounds such as the tetracyclines. One challenge is administering such stains to humans in a screening scenario. While Bone Tag and OsteoSense stains perform well in vivo in animal models, they are not yet approved for human use. By comparison, humans have been treated with oral tetracyclines for decades, and their pharmacokinetics are known. Thus we sought to stain HAP deposits in situ by infusion through the vasculature.
All experiments were performed with oversight and approval from the UMB IRB. For labeling we perfused donor cadavers (post mortem time < 6 hours, >55 years old) with chlortetracycline (Cl-Tet) in PBS for one hour at therapeutic levels by infusion through the left interior carotid artery, followed by a flush with PBS. Entry of Cl-Tet was observed in scleral blood vessels visually. Eyes were enucleated, the RPE-choroid complex flat mounted on slides, and sub-RPE deposits imaged by brightfield and fluorescence lifetime imaging microscopy (FLIM) as described (Szmacinski, et al., Proc. SPIE 10484 (2018)).
Drusen in the macular region were imaged under 20x magnification. In initial trials drusen were readily visible by fluorescence on the slide, but compared with flatmounted retinas stained following dissection, the lifetime contrast was somewhat less marked, and the staining was less discernable over background.
We conclude that brief infusion staining is not adequate, but note that Cl-Tet is slowly absorbed and cleared over several hours in vivo, and so reproducing such pharmacokinetics with an unpreserved cadaver is infeasible. Future experiments will match the area under the curve of drug concentration vs time to mimic oral administration.<!--[if !supportAnnotations]--><!--[endif]-->
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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