Imaging hydroxyapatite in sub-retinal pigment epithelial deposits by fluorescence lifetime imaging microscopy with tetracycline staining

Abstract

Significance: Recent evidence suggests that hydroxyapatite (HAP) in sub-retinal pigment epithelial (sub-RPE) deposits in aged human eyes may act to nucleate and contribute to their growth to clinically detectable size. Sub-RPE deposits such as drusen are clinical hallmarks of agerelated macular degeneration (AMD), therefore enhanced and earlier detection is a clinical need. We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, can also label the HAP in sub-RPE deposits. However, HAP-bound tetracycline fluorescence excitation and emission spectra overlap with the well-known autofluorescence of outer retinal tissues, making them difficult to resolve.Aim: In this initial study, we sought to determine if the HAP-bound tetracyclines also exhibit enhanced fluorescence lifetimes, providing a useful difference in lifetime compared with the short lifetimes observed in vivo in the human retina by the pioneering work of Schweitzer, Zinkernagel, Hammer, and their colleagues, and thus a large enough effect size to resolve the HAP from background by fluorescence lifetime imaging.Approach: We stained authentic HAP with tetracyclines and measured the lifetime(s) by phase fluorometry, and stained aged, fixed human cadaver retinas with drusen with selected tetracyclines and imaged them by fluorescence lifetime imaging microscopy (FLIM).Results: We found that chlortetracycline and doxycycline exhibited substantial increase in fluorescence lifetime compared to the free antibiotics and the retinal background, and the drusen were easily resolvable from the retinal background in these specimens by FLIM.Conclusions: These findings suggest that FLIM imaging of tetracycline (and potentially other molecules) binding to HAP could become a diagnostic tool for the development and progression of AMD.

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium

“…The effects on protein processing and protein export/import in this study suggest that zinc supplementation might modulate directly sub-RPE accumulation. The effects of zinc in sub-RPE deposit formation in clinical studies are varied (see review [ 73 , 74 ]) and will require further investigations, probably with the use of methods that are able to specifically identify sub-RPE deposits in a clinical setting [ 75 , 76 , 77 ]. Changes in cell adhesion to polarity [ 78 ] and modulation of extracellular matrix organization had also been reported in AMD [ 31 , 79 ] which highlight another layer of complexity of zinc action on the RPE.…”

Optical Microscopy and the Extracellular Matrix Structure: A Review

“…They demonstrated the use of FLIM in imaging biosensors that have a high potential for use in vivo, in organoid models, or for further study of tissue processes, such as healing [ 95 ]. FLIM was also used by Szmacinski et al in their 2020 study of extracellular hydroxyapatite deposits in the retina [ 96 ]. A hallmark of age-related macular degeneration, hydroxyapatite deposits (known as drusen) are extracellular deposits found under the retina [ 96 ].…”

“…FLIM was also used by Szmacinski et al in their 2020 study of extracellular hydroxyapatite deposits in the retina [ 96 ]. A hallmark of age-related macular degeneration, hydroxyapatite deposits (known as drusen) are extracellular deposits found under the retina [ 96 ]. Using a tetracycline stain, Szmacinski et al were able to demonstrate the use of FLIM in resolving the locations of hydroxyapatite deposits in the subretinal layer and its potential use as a tool in the diagnosis of other age-related eye diseases [ 96 ].…”

Bruch's membrane calcification in pseudoxanthoma elasticum

“…43 In ABCC6 -/mice, intraperitoneal supplementation of PP i did indeed prevent ectopic calcification. 106 However, the pharmaceutical properties of PP i impede the use of PP i as a therapeutic supplement in humans up to now; oral supplementation is ineffective so it needs to be administered intravenously. 107 Bisphosphonates, a common drug type, are synthetic and stable PP i analogues and may therefore substitute as PP i supplementation.…”

“…46,108 In ABCC6 -/mice, the bisphosphonate etidronate was found to reduce ectopic calcification in mice that were treated before the onset of ectopic calcification. 106,109,110 Together with the evidence that bisphosphonates reduce arterial wall calcification in human subjects, 111 this led to the design of a randomised double-blind placebocontrolled trial to test the effect of etidronate in PXE patients. In this trial, etidronate was associated with reduced arterial calcification in the femoral arteries and fewer excessive CNV activity that necessitated prompt treatment.…”