Clinical and nano-analytical imaging identify calcified nodules as progression markers for age-related macular degeneration

Abstract

Purpose : Heterogeneous internal reflectivity within drusen (HIRD) is an optical coherence tomography (OCT) biomarker associated with progression to late age-related macular degeneration (AMD). Multilobular refractile nodules (5-20 µm) in drusen contain phosphates. To test whether nodules contain calcium in a specific mineralization state that might account for HIRD, we sought a longitudinal, multimodal imaging clinical description and molecular/physical differentiation from small reflective hydroxyapatite (HAP) spherules and Bruch’s membrane plaques.

Methods : Two clinical cohorts were followed for 1 and 6 years, respectively. Human donor eye tissues were subject to in vivo and ex vivo OCT, other multi-modal imaging modalities, histology, and several nano-analytical imaging techniques from material sciences.

Results : HIRD are associated with hypoautofluorescence of overlying retinal pigment epithelium (RPE) and 4-fold increased risk of progression to geographic atrophy at 1 year through stages outlined below (Figure 1). Histology of clinically imaged eyes linked HIRD to nodules. By energy dispersive x-ray spectroscopy, time of flight secondary ion mass spectrometry and specific histochemistry, and transmission electron microscopy with selected area electron diffraction, respectively, we found that 1) nodules, spherules, and plaques contain calcium and phosphate, and uniquely, nodules lack magnesium; 2) HAP is the major nodule constituent; 3) nodules are polycrystalline HAP, spherules diffracts as single crystal whitlockite, and plaques are HAP of amorphous crystallinity.

Conclusions : Nodules signify risk by association with the agonal state of the RPE (Figure 1). They may form due to a shift in extracellular magnesium-calcium balance and lowering of environmental pH as RPE degenerates. Results highlight the role of RPE in outer retinal mineral regulation and the potential of clinical imaging for molecular discovery.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

<!--StartFragment-->Figure 1: Multi-modal imaging representing the different stages of progression of a calcified nodule and HAP spherule as seen in different patients on color fundus photo (CFP) (far left), fundus autofluorescence (FAF) (middle left), near infra-red (NIR) (middle right) and optical coherence tomography (OCT) (far right). <!--EndFragment-->