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Matthew Glynn Pilgrim, Lajos Csincsik, Sarah Fearn, David S McPhail, Jonathan C Knowles, Imre Lengyel; High-resolution molecular imaging of hydroxyapatite associated with sub-RPE deposits. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2376.
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We have shown recently that sub-RPE deposits, a feature of ageing and age-related macular degeneration (AMD), contain hydroxyapatite (HAP), the mineral component of bone and teeth. However, the molecular mechanism by which HAP deposition occurs is not yet known. We hypothesize that the molecular events underlying deposit formation will be the key to understanding the early molecular events leading to AMD. Here, using high-resolution molecular imaging, we analysed the composition of sub-RPE deposits in sections of human eyes.
After obtaining local ethical permission, human donor eyes with sub-RPE deposits were obtained from the UCL Institute of Ophthalmology tissue depository. Multiple high-resolution imaging modalities were used including scanning electron microscopy (SEM), transmission electron microscopy (TEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Molecular analysis was performed using energy dispersive X-ray (EDX), X-ray diffraction (XRD) and mass spectrometry. This was correlated with Von Kossa staining and fluorescent HAP labelling of adjacent tissue sections and visualized under light, fluorescence and confocal microscopy.
High-resolution correlative imaging identified spherules of 0.5μm - 6.0μm in diameter. The spherules were numerous, were observed in clusters and were distributed throughout all the examined sub-RPE deposits. SEM images showed a wide variation in the spherule surface structure; subsequent EDX analysis and SIMS imaging confirmed the presence of calcium and phosphate within these structures. XRD patterns obtained using TEM were consistent with the presence of HAP.
Using multiple complementary high-resolution methodologies, we confirmed that sub-RPE deposits contain spherules with HAP shells. Determining how these HAP spherules develop will be important for fully understanding the molecular changes underlying deposit formation and progression to diseases like AMD. This is the first step in curing, or at least slowing down, progression of this age-related disease.
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