Research ArticleRetinal Disease

Calcified nodules in retinal drusen are associated with disease progression in age-related macular degeneration

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Science Translational Medicine  07 Nov 2018:
Vol. 10, Issue 466, eaat4544
DOI: 10.1126/scitranslmed.aat4544
  • Fig. 1 Multimodal imaging of three types of calcified structures associated with advanced AMD.

    HIRD, reflective dots, and plaques seen on (A) OCT, (B) CFP, (C) FAF, and (D) NIR. HIRD with a hyporeflective core (red arrow) and a hyperreflective cap (pink arrowhead), hyperreflective dots (green circle), and hyperreflective areas (blue arrowhead) are indicated.

  • Fig. 2 Imaging of HIRD by OCT and nodules by histology.

    (A to D) Eye of an 86-year-old white female with GA imaged in vivo 4 months before death. (A) NIR image shows translucent large drusen. Green line, level of OCT scan in (B). Yellow frame, druse on (B) to (D). (B) OCT image showing HIRD (yellow frame). (C) Druse corresponding to the HIRD. Large refractile nodules are surrounded by lipid-rich soft druse material (gray). SDD, subretinal drusenoid deposits. (D) Panoramic light microscopy. The HIRD of (B) and the druse are at the same distance from the optic nerve head, as determined by comparing corresponding OCT scans and histology at the same dimensions. (E to H) Eye of a 94-year-old white female with GA imaged ex vivo. (E) CFP shows refractile drusen in the superior temporal quadrant. Green line, level of OCT scan in (F). (F) Yellow frame denotes an HIRD. (G) Calcific nodules are surrounded by processes of Müller cells. Overlying BLamD is thin and fragmented. (H) Context of calcified druse showing degeneration of outer retina including gliotic Henle fiber layer and absent photoreceptors. Submicrometer epoxy resin sections of osmium tannic acid paraphenylenediamine post-fixed specimens stained in toluidine blue. Separation of retina from RPE (B) and choroid from sclera (H) is artifactual. NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; HFL, Henle fiber layer; OPL, outer plexiform layer; ONL, outer nuclear layer; BLamD, basal laminar deposit (a thickening of basement membrane material between the RPE and its basement lamina); Ch, choroid; Sc, sclera. Yellow arrowheads, calcific nodules; green arrowheads, BLamD; black arrowheads, BrM.

  • Fig. 3 Histological variants of calcific nodule formation and RPE degeneration in GA.

    (A) Small soft druse (d) containing lipoprotein-derived debris (artifactually fractured) and intact overlying RPE. (B) Small soft druse with partial contents, refractile nodules, and intact overlying RPE. (C) Calcific nodules and spherules and RPE-derived granule aggregates, with partial covering of RPE and photoreceptors, and lacking BLamD. (D) Druse with calcific nodules and nucleated “subducted” RPE with thick overlying BLamD and nearly complete coverage by RPE. (E) Calcific nodules with very thin BLamD and no RPE. (F) Calcific nodules with thin BLamD and subducted RPE. (G) Calcific nodules are found with thin overlying BLamD. Submicrometer epoxy resin sections of osmium tannic acid paraphenylenediamine post-fixed specimens stained in toluidine blue. Green arrowheads, BLamD; c, calcific nodule; black circle, calcific spherules; black arrowheads, calcification within BrM; red arrowheads, RPE/RPE-derived material as indicated.

  • Fig. 4 SEM and EDX spectroscopy of nodules, spherules, and plaques.

    (A to D) High-magnification electron micrographs of calcific lesions. (A) An isolated nodule (asterisk) is composed of lobed units ~1 to 5 μm in diameter. Cracks present on the nodule surface were formed during sectioning. (B) Spherules (asterisk) about 0.5 to 5.0 μm in diameter were located between the basal lamina of the RPE and the inner collagenous layer of BrM, along with extracellular material (arrowheads). (C) Plaques (arrowheads), tens of micrometers in length and with cracks suggestive of mineralization, appeared visually similar to nonmineralized BrM. Arrows indicate surrounding tissues. (E to J) Elemental maps of nodules, spherules, and plaques generated using EDX spectroscopy. (E to G) X-ray emission maps specific to calcium. (H to J) X-ray emission maps specific to phosphate. (K to M) Representative EDX spectra of x-ray emissions for nodules, spherules, and plaques. (N to P) HAP-specific staining confirmed the presence of apatite in nodules, spherules, and plaques (arrowheads delimit the BrM).

  • Fig. 5 TEM and SAED of nodules, spherules and plaques.

    (A to C) Low-magnification electron micrographs of nodules, spherules, and plaques. (A) Nodules were composed of multiple subunits (lobes) (1 to 20 μm in diameter). (B) Spherules (0.5 to 5 μm in diameter) exhibited increased electron density. (C) Plaques exhibited increased electron density and appeared crossed over and fibrous (inset image). (D to F) High-magnification electron micrographs of calcific lesions associated with AMD. (D) Single “lobe” subunit of a nodule. Each lobe has an electron-dense crust and a less electron-dense core. (E) Spherules displayed a heterogeneous structure; needle crystal-like structures formed a shell surrounding a less electron-dense core. (F) High-resolution micrograph of a plaque. White arrowheads indicate regions where the lattice is visible. (G to I) SAED of nodules, spherules, and plaques. A typical diffraction pattern obtained from each lesion is shown. Table S2 details additional reflexions identified by SAED but not indicated on figures. (G) Nodules were indexed to HAP. A sharp diffraction ring for (002) was present, whereas a single diffuse ring was present for reflexions (211), (112), and (300). Rings characteristic of (221) and (004) were also observed. Diffraction patterns for the crust and the core were similar. (H) The mineral component of spherules was identified as whitlockite. Zone axes (010) of whitlockite were identified with vector points for (−102), (006), and (104). (I) Diffraction patterns obtained for BrM plaques produced a diffuse ring characteristic of apatite.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/466/eaat4544/DC1

    Fig. S1. Appearance of HIRD as seen on multimodal imaging during progression.

    Fig. S2. Refractile drusen with nodules and RPE atrophy in the index case for microanalysis.

    Fig. S3. SEM of isolated nodules.

    Fig. S4. Density-dependent SEM of calcific lesions.

    Fig. S5. Synchrotron microfocus x-ray fluorescence confirms the presence of Ca in large nodules.

    Fig. S6. TOF-SIMS confirms the presence of HAP within nodules.

    Fig. S7. Proposed progression stages of calcified structures in AMD.

    Fig. S8. Two variants of HIRD.

    Table S1. Composition and appearance of calcified structures in eyes with GA as revealed by multimodal clinical and molecular imaging.

    Table S2. Table of sample sources.

    Table S3. D-spacings and I/Imax (%) of candidate calcium phosphate compounds.

    Dataset S1. Primary data from cohort 1.

    Dataset S2. Primary data from cohort 2.

    Movie S1. Progression of nodules, spherules, and BrM plaques.

  • The PDF file includes:

    • Fig. S1. Appearance of HIRD as seen on multimodal imaging during progression.
    • Fig. S2. Refractile drusen with nodules and RPE atrophy in the index case for microanalysis.
    • Fig. S3. SEM of isolated nodules.
    • Fig. S4. Density-dependent SEM of calcific lesions.
    • Fig. S5. Synchrotron microfocus x-ray fluorescence confirms the presence of Ca in large nodules.
    • Fig. S6. TOF-SIMS confirms the presence of HAP within nodules.
    • Fig. S7. Proposed progression stages of calcified structures in AMD.
    • Fig. S8. Two variants of HIRD.
    • Table S1. Composition and appearance of calcified structures in eyes with GA as revealed by multimodal clinical and molecular imaging.
    • Table S2. Table of sample sources.
    • Table S3. D-spacings and I/Imax (%) of candidate calcium phosphate compounds.
    • Legends for datasets S1 and S2
    • Legend for movie S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Dataset S1 (Microsoft Excel format). Primary data from cohort 1.
    • Dataset S2 (Microsoft Excel format). Primary data from cohort 2.
    • Movie S1 (.mov format). Progression of nodules, spherules, and BrM plaques.

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