Sub-retinal drusenoid deposits in human retina: organization and composition

Abstract

We demonstrate histologically sub-retinal drusenoid debris in three aged human eyes, two of them affected by age-related maculopathy. By postmortem fundus examination, the lesions were drusen-like, i.e., they were pale spots apparently at the level of the retinal pigment epithelium (RPE). Light and electron microscopy revealed aggregations of membranous debris, the principal constituent of soft drusen, in the sub-retinal space. Immunohistochemistry and confocal microscopy confirmed the presence of molecules typically associated with drusen (positive for unesterified cholesterol, apoE, complement factor H, and vitronectin) without evidence for molecules associated with photoreceptors (lectin-binding disaccharide bridges and opsins), Müller cells (glial fibrillary acid protein and cellular retinal binding protein, CRALPB), or RPE (CRALPB). The fact that a drusenoid material, sharing some markers with conventional drusen, can occur on opposite faces of the RPE, suggests deranged polarity of normally highly vectorial processes for basolateral secretion from RPE, and that overproduction of secreted materials and direction of secretion are independently specified processes. In the future, drusenoid sub-retinal debris might be more frequently revealed by emerging high-resolution imaging techniques.

Figure 1. Maculas of eyes with sub-retinal drusenoid debris

A-1, B-1: post-mortem fundus appearance, produced using a stereomicroscope and epi- and trans-illumination of eyecups after removal of the anterior segments. White bars, 1 mm. A-2, B-2, C: One μm-thick sections stained with toluidine-O-blue and photographed using a 40X plan fluor objective. ONL, outer nuclear layer; RPE, retinal pigment epithelium; Ch, choroid. RPE appears thicker in A-2 and C than B-2, because it was sectioned at a slightly oblique angle. Black bars, 50 μm. A-1: Case 1, left eye. Optic nerve head is at left edge of the panel. Arrow indicates several large drusen. A-2: Case 1. Sheet of retina with attached RPE was removed from Bruch's membrane. Arrowheads point to two domes of SRD. Little of the druse contents remain at the base of the RPE in this eye (arrows). B-1: Case 3, left eye. Arrows indicate some regularly scattered small drusenoid spots. B-2: Case 3. Arrowheads indicate two areas of SRD. More striking examples from Case 3 are shown in Figure 3. C: Case 2. Arrowheads indicate two domes of SRD. Some dislodged druse contents are visible (d).

Figure 2. Ultrastructure of sub-retinal drusenoid debris

RPE, retinal pigment epithelium. OS, outer segments. Bar in each panel, 10 μm. A. Case 1. Aggregation of SRD contains flocculent material with interspersed globules of homogeneous material bounded with electron-dense lines (arrowheads). Retina is at the top and choroid at the bottom of the panel. B. Case 3. Aggregation of SRD, delimited by dashed lines, contains abundant membranous loops (arrowheads) resembling similar material within basal laminar deposit (arrowhead) external to the RPE. Photoreceptor OS also appear full of expanded membranous profiles, of a much lesser electron density. The RPE cell layer, which spans the panel, is not uniform in either morphology or pigmentation. A true basal lamina is located external to the RPE (paired arrows), but not in relation to the SRD. C. Case 3. Within SRD (between the dotted line and the RPE), bundles of apical microvilli are apparent (pairs of wavy arrows).

Figure 3. Morphology of sub-retinal drusenoid debris in Case 3

A-D: Cryo-sections, 10 μm-thick, stained with Gill's hematoxylin #3. Bar in A, 50 μm, applies to all panels. OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segments; RPE, retinal pigment epithelium; C, choroid. A. Lesions (arrowhead) were typically located in the sub-retinal space sandwiched between the photoreceptor OS and the RPE. B-D. Occasionally the lesions penetrated the OS layer, IS layer, and ONL. In C, arrowheads delineate the outer border of the lesion, and the arrow indicates a tiny druse. C and D are serial sections demonstrating the progression of the debris through the photoreceptors.

Figure 4. Composition of sub-retinal drusenoid debris in Case 3

Cryo-sections, 10 μm-thick. OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium; Ch, choroid. A-C: Wide-field epi-fluorescence. Bar in C, 50 μm, applies to panels A-C. A. Filipin-stained unesterified cholesterol is abundant, in amorphous and reticulated staining patterns. SRD is delineated by yellow arrowheads. The autofluorescent RPE is out of focus. B. SRD is apolipoprotein E-immunoreactive. C. Non-immune serum control section shows no fluorescence in the SRD. D-F: Confocal fluorescence. Bar in D, 100 μm, applies to panels D-F. D. SRD is CFH-immunoreactive. Section is closer to the fovea than the sections shown in the other panels, and the Henle fiber layer of the OPL is very thick. E. SRD is vitronectin-immunoreactive. F. SRD shows no reactivity for PNA (red), which stains cone matrix sheaths, or GFAP (green), which localizes exclusively to Müller cell end feet. G-I: Epifluorescence. Bars in G and I (applies to H as well), 100 μm. G. CRALBP (green) staining is present throughout Müller cells and in the apical processes of the RPE cells, but is absent from the SRD. SRD shows no reactivity for PNA (red). H. Higher magnification of the SRD in panel G, demonstrates CRALBP immunoreactivity within apical processes of RPE cells. I. Brightfield image of SRD depicted in H. SRD is delineated by yellow arrowheads. Nuclei in panels D-H were stained with 1 μg/ml Hoechst (blue).