A histological study of atherosclerotic characteristics in age-related macular degeneration

Abstract

This study investigated the pathogenesis of age-related macular degeneration (AMD) using histological methods that are commonly used for atherosclerotic vascular disease (ASVD). 1 normal, 3 early dry AMD, and 1 late dry AMD eyes were obtained from the Lions Eye Bank of Oregon and systematically dissected. They were stained with hematoxylin and eosin, Oil red O, Masson, Elastica van Gieson, Alizarin red, and Prussian blue. Additionally, the normal and late dry AMD eyes were immunostained for a-smooth muscle actin, CD45, and CD68 with Nile red and DAPI. Correlations were found between severity of AMD and lipid accumulation in the deep sclera (+), numbers of drusen between the Bruch's membrane and retinal pigment epithelium (RPE) (+), amount of collagen in the deep sclera (+), and amount of elastin in the deep sclera (-) (P < 0.1). Geographic atrophy, RPE detachment, and abnormal capillary shape and distribution in the choriocapillaris were observed in the fovea of late AMD. There were no stenosis, plaque, hemorrhage, and calcification. Additionally, late AMD tended to have higher smooth muscle thicknesses of the choroidal vascular walls, lower numbers of T lymphocytes in the choroid, and higher numbers of macrophages near the RPE and in the choroid relative to normal (P < 0.1). Macrophages-derived foam cells were detected near the Bruch's membrane in late AMD. Therefore, the present study showed many histological characteristics of ASVD in AMD, which suggests an association between them; however, there were also some histological characteristics of ASVD that were not found in AMD, which indicates that there exist pathogenic differences between them. The results generally support the vascular model of AMD, but some details still need clarification.

Keywords: Atherosclerosis; Atherosclerotic vascular disease; Blood vessel; Histology; Staining.

Figure 1

Eye dissection. After removing the anterior tissues and the vitreous, the posterior landmarks became visible (a). Two tissue segments were dissected out based on the landmarks, and tissue sections were collected from four different areas (b).

Figure 2

Representative images of normal and atherosclerotic blood vessel tissues of human coronary artery intima with different stains and immunostains. In atherosclerotic tissue, intimal thickening and plaque rupture were present in atherosclerotic lesions (b, scale bar = 400 μm) (b). Large amount of lipid droplets (blue arrow) and cholesterol crystals (black arrow) accumulated especially in plaque (d, scale bar = 200 μm). Fibroproliferation and fibrous cap (black arrow) (f, scale bar = 200 μm), elastin reduction (h, scale bar = 200 μm), calcium salt deposition (black arrows) (j, scale bar = 300 μm), and hemorrhage (black arrow) (l, scale bar = 100 μm) also clearly appeared in atherosclerotic tissue. Additionally, smooth muscle cells (n, scale bar = 100 μm), T lymphocytes (p, scale bar = 40 μm), and macrophages (r, scale bar = 40 μm) visibly increased in atherosclerotic tissue. Furthermore, macrophage-derived foam cells (white arrow) were detected by triple staining of CD68, Nile red, and DAPI (r). These characteristics were not seen in normal tissue (a, scale bar = 500 μm; c, scale bar = 100 μm; e, scale bar = 100 μm; g, scale bar = 200 μm; i, scale bar = 200 μm; k, scale bar = 100 μm; m, scale bar = 100 μm; o, scale bar = 40 μm; q, scale bar = 40 μm).

Figure 3

Representative images of Oil red O-stained ocular tissue in deep sclera. Lipids mostly accumulated in the deep sclera of the posterior eyes. The amount of lipid accumulation was visibly different between normal (a), early AMD (b), and late AMD (c). Furthermore, there appeared to be a gradual increase in the amount of lipid accumulation with distance from the choroid. DS, deep sclera; SS, superficial sclera. Scale bars = 200 μm.

Figure 4

Ratio of lipid accumulation versus severity of AMD in deep sclera.

Figure 5

Representative images of Oil red O-stained ocular tissue between BrM and RPE. Drusen could hardly be seen in normal eye, and the RPE was intact (a). Hard drusen (red arrow), compound drusen (green arrow), and mild RPE disruption (black arrow) were seen in early AMD eyes (b). In addition to hard and compound drusen, empty spaces (asterisk), which were most likely soft drusen that had lost its lipid content during the fixation and dehydration procedures as previously reported, were detected only in late AMD eye (c) [57, 58]. Moreover, severe RPE disruption was seen in non-atrophic regions of late AMD (black arrow) (c). Geographic atrophy, RPE detachment (black arrow), and abnormal capillary shape (non-circular; blue arrow) and distribution (uneven) in the choriocapillaris were seen in the fovea of late AMD (d). IS/OS, inner and outer segments of photoreceptors; CC, choriocapillaris. Scale bars = 50 μm.

Figure 6

Numbers of drusen versus severity of AMD between BrM and RPE.

Figure 7

Representative images of Masson-stained ocular tissue in deep sclera. The amount of collagen was visibly different between normal (a), early AMD (b), and late AMD (c). Scale bars = 40 μm.

Figure 8

Ratio of collagen versus severity of AMD in deep sclera.

Figure 9

Representative images of EVG-stained ocular tissue in deep sclera. Elastin fibers (black arrows) were distributed in the space between collagen bundles, and the amount of elastin was visibly different between normal (a), early AMD (b), and late AMD (c). Scale bars = 40 μm.

Figure 10

Ratio of elastin versus severity of AMD in deep sclera.

Figure 11

Representative images of vascular smooth muscle fibers in choroid. The vascular walls were visibly thinner in normal (a) compared to late AMD (b). Sections were labelled with a-SMA (green), Nile red (red) and DAPI (blue). Scale bars = 50 μm.

Figure 12

Smooth muscle thickness of vascular walls in normal and late AMD eyes in choroid.

Figure 13

Representative images of T lymphocytes in choroid. There were visibly more T lymphocytes (white arrows) in normal (a) compared to late AMD (b). Sections were labelled with CD45 (green), Nile red (red) and DAPI (blue). Scale bars = 20 μm.

Figure 14

Number of T lymphocytes in normal and late AMD eyes in choroid.

Figure 15

Representative images of macrophages near RPE and in choroid. In normal, macrophages were uniformly distributed in the choroid (white arrows), and they were rarely seen near the RPE (a). In late AMD, macrophages concentrated near the RPE (or where it used to be) and in the choroid in the atrophic region (white arrows) and non-atrophic region (orange arrows) (b). Sections were labelled with CD68 (green), Nile red (red) and DAPI (blue). Chor, choroid. Scale bars = 50 μm.

Figure 16

Numbers of macrophages in normal and late AMD eyes near RPE (a) and in choroid (b).

Figure 17

Macrophage-derived foam cells (white arrows) near BrM in late AMD. The cells were co-stained with CD68 (green), Nile red (red), and DAPI (blue). Scale bar = 20 μm.