Age-related changes in human macular Bruch's membrane as seen by quick-freeze/deep-etch

Abstract

Lipid-containing inclusions have been observed in human Bruch's membrane (BrM) and are postulated to be associated with age-related maculopathy (ARM), a major cause of legal blindness in developed countries. The dehydration associated with specimen preparation for thin-section transmission electron microscopy causes loss of these inclusions. Better preservation of the ultrastructure of the inclusions and tissue is achieved by using a quick-freeze/deep-etch preparation. We use this technique to examine normal human macular BrM in order to characterize the deposition of the lipid-rich inclusions and their age-related accumulation within different layers of the tissue. We find that various inclusions mentioned in other studies can be formed by combinations of three basic structures: lipoprotein-like particles (LLPs), small granules (SGs) and membrane-like structures. These inclusions are associated with collagen and elastic fibrils by fine filaments. In younger eyes, these inclusions are found mostly in the elastic (EL) and outer collageneous layer (OCL) and occupy a small fraction of the interfibrillar spacing. As age increases, LLPs and SGs gradually fill the interfibrillar spacing of the EL and inner collageneous layer (ICL) of the tissue, and later form a new sublayer, the lipid wall, within the boundary region between the basal lamina of retinal pigment epithelium (RPE) and ICL. Because the formation of the lipid wall only occurs after these inclusions fill the ICL, and it seems unlikely that the LLPs can pass through the packed layer, this result suggests a possible RPE origin of the LLPs that make up the lipid wall.

Figure 1

An oblique QFDE section of the RPE-BrM-choriocapillaris complex of a 45 yr eye. Different layers of BrM are: RPE, retinal pigment epithelium; BL-RPE, basal lamina of the RPE; ICL, inner collageneous layer; EL, elastic layer; OCL, outer collageneous layer; BL-CC, basal lamina of choriocapillaris; and CC-endo, choriocapillaris endothelium marked in the figure. Bar = 2 μm.

Figure 2

Higher magnification images of different layers of the oblique section seen in Figure 1. (A) The basal lamina of RPE cells. Notice that the BL-RPE was divided into two sublayers. On the RPE side was the lamina lucida and on the other side was the lamina densa. Arrows indicate the boundary of the BL-RPE. (B) The ICL was composed by collagen fibrils (arrows) and fine filaments, on which the lipoprotein-like particles (LLPs) and small granules (SGs) were accumulated. (C) EL was marked by the wide, smooth elastin fibrils. This layer also contained the collagen fibrils and fine filaments. E, the elastin fibrils. (D) The OCL and the thin BL-CC. The OCL was composed by the same components as the ICL. The thin layer of BL-CC, indicated by arrows, was located adjacent to the vessel wall of choriocapillaris. Star, the vessel wall of choriocapillaris. Bar = 500 nm.

Figure 3

(A) LLPs (arrows) were the major inclusions seen in BrM of a 45 yr eye. The particle was usually 60 to 100 nm in diameter and composed of a core surrounded by a thin surface layer. Bar = 200 nm. (B) Two LLPs fused to form a dumbbell-shaped particle in a 78 yr eye. Bar = 50 nm.

Figure 4

(A) LLPs were associated with fine filaments as indicated by arrowheads. Eye, 34 yr. Bar = 150 nm. (B) SGs (arrows) surrounded the LLPs (stars). Bar = 150 nm.

Figure 5

(A) SGs were small round solid particles deposited among LLPs. Eye, 29 yr. Bar = 50 nm. (B) They were usually seen attached to the fine filaments (arrowheads) or LLPs in the tissue. Eye, 34 yr. Bar = 25 nm. (C) Cluster of tightly packed SGs (arrows) were also observed. Eye, 34 yr. Bar = 200 nm.

Figure 6

The ultrastructure of the LLP-SGs complex as revealed by fracturing of four LLPs (34 yr eye). Under each image is a sketch depicting the observed LLP-SGs complex. (A) A complete complex. Short struts were seen radiating from the complex and associating with other extracellular matrix components. (B) A complex with the upper half of SGs surrounding layers removed. (C) A complex with both the upper SGs and the core of LLP removed. (D) A complex with only the lower half SGs surrounding layers remained. All four conformations of the complex were often observed. Bar = 100 nm.

Figure 7

(A) A CMBB is shown in the center of the image of a 34 yr eye. Both the LLP and SG could be found within the membrane-like structure (arrows). These components seemed to be associated with each other via thin fibrils. Bar = 300 nm. (B) Higher magnification view of the membrane-like structure (arrows). Short struts extended from the membrane and associated with other components in the tissue (arrowhead). Bar = 150 nm.

Figure 8

Drusenoid structures (D) seen in (A) 29 and (B) 50 yr eyes prepared by OTAP method. CMBBs were present in panel B (arrows). Arrowheads, BL-RPE. Bar = 1μm.

Figure 9

Drusenoid structure (D) seen in a 59 yr eye. The dome-shaped deposit was located between the BL-RPE and ICL. Arrows indicate the location of the BL-RPE. Bar = 2 μm.

Figure 10

Higher magnification views of the drusenoid structure in Fig. 9. (A) Components of the drusenoid structure. All three types of inclusions, associated with each other via fine fibrils, were present. CMBBs seemed to be major component of the structure. Bar = 200 nm. (B) No collagen fibrils (arrowheads) of the ICL were found within the drusenoid structure (D). Bar = 800 nm. (C) Thin fibrils (arrowheads) were seen extended from the BL-RPE into the drusenoid structure. Bar = 200 nm.

Figure 11

Oblique sections of BrM of (A) 34 and (B) 77 yr eyes. Arrowheads indicate the BL-RPE. The EL and OCL of the younger eyes were clearly denser than the ICL. In the older eyes, the EL and ICL were packed with inclusions thus appeared more crowded than the OCL. Bars = 1μm.

Figure 12

Age-related changes of OCL. The inclusions did not seem to fill the interfibrillar spacing of the OCL. Images of younger eyes as shown in (A) to (C) resembled older ones as (D) to (F). Bar = 500 nm.

Figure 13

Age-related changes of EL. Eyes > 34 yr were packed with inclusions in this layer. Bar = 300 nm.

Figure 13

Age-related changes of EL. Eyes > 34 yr were packed with inclusions in this layer. Bar = 300 nm.

Figure 14

Age-related changes of ICL. Eyes > 60 yr were packed with inclusions in this layer. Bar = 300 nm.

Figure 14

Age-related changes of ICL. Eyes > 60 yr were packed with inclusions in this layer. Bar = 300 nm.

Figure 15

Age-related changes of the boundary between the BL-RPE and ICL in eyes < 60 yr. The lamina lucida and lamina densa of the BL-RPE can be clearly distinguished as the former has much more interfibrillar spacing. Bar = 500 nm.

Figure 16

The formation of lipid walls in older eyes. Patches of the lipid wall were seen in the boundary between the BL-RPE and ICL in eyes > 60 yr. It appeared that the lipid wall formed at different ages for different eyes. The lipid wall was found in some eyes such as (C) and (F), while in others (D, E) fewer LLPs were observed. Bar = 500 nm.

Figure 17

Comparison between the ultrastructure of the (A) lipid wall and (B) inclusions in the ICL of an 82 yr eye. The lipid wall of this eye was mainly composed of tightly packed LLPs, while both the SGs and LLPs filled interfibrillar spacing in the ICL. Large and deformed LLPs were usually seen in the lipid wall. Bar = 200 nm.