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Histopathological Characteristics of Age-Related Macular Degeneration
Ehud Zamir
Hadassah–Hebrew University Medical School, Jerusalem, Israel
Narsing A. Rao
Doheny Eye Institute, University of Southern California Keck School of Medicine, Los Angeles, California
I.INTRODUCTION
Age-related macular degeneration (AMD) is common among the elderly, and its incidence increases progressively with advancing age. According to the Beaver Dam Eye Study, 11% of the population are affected by AMD at age 65–74, and this number increases to 28% after age 74. Therefore, postmortem studies have been a source of extensive histopathological information on this disease. Large series have been presented and analyzed in multiple studies, and the result is a significant body of well-established histopathological data that is available at present. Additional information derives from the availability of subretinal neovascular membranes removed by a variety of increasingly popular subretinal surgery techniques (1). AMD is currently well characterized at both the light microscopic and the electron microscopic level.
The histopathological hallmark of early, nonexudative AMD is the occurrence of drusen, as well as basal deposits along the basement membrane of the retinal pigment epithelium, with degeneration of the overlying retinal pigment epithelium (RPE) and photoreceptor cells. Drusen are classified into three main categories: small, hard drusen (also termed nodular drusen), soft drusen, and large, confluent drusen. Small, hard drusen are the most common type. They are, by definition, smaller than 63 microns, and may be present in the eyes of patients with or without AMD, including young individuals. They are not considered to be a risk factor for AMD (2–5). Hard drusen can be detected clinically when they reach the size of 25–30 microns (2), and they tend to hyperfluoresce on fluorescein angiography. Histologically, they appear as globular, hyaline structures external to the RPE basement membrane. The overlying RPE cells may be atrophic (5) (Fig. 1). Ultrastructurally, hard drusen contain membrane-bound bodies that are found in what Sarks and associates have described as “entrapment sites,” in which coated, membrane-bound bodies are “trapped” between the RPE basement membrane and the inner collagenous layer of Bruch’s membrane (2). This material is currently presumed to derive from the RPE (5).
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Figure 1 A hard druse. A globular hyaline deposit with overlying RPE atrophy. The retina is artifactually detached, thus not shown in this picture (H&E). See also color insert, Fig. 2.1.
Bruch’s membrane normally contains five layers, in the following order: Innermost is the basement membrane of the RPE, followed by the inner collagenous layer of Bruch’s membrane, the elastic lamina, the outer collagenous layer, and the basement membrane of the choriocappillaris. Basal deposits (6) are collections of acellular debris in different planes in and along Bruch’s membrane (Fig. 2, 3). Most authors define two different types of basal deposits as follows: Amorphous, acellular debris accumulating between the basal plasma membrane of the retinal pigment epithelium and the basement membrane of the RPE is referred to as basal laminar deposits (BLamD) (3). In contrast, deposition of material external to the BM of the RPE, e.g., between the latter and the inner collagenous layer of Bruch’s membrane, is termed basal linear deposits (BlinD). The two types differ not only in their anatomical distribution, but also with regard to their chemical characteristics and pathological significance, as will be explained below. Differentiation of these two findings on light microscopic grounds is difficult, and there may be significant morphological overlap (4). Transmission electron microscopy is the main tool in detecting and analyzing those deposits. It demonstrates that BLamD are composed of fibrous long-spacing collagen (possibly type IV collagen); amorphous, basement membrane-like deposits are features of normal aging that appear after age 60 and are not markers of AMD.
BlinD, also termed diffuse or confluent drusen (3,7,8), are composed of membraneous material and this was found to be a sensitive and specific feature of AMD, although the association between the two does not necessarily indicate causality. Rather, both could reflect damage to the RPE (6). While BLamD only appear after age 60, they are not specific to AMD, and are also present in non-AMD, aging eyes. In contrast, the prevalence of BlinD and large, soft drusen is 24 times higher in eyes with AMD compared to age-
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Figure 2 Basal laminar deposit (bld) lies between the retinal pigment epithelium (rpe) and the inner aspect of Bruch’s membrane in this choroidal neovascular membrane excised from a patient with age-related macular degeneration. Wide-spaced collagen is in the basal laminar deposit and scarred choroid versus a component of the choroidal neovascular membrane (asterisk) are present within Bruch’s membrane (original magnification, 10,440; inset, original magnificaton, 400). Reprinted with permission from Ref. 14.)
matched controls (6). BlinD are composed of granular, vesicular, or membranous material, with foci of wide-spaced collagen. The origin of that membranous debris is thought to be membranes of photoreceptor outer segments, delivered by the RPE in the form of vacuoles and vesicles (6,7,9). Soft drusen (large, poorly delineated drusen) may represent focal accentuation of that basal linear material, while its diffuse deposition forms BlinD (3,8). Soft drusen are seen by light microscopy as localized deposition of granular, pale material with sloping edges (4). They can also represent areas of detachment of BlinD or BLamD by proteinatious material. Larger detachments of basal deposits correspond to serous pigment epithelial detachments. Unlike hard drusen, large, soft, and confluent drusen (Fig. 4) are a sign of AMD (4). In Green and Enger’ s series of 760 globes with AMD, 10.9% showed soft drusen by histopathology, and 27.6% had basal linear deposits (3).
All forms of drusen may show calcification in the process of regression (5). It was shown to be present in nodular (hard) and soft drusen in 0.8% and 7.7% of eyes with AMD, respectively (3).
Calcification and fragmentation of Bruch’s membrane were shown to be associated with AMD, and to be more severe in eyes with exudative AMD compared to dry AMD (4). Calcification was mostly found in the elastic layer of Bruch’s membrane, and both calcification and fragmentation were more common in the macular, compared to the extramacular,
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Figure 3 A retinal pigment epithelial cell with a bland nuceus (N) displays basal laminar deposit (between arrows) and basal linear deposit (between arrowheads) (transmission electron microscopy, original magnification 3610). Close inspection shows that the basal laminar deposit (lam, upper inset, transmission electron microscopy, original mangnification 5365) is located between the plasma membrane and basement membrane (arrowheads) of the retinal pigment epithelium, and the basal linear deposit (lin, lower inset, transmission electron microscopy, original magnification 2600) is external to the basement membrane. (Reprinted with permission from Ref. 17).
Figure 4 Basal deposits (“diffuse drusen”). There is a marked sub-RPE thickening of the Bruchs’ membrane (arrows) (PAS). See also color insert, Fig. 2.4.
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Figure 5 Diskiform scarring, low magnification. Notice dystrophic calcification. Calcium crystals are seen within the substance of the sub-RPE fibrous plaque (arrow). See also color insert, Fig. 2.5.
area (4). The authors suggested that such changes in Bruch’s membrane play a role, together with diseased RPE and basal deposits, in facilitating growth of fibrovascular membranes.
II.GEOGRAPHICAL (AREOLAR) AGE-RELATED MACULAR DEGENERATION
This form, also called “dry AMD,” is more common than the “wet,” neovascular form. Its main features include atrophy, migration, and degeneration of the RPE cells. There is a tendency for sparing of the foveal area early on, with eventual foveal involvement and more severe visual loss. This fact was attributed by Weiter et al. (10) to the higher concentration of xanthophyll pigment. There is secondary degeneration and loss of the photoreceptors that overlie the degenerating RPE cells, including their inner segments in severe cases (Fig. 5). This condition may follow serous RPE detachments or large drusen (11). In Green and Enger’s series (3), 37% of the eyes had RPE atrophy.
III.THE “WET” (NEOVASCULAR) FORM OF AMD
This form is characterized by growth of choroidal neovascular membranes into the subRPE or subretinal areas. These membranes can distort the macular topography and later produce hemorrhagic or serous RPE and retinal detachments. The latter are the major sources of visual loss in AMD. Later in the process, these lesions may scar to form a fi-
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brovascular, “diskiform” scar (Fig. 5–7). In the early stages, choroidal capillaries are seen growing into the Bruch’s membrane. The proliferating choroidal vessels start as capillaries and develop into arteries and veins over time. Exudation of lipid under the RPE or into the subretinal space may be seen. Bleeding into the sub-RPE space is common, and blood may gain access into the subretinal space, and even into the vitreous cavity. At a later stage, this serous or hemorrhagic detachment evolves into a dense fibrous scar, clinically described as a “diskiform” scar. Such lesions were found in 40.6% of 760 globes studied by Green and Enger (3). The scar may contain hyperplastic RPE cells and basement membrane, as well as hemosiderin and calcifications. It may have a single subretinal component, or three components separated by RPE basement membrane and basal deposits. The larger the diskiform scar, the more likely it is to find degenerated photoreceptors in the overlying retina. RPE tears were found in 6.8% of eyes (3). A rare finding in diskiform scars is the occurrence of granulomatous reaction with giant cells (12,13). Dastgheib and Green (12) described a diskiform scar of at least 11 years’ duration, which had a prominent foreign-body giant cell response located at the Bruch’s membrane, with cytoplasmic extensions from some of the cells in the inner and outer layers of Bruch’s membrane. These cells seemed to be actively breaking down Bruch’s membrane and engulfing it. The authors hypothesized that inflammatory response may take part in the pathogenesis of breaks in Bruch’s membrane and the ensuing choroidal neovascularization. By this theory, the diseased RPE as well as the basal deposits induce a local inflammatory response that includes macrophages, T-cell activation, and formation of multinucleated giant cells. These in turn may cause damage to the Bruch’s membrane and secrete angiogenic factors, both contributing to the development of choroidal neovascular membranes (CNVM). Excised neovascular membranes from eyes with AMD, but not from other underlying etiologies, also contained foreign-body giant
Figure 6 Same case as in Figure 5, higher magnification. Notice degenerated outer segments of photoreceptors and atrophic RPE. There is a sub-RPE fibrous sheet. See also color insert, Fig. 2.6.
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Figure 7 Diagram of the stages of development and surgical excision of type 2 choroidal neovascularization. Closed arrows indicate growth of new capillaries from the choriocapillaris (cc), through a defect in the focally damaged Bruch’s membrane (Bm), and into the subsensory retinal space accompanied by proliferating retinal pigment epithelial cells (RPE) at their advancing border and along their posterior surface. Open arrow shows how the neovascular membrane was removed through retinotomy site using forceps. (Reprinted with permission from Ref. 21.)
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cells and basal deposits (14). These findings have been suggested as evidence for CNVM representing a stereotypical, nonspecific wound repair-like process that is analogous to granulation tissue (15).
IV. EXCISED SUBRETINAL MEMBRANES AND
ANGIOGRAPHIC CORRELATIONS
Studies of excised subretinal membranes reveal that their most common constituents are RPE cells, vascular endothelium, and fibrocytes. Less common are inflammatory cells, macrophages, photoreceptors, myofibroblasts, pericytes, and choroidal tissue (14,16,17). The anatomical level at which CNVM grow has been a subject of interest of a large number of studies. Evidence from different authors has suggested a predominant sub-RPE growth pattern (3,18–20). In Green and Enger’s series, 39/44 early (small) membranes grew under the RPE, and only 5/44 were subretinal (3). However, of 310 disciform scars, 80% had subretinal involvement. Gass (21) has suggested two types of CNVM: Type I, common in AMD, grows under the RPE, while type 11, seen in other pathologies such as the presumed ocular histoplasmosis syndrome (POHS), grows under the neurosensory retina (Fig. 8,9). Gass postulated that, since there is a generalized weakening of normal adhesion between the RPE and Bruch’s membrane in AMD, the proliferating choroidal vessels are growing unimpeded in the sub-RPE space. In contrast, POHS includes only focal defects in the RPE–Bruch’s membrane integrity, and therefore, the vascular proliferation cannot easily grow under the RPE. According to this theory, these anatomical differences would explain the less favorable visual results after excision of membranes in AMD, compared to POHS. In the former, the membrane does not grow in a potential space, but rather within the inner layers of Bruch’s membrane. Therefore, its removal would cause more destruction to the overlying RPE, limiting final visual acuity.
Evidence from histopathological studies of excised membranes has been accumulating in recent years. Grossniklaus et al. (14) studied 90 excised membranes from eyes with AMD, and correlated their anatomical localization and other histopathological features with their fluorescein angiographic characteristics (all membranes with interpretable FA were classic, and were subdivided into well-demarcated versus poorly demarcated). Approximately 60% of the membranes were classic, well demarcated, while the other 40% were classic and poorly demarcated. Well-demarcated membranes had a “bull’s-eye” appearance on FA, with a hyperfluorescent center that histologically corresponded to the sub-RPE fibrovascular core of the membrane. The rim of blocked fluorescence corresponded to hypertrophied RPE, and the outer rim of faint hyperfluorescence corresponded to subretinal fibrin. Conversely, membranes that histologically seemed to break through the RPE into the subretinal space were mostly poorly demarcated in the preoperative FA, showing a pattern of scalloped net with late obscuration. The authors suggest that well-demarcated, sub-RPE membranes may evolve into “breakthrough,” poorly demarcated, subneurosensory retinal membranes. Results from the Submacular Surgery Trial, reported by Grossniklaus and Green (17), showed that of 32 excised AMD membranes that could be anatomically oriented, 16 were subretinal. Sixteen had a sub-RPE component, and all the non-AMD membranes were subretinal. Surpisingly, this study showed that a significant number (50%) of AMD membranes could be localized to the subretinal space and had no sub-RPE component. This is in contrast to earlier reports showing predominantly sub-RPE location of CNVM (3,18–21).
Lafaut et al. (22) described a different correlation between the location of membranes relative to the RPE and their fluorescein angiographic features. The study included 35 AMD membranes that were preoperatively classified as classic or occult, based on the definitions of
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Figure 8 Diagram of stages of development and surgical excision of type 1 choroidal neovascularization. Closed arrows indicate growth of new capillaries from choriocapillaris (cc) through Bruch’s membrane (Bm) and between the basement membrane of the retinal pigment epithelium (RPE) and the thickened and degenerated inner collagenous zone of Bruch’s membrane. Open arrow shows how the neovascular membrane was removed through a retinotomy by using forceps. (Reprinted from Ref. 21.)
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Figure 9 Surgically excised subfoveal choroidal neovascular membrane specimen from an 82- year-old man with AMD. Outer retinal elements were included in the excised specimen. Notice a dense fibrovascular membrane above the RPE and underneath the photoreceptor nuclei. This is a type II, or subretinal, membrane, which, according to Gass’ theory, is more typical of non-AMD membranes (21). See also color insert, Fig. 2.9.
the Macular Photocoagulation Study (23). This is in contrast to earlier studies that only included classic membranes, as described above. Histopathological features were studied by light microscopy. Classic membranes had a predominant subretinal fibrovascular component, while occult membranes predominantly involved the sub-RPE space. Mixed (classic and occult) membranes had both subretinal and sub-RPE histopathological components. It was also found that the vascular pattern of classic membrane included presence of both capillaries and large-caliber vessels, whereas occult membranes contained mostly capillaries. The authors therefore suggested that CNVM usually starts at the sub-RPE level, and appears classical on fluorescein angiography (FA) if it breaks through the RPE, into the subretinal space. Otherwise, if it remains sub-RPE, it has FA features of “occult” membranes. Part of this inconsistency of results between different studies stems from the inherent difficulty in properly orienting the excised specimens to determine whether they are from the sub-RPE or the subretinal space. Grossniklaus et al. (14) have noted that the presence of RPE in the membrane, although usually used to indicate a sub-RPE location, may be misleading. Membranes removed from the subretinal space also contain RPE cells, lining their external surface (Fig. 10). This may simulate a sub-RPE membrane and hence prohibit proper orientation. Overall, only about half the specimens studied in another publication from the same group could be oriented, owing to folds or lack of landmarks (17).
Grossniklaus et al. have suggested that the lack of vascular endothelium in excised membranes may represent inadequate surgical removal and may be a predictor of recurrence (14). This was based on the observation that approximately 50% of the membranes that recurred did not show vascular endothelium.
A study by Lee et al. (24) examined the light microscopic features of 14 surgically
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Figure 10 A choroidal neovascular membrane breaking through the Bruch’s membrane and into the sub-RPE plane (“type II” membrane). Notice intact Bruch’s membrane at the right side of the picture (arrow). Elements seen in the membrane include a capillary, a few mononuclear leukocytes, endothelial cells, RPE cells, and collagen (PAS). See also color insert, Fig. 2.10.
removed subretinal neovascular membranes related to AMD. One membrane was studied by TEM. All were well-defined membranes smaller than 3.5 disk areas as seen by preoperative FA. In addition, ICG angiography was performed in all patients before and after surgery. Cellular elements found in the excised membranes included RPE cells, vascular endothelium, inflammatory cells (including rare foreign-body giant cells), red blood cells, smooth muscle cells, and fibrocytes (spindle-shaped cells). Acellular constituents included basal laminar deposits, Bruch’s membrane, collagen, and fibrin. No correlation was found between the anatomical location of the membranes (sub-RPE vs. subretinal) and the ICG angiographic features (well demarcated vs. poorly demarcated). However, this study only included membranes with well-demarcated membranes on FA, while ICG angiography is more useful in cases of occult membranes, which are often poorly demarcated.
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