THE ANATOMICAL RECORD 226:295-306 (19901
SEM and TEM Analyses of Isolated Human Retina1 Microvessel Basement Membranes in Diabetic Retinopathy EDWARD C. CARLSON
AND
NANCY J. BJORK
Department of Anatomy and Cell Biology, School of Medicine, University of North Dakota, Grand Forks, North Dakota 58202
ABSTRACT Human retinas from persons with diabetic retinopathy and agematched controls were rendered acellular by sequential detergent treatment. The resulting network of microvascular extracellular matrix (ECM) materials, including basement membranes (BMs), was compared by TEM and, following cryofracture, by SEM. Our study demonstrates that in diabetics, retinal capillmy BM complexes are generally thickened and that their ECM subcomponents, including BM leaflets and BM-like pericytic matrix (PCM), are differentially altered. Two diabetic microvessel types were identified. In type A vessels, ECM expansion is manifested by loosely arranged combinations of concentric PCM layers and collagen fibrils with thickened subendothelial (EBM) and pericyte (PBM) BM leaflets. Type B vessels show densely compact central PCM masses and poorly recognizable EBMs and PBMs. In both types, Muller cell BMs (MBMs) are relatively unaffected. High-resolution SEM shows tissue-specific features in normal EBM and MBM surfaces, but disease-related topographic changes are not evident. It is possible that the ECM arrangements identified in our study relate to different microvessel domains and that their specific morphological features may play important roles in the pathogenesis of diabetic retinopathy including capillary closure and neovascularization. Light (LM) and transmission electron microscopic (TEM) observations of human retinal microvessels and their associated extracellular matrix (ECM) components in late stages of diabetic retinopathy have been described in numerous reports (Toussaint and Dustin, 1963; Ashton, 1965; Oliveira, 1966; Cogan and Kuwabara, 1967; Bloodworth, 1967; Speiser et al., 1968; Ashton, 1974; Henkind, 1978; Ashton, 1983; Fryczkowski and Sato, 1986). In these studies, the most commonly cited disease-related alterations relate to basement membrane (BM) thickening. Concomitant with this inexorable BM accumulation is a series of cellular events the pathogenetic chronology of which is unclear. These include breakdown of the blood-retina1 barrier, formation of microaneurysms, capillary closure, selective intramural pericyte degeneration, vessel shunting, exudate formation, and neovascularization (see Ashton, 1974, for review). Most TEM descriptions of diabetic retinal vessels were carried out on intact tissues derived from autopsy or enucleation and were important because they demonstrated relationships of retinal vessel cells to their associated BMs in chronic stages of diabetic retinopathy. Moreover, because they required a re-examination of nondiabetic retinal capillaries as controls, several comparative ultrastructural studies of normal and diabetic human retinal microvessels were carried out (Toussaint and Dustin, 1963; Ashton, 1965; Cogan and Kuwabara, 1967; Bloodworth, 1967). The presence of cells in these preparations, however, inhibited morpho0 1990 WILEY-LISS, INC.
logical analyses of the distributions of noncellular materials and prevented three-dimensional demonstrations of their topographical (surface) features by scanning electron microscopy (SEM). The development of a method for selectively solubilizing cellular materials from organ subfractions and leaving ECM components (including BMs) intact (Meezan et al., 1975; Carlson et al., 1978) provided a n opportunity for morphological investigations of isolated retinal microvessel BMs. In a n effort to study the morphological specificities of retinal capillary BMs, we recently applied this technique to bovine (Carlson et al., 1988; Carlson, 1988) and human (Carlson, in pressaSb)tissues. Our studies showed that following sequential detergent treatment, the histoarchitectural relationships of all retinal capillary BM leaflets and associated pericytic matrix (PCM) were preserved. Furthermore, these structures were easily prepared for LM, TEM, and SEM analysis. In the current study, isolated retinal microvessel BMs from patients with clinically identified diabetic retinopathy were examined. The purpose of the study was to demonstrate disease-related morphological alterations in BMs from diabetic patients. Efforts were made to localize these changes to specific retinal capillary BM leaflets or to PCM. We show by correlated
Received November 14,1988; accepted June 21, 1989.
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E.C. CARLSON AND N.J. BJORK
Figs. 1-4.
RETINAL MICROVESSEL BASEMENT MEMBRANES IN DIABETES
TEM and SEM analyses, fresh new views of acellular human retinal microvessel BMs in normal and diabetic retinal microvessels and emphasize the relationships of altered BM leaflets to the entire capillary BM complex. MATERIALS AND METHODS
Fifty human eyes, posterior eye cups, or isolated retinas from insulin-dependent diabetic patients (aged 50-69) and more than 100 normal eyes were received from the National Disease Research Interchange (NDRI), Philadelphia, Pennsylvania, and from the Department of Pathology, the University of North Dakota, Grand Forks, North Dakota. The tissues were derived from autopsy or enucleation procedures and were received on ice within 72 h r of surgical removal or death. They were shipped to our laboratory in 0.1 M Tris-buffered saline with protease inhibitors (0.025 M ethylenediaminetetraacetic acid [EDTA], 0.001 M benzamidine hydrochloride, 0.001 M phenylmethylsulfonyl fluoride [PMSF], 0.01 M N-ethylmaleimide “EM]) on wet ice. All tissues used in the current study were from patients with known diabetic retinopathy or age-matched controls (individuals with no prior history of eye disease). Preparation of Acellular Retinas Retinas were removed from whole eyes and posterior eye cups and either fixed immediately for TEM or placed in individual 50 ml tubes with at least 40 ml of 0.01 M EDTA and 0.05% sodium azide per retina. Osmotic lysis was allowed to proceed for several hours at 4°C following which the fluids were replaced sequentially by 3% Triton X-100, several rinses of distilled water, 0.025% DNAase in 1M NaC1,4% sodium deoxy-
297
cholate, and numerous final rinses of distilled water to remove the detergent. The conditions of the steps in this procedure were identical to those previously published (Carlson, 1988). This revealed a n arborizing network of acellular retinal microvessel BMs and associated ECM. Occasionally sheets of acellular internal limiting membrane were found in the preparation but were easily removed with forceps or drawn pipet. Acellular retinas resulting from this procedure were photographed in a n Olympus BH-2 compound photomicroscope equipped with darkfield illumination and fixed for TEM and SEM as described below. Electron Microscopy
Primary fixation was carried out in paraformaldehyde/glutaraldehyde (Karnovsky, 1965) buffered at pH 7.4 with 0.2 M sodium cacodylate/HCl for 1h r at room temperature. Tissue samples were post-fixed 90 min (4°C) in 2% Os04 buffered with 0.15 M sodium cacodylate/HCl. These were rinsed in distilled water and prepared for TEM a s previously described (Carlson et al., 1988). Samples to be studied by SEM were cryofractured prior to observation (Carlson and Hinds, 1983). In brief, acellular retinas were transferred from absolute ethanol to Freon 22 (cooled with liquid N2) and then plunged directly into liquid N2. These were cleaved with a chilled single-edged razor blade and returned to absolute ethanol prior to critical point drying, mounting, and coating with gold-palladium as described previously (Carlson et al., 1988). Coated preparations were observed and photographed in a n Hitachi S-800 field emission SEM at original magnifications of 100-70,000 diameters. RESULTS
At the level of TEM, cross sections through normal adult human retinal capillaries show they are comprised of inner continuous layers of endothelial cells Fig. 1. Low-magnification transmission electron micrograph of surrounded by discontinuous processes of intramural section through normal human retinal capillary. The vessel wall consists of continuous endothelium (EC) surrounded by an interrupted pericytes (Fig. 1).These cell types are invested by a (arrows) subendothelial basement membrane (EBM).More externally complex system of BM leaflets which together with are numerous processes of intramural pericytes (P) surrounded by PCM collectively constitutes the retinal capillary BM. pericytic basement membrane (PBM) except where pericytes and en- The system consists of: 1) a n outer Muller cell BM dothelial cells contact. The vessel wall is surrounded by Muller cells (MC) which lie subjacent to their own basement membrane (MBM). (MBM) which is uninterrupted and closely applied to Pericytic matrix (PCM) comprised of collagen fibrils and fibrillogran- basal surfaces of surrounding Muller cells, 2) a n inner ular basement-membrane-like material fills in the perivascular subendothelial BM (EBM) which is discontinuous at space. x 8,300. points where endothelial cells and pericytes are in close contact, and 3) pericytic BM (PBM) which surrounds Fig. 2. Darkfield light micrograph of isolated normal human retcytoplasmic processes of pericytes, and which may be inal microvessel basement membranes (RMBM). Following detergent treatment a n arborizing network of vessel basement membranes com- fused to MBM or EBM. In addition, a BM-like pericytic prised mainly of capillaries retains its in situ histoarchitecture. matrix (PCM) similar to glomerular mesangial matrix x 145. (MM), and numerous collagen fibrils, occupy the perivascular connective tissue space. Fig. 3. Transmission electron micrograph of detergent-treated normal human retinal capillary. All cellular elements of the vessel wall When retinas are dissociated from the underlying are solubilized leaving only the component basement membranes and retinal pigment epithelium and sequentially extracted associated extracellular matrix (compare with Fig. 1). Detail of area similar to that shown in rectangle is shown in Figure 5. EBM, suben- with detergents and endonuclease, all cellular elements are solubilized leaving only the internal limitdothelial basement membrane; PBM, pericyte basement membrane; MBM, Muller cell basement membrane; PCM, pericytic matrix; (PI, ing membrane (which usually can be removed a s a sin“pockets” occupied by pericytes in vivo. X 9,700. gle acellular sheet) and a network of translucent “tubes” clearly demonstrated by darkfield microscopy Fig. 4. Transmission electron micrograph of detergent-treated hu(Fig. 2). The network consists primarily of acellular man retinal capillary basement membrane in diabetic retinopathy. The layer of fibrillar collagen (COL) at left is related to a large adja- capillary-sized vessels which freely anastomose and cent vessel. Individual BM leaflets including subendothelial (EBM) branch from larger arteriolar and venular structures. and Muller (MBM) are recognizable. A massive increase in pericytic Cross sections show that these microvessel “ghosts” do matrix (PCM) is obvious (compare with Fig. 3). Detail of area similar not collapse and retain their in situ cylindrical shapes to that shown in rectangle is shown in Figure 6. x 9,400.
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Figs. 5, 6.
RETINAL MICROVESSEL BASEMENT MEMBRANES IN DIABETES
(Fig. 3 ) . Moreover, they maintain their morphological integrity and all subcomponents of the retinal capillary BM are recognizable in their in vivo histoarchitectural arrangements (compare Figs. 1 and 3). However, in these specimens cells are conspicuously absent and discontinuous EBMs border vessel lumens. Furthermore, spaces previously occupied by pericytes are represented by empty “pockets” surrounded by PBM. When similar preparations are made from retinas derived from patients with clinically confirmed diabetic retinopathy, some of the retinal capillary BMs are dramatically altered (Fig. 4). Although it is difficult to show micrographs representative of all acellular diabetic vessels, most show increased thickness of one or more BM subcomponents while many exhibit considerable PCM redundancy, often in concentric layers. Increased electron density of the PCM is also a feature that is common to nearly all. At increased magnifications, normal acellular walls show interrupted EBMs while MBMs are continuous and more compact with collagen fibrils frequently inserted into their adventitial surfaces (Fig. 5). PBMs surround empty pericytic “pockets” while PCM fills in much of the perivascular space and surrounds “nests” of collagen fibrils which usually are located adjacent to the MBM and rarely intermingle with PCM. Thicknesses of entire capillary BM complexes are highly variable and are related to the number and distribution of cellular elements in various portions of the vessel wall. Our data indicate, however, that when measurements are taken a t the thinnest points from circular samples (i.e., specimens least likely to be sectioned obliquely) they range from 750-900 nm (Table 1). When acellular capillary BMs in diabetic retinopathy are carefully examined, at least two types are evident. One (type A) shows obvious EBM and PBM thickening (up to 300 nm) and concentric layers of electron-dense BM-like material which occupy most of the vessel wall (Figs. 4, 6A). In a second type, (type B) EBMs and PBMs are poorly recognizable and the majority of the capillary wall is replaced by electron-dense PCM (Fig. 6B). The latter is not distinctly layered, however, and may represent a massively thickened
Fig. 5. Transmission electron micrograph of portion of acellular normal human retinal capillary basement membrane in area similar to that shown in rectangle in Figure 3. The capillary BM complex is comprised of three morphologically distinct basement membranes including a discontinuous subendothelial (EBM), a more sharply delineated Muller (MBM), and a pericytic (PBM) which surround “pockets” (P) previously occupied by pericytes. The pericytic matrix (PCM) is loosely distributed in the perivascular space. COL, collagen fibrils. x 32,300. Fig.6. Transmission electron micrographs of portions of acellular human retinal capillary basement membranes taken from patients with advanced diabetic retinopathy. The areas shown are similar to that indicated by rectangle in Figure 4. A Type A vessel in which individual EBM and PBM leaflets are variously thickened. Bracket indicates area of increased pericytic matrix (PCM) electron density (compare with Fig. 5). COL, collagen fibrils; (P), pericytic pocket. B Type B vessel with indistinct subendothelial basement membrane. Miiller (MBM) and pericyte (PBM) basement membranes are recognizable. Bracket shows area where pericytic matrix (PCM) appears greatly increased over normal counterparts (compare with Fig. 5). (P), pericytic pocket; COL, collagen fibrils. A, x 20,500. B, X 26,600.
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EBM which extends up to, but does not replace, the sub-MBM collagen layer. In both types MBMs seem relatively unchanged from their normal counterparts, averaging about 100 nm in thickness. As with normal specimens, thickness measurements of diabetic retinal BM complexes are only marginally accurate because sampling is difficult. Nevertheless, points taken randomly from the thinnest areas of more than 100 cross sections indicate that overall thicknesses of retinal capillary BM complexes ranged from 1,200-3,000 nm in diabetic retinopathy (Table 1). Although TEM sections demonstrate that human retinal capillary BMs from both normal and diabetics maintain their cylindrical shapes following cell removal, it may be argued that such intrinsic structural rigidity is related to epoxy support following fixation. To test this hypothesis, fixed acellular vessels were cryofractured and then prepared for SEM observation. Despite the absence of liquids or resins these samples did not collapse but remained intact, and they demonstrated tubular shapes in which surfaces of external MBMs and internal EBMs could be identified clearly (Fig. 7). Higher magnifications demonstrated that at least three distinct layers (MBM, EBM, and PCM) could be delineated a t the cut edge of normal capillary BMs (Fig. 8). All layers were comprised of closely packed (20-120 nm) spheroidal particles. Compared to their normal counterparts, cross sections through acellular retinal capillaries from persons with diabetic retinopathy exhibited remarkably thick walls and narrowed lumens (Figs. 9-11). Although increased wall thickness was a common feature among these vessels, their substructural features differed. For example, some were comprised mainly of fibrillar material (Fig. 9) which, a t higher magnifications, was identified as collagen fibrils with attached solitary and aggregate spheroids (Fig. 10). Others demonstrated a single, thick, and remarkably regular BM (Fig. 11A) or poorly defined lamellae of highly dense BM-like material (Fig. 11B) with both types displaying occasional pericytic pockets. The morphological features of these vessels corresponded closely to their TEM counterparts (compare Fig. 9 with Fig. 6A and Fig. 11A and 11B with Fig. 6B). When acellular retinal vessels fortuitously fractured longitudinally were viewed by SEM, their threedimensional shapes and the distributions of their subcomponent BMs were appreciated. Normal vessels showed internal EBM surfaces thrown into circumferential folds (Fig. 12). Moreover, these surfaces exhibited numerous fenestrations (Carlson, in pressb) which corresponded to areas where endothelial cells and pericytes formed close junctions in sitd. Higher-magnification views of longitudinally fractured edges (Fig. 13) demonstrate clearly their tripartite wall structure with irregular “pocket-laden” PCM interposed between distinct MBM and EBM layers. In contrast, similarly fractured acellular retinal vessel walls from diabetics often were massively thickened. This accumulation of ECM was not focal but extended for considerable distances parallel to the long axis of the vessel. At low magnifications PCM accumulations appeared dense and homogeneous (Fig. 14). High-resolution SEM showed, however, that they were comprised of densely packed spheroidal subunits which
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Figs. 7-1 1,
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RETINAL MICROVESSEL BASEMENT MEMBRANES IN DIABETES
TABLE 1. Morphological features of extracellular matrix components of normal and diabetic human retinal capillaries
Thickness (nm) Diabetic
ECM component Entire capillary BM complex Miiller BM
Pericyte BM Subendothelial BM PCM Collagen fibril location
Normal 750-900
n
>100 >loo >loo >loo
90 (80-100) 90 (80-100) 70 (60-90) Loosely organized
BM-like material Sub-MBM
averaged 80 nm in diameter and extended between poorly distinguishable EBM and MBM layers (Fig. 15). Although total wall thickness was greatly increased in diabetics, spheroidal subunits closely resembled those seen in normal acellular vessels. High-resolution SEM studies of normal external MBM and internal EBM surfaces showed t h a t both were composed of spheroidal particles, the size and density of which were apparently specific for individual BM leaflets (Fig. 16A,B). MBM external surfaces showed large 50-120 nm particles and were tightly packed with little interparticulate material (Fig. 16A), while internal EBM surface particles appeared less distinct (Fig. 16B). It is possible, however, that this latter feature may be related to specimen coating or sampling error since in other areas spheroids were more crisply demarcated (data not shown). A common observation in these specimens was circular to oval fenestrations some of which were shallow (Fig. 16B) while others
Fig. 7. Scanning electron micrograph showing cryofractured normal human acellular retinal capillary basement membrane similar to that shown in Figure 3. MBM,, external surface of Miiller basement membrane; EBM,, internal surface of subendothelial basement membrane. Detail of area similar to that in rectangle is shown in Figure 8. x 17,000. Fig. 8. Transmission electron micrograph of area similar to that shown in rectangle in Figure 7. Subendothelial (EBM) and Miiller (MBM) basement membranes are comprised of layers of spheroids. Similar materials comprise the pericytic matrix (PCM) which is loosely arranged with spaces previously occupied by pericytes. x 50,000. Fig. 9. Scanning electron micrograph showing cryofractured type A human acellular retinal capillary basement membrane in diabetic retinopathy. The vessel wall is thickened (compare with Fig. 7) and is comprised of loosely arranged fibrillar material. Detail of area in rectangle is shown in Figure 10. EBMi, internal surface of subendothelial basement membrane. x 7,000. Fig. 10. Higher magnification of area shown in rectangle in Figure 9. Numerous collagen fibrils (COL) and associated spheroids are loosely arranged between more compact layers of subendothelial (EBM) and Miiller cell (MBM) basement membranes. x 50,000. Fig. 1 1 , Scanning electron micrographs of cryofractured type B human acellular retinal capillary basement membranes in diabetic retinopathy. A Vessel wall is massively thickened and is comprised of basement membrane material (bracket) similar to that shown by TEM in Figure 6A. B Vessel wall is comprised mainly of homogeneous, densely packed basement-membrane-like material (bracket). (P), space occupied by pericyte in vivo. A, x 15,000. B, X 17,000.
Type A 1,200-3,000
Type B 1,200-3,000
100
SEM and TEM Analyses of Isolated Human Retina1 Microvessel Basement Membranes in Diabetic Retinopathy EDWARD C. CARLSON
AND
NANCY J. BJORK
Department of Anatomy and Cell Biology, School of Medicine, University of North Dakota, Grand Forks, North Dakota 58202
ABSTRACT Human retinas from persons with diabetic retinopathy and agematched controls were rendered acellular by sequential detergent treatment. The resulting network of microvascular extracellular matrix (ECM) materials, including basement membranes (BMs), was compared by TEM and, following cryofracture, by SEM. Our study demonstrates that in diabetics, retinal capillmy BM complexes are generally thickened and that their ECM subcomponents, including BM leaflets and BM-like pericytic matrix (PCM), are differentially altered. Two diabetic microvessel types were identified. In type A vessels, ECM expansion is manifested by loosely arranged combinations of concentric PCM layers and collagen fibrils with thickened subendothelial (EBM) and pericyte (PBM) BM leaflets. Type B vessels show densely compact central PCM masses and poorly recognizable EBMs and PBMs. In both types, Muller cell BMs (MBMs) are relatively unaffected. High-resolution SEM shows tissue-specific features in normal EBM and MBM surfaces, but disease-related topographic changes are not evident. It is possible that the ECM arrangements identified in our study relate to different microvessel domains and that their specific morphological features may play important roles in the pathogenesis of diabetic retinopathy including capillary closure and neovascularization. Light (LM) and transmission electron microscopic (TEM) observations of human retinal microvessels and their associated extracellular matrix (ECM) components in late stages of diabetic retinopathy have been described in numerous reports (Toussaint and Dustin, 1963; Ashton, 1965; Oliveira, 1966; Cogan and Kuwabara, 1967; Bloodworth, 1967; Speiser et al., 1968; Ashton, 1974; Henkind, 1978; Ashton, 1983; Fryczkowski and Sato, 1986). In these studies, the most commonly cited disease-related alterations relate to basement membrane (BM) thickening. Concomitant with this inexorable BM accumulation is a series of cellular events the pathogenetic chronology of which is unclear. These include breakdown of the blood-retina1 barrier, formation of microaneurysms, capillary closure, selective intramural pericyte degeneration, vessel shunting, exudate formation, and neovascularization (see Ashton, 1974, for review). Most TEM descriptions of diabetic retinal vessels were carried out on intact tissues derived from autopsy or enucleation and were important because they demonstrated relationships of retinal vessel cells to their associated BMs in chronic stages of diabetic retinopathy. Moreover, because they required a re-examination of nondiabetic retinal capillaries as controls, several comparative ultrastructural studies of normal and diabetic human retinal microvessels were carried out (Toussaint and Dustin, 1963; Ashton, 1965; Cogan and Kuwabara, 1967; Bloodworth, 1967). The presence of cells in these preparations, however, inhibited morpho0 1990 WILEY-LISS, INC.
logical analyses of the distributions of noncellular materials and prevented three-dimensional demonstrations of their topographical (surface) features by scanning electron microscopy (SEM). The development of a method for selectively solubilizing cellular materials from organ subfractions and leaving ECM components (including BMs) intact (Meezan et al., 1975; Carlson et al., 1978) provided a n opportunity for morphological investigations of isolated retinal microvessel BMs. In a n effort to study the morphological specificities of retinal capillary BMs, we recently applied this technique to bovine (Carlson et al., 1988; Carlson, 1988) and human (Carlson, in pressaSb)tissues. Our studies showed that following sequential detergent treatment, the histoarchitectural relationships of all retinal capillary BM leaflets and associated pericytic matrix (PCM) were preserved. Furthermore, these structures were easily prepared for LM, TEM, and SEM analysis. In the current study, isolated retinal microvessel BMs from patients with clinically identified diabetic retinopathy were examined. The purpose of the study was to demonstrate disease-related morphological alterations in BMs from diabetic patients. Efforts were made to localize these changes to specific retinal capillary BM leaflets or to PCM. We show by correlated
Received November 14,1988; accepted June 21, 1989.
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E.C. CARLSON AND N.J. BJORK
Figs. 1-4.
RETINAL MICROVESSEL BASEMENT MEMBRANES IN DIABETES
TEM and SEM analyses, fresh new views of acellular human retinal microvessel BMs in normal and diabetic retinal microvessels and emphasize the relationships of altered BM leaflets to the entire capillary BM complex. MATERIALS AND METHODS
Fifty human eyes, posterior eye cups, or isolated retinas from insulin-dependent diabetic patients (aged 50-69) and more than 100 normal eyes were received from the National Disease Research Interchange (NDRI), Philadelphia, Pennsylvania, and from the Department of Pathology, the University of North Dakota, Grand Forks, North Dakota. The tissues were derived from autopsy or enucleation procedures and were received on ice within 72 h r of surgical removal or death. They were shipped to our laboratory in 0.1 M Tris-buffered saline with protease inhibitors (0.025 M ethylenediaminetetraacetic acid [EDTA], 0.001 M benzamidine hydrochloride, 0.001 M phenylmethylsulfonyl fluoride [PMSF], 0.01 M N-ethylmaleimide “EM]) on wet ice. All tissues used in the current study were from patients with known diabetic retinopathy or age-matched controls (individuals with no prior history of eye disease). Preparation of Acellular Retinas Retinas were removed from whole eyes and posterior eye cups and either fixed immediately for TEM or placed in individual 50 ml tubes with at least 40 ml of 0.01 M EDTA and 0.05% sodium azide per retina. Osmotic lysis was allowed to proceed for several hours at 4°C following which the fluids were replaced sequentially by 3% Triton X-100, several rinses of distilled water, 0.025% DNAase in 1M NaC1,4% sodium deoxy-
297
cholate, and numerous final rinses of distilled water to remove the detergent. The conditions of the steps in this procedure were identical to those previously published (Carlson, 1988). This revealed a n arborizing network of acellular retinal microvessel BMs and associated ECM. Occasionally sheets of acellular internal limiting membrane were found in the preparation but were easily removed with forceps or drawn pipet. Acellular retinas resulting from this procedure were photographed in a n Olympus BH-2 compound photomicroscope equipped with darkfield illumination and fixed for TEM and SEM as described below. Electron Microscopy
Primary fixation was carried out in paraformaldehyde/glutaraldehyde (Karnovsky, 1965) buffered at pH 7.4 with 0.2 M sodium cacodylate/HCl for 1h r at room temperature. Tissue samples were post-fixed 90 min (4°C) in 2% Os04 buffered with 0.15 M sodium cacodylate/HCl. These were rinsed in distilled water and prepared for TEM a s previously described (Carlson et al., 1988). Samples to be studied by SEM were cryofractured prior to observation (Carlson and Hinds, 1983). In brief, acellular retinas were transferred from absolute ethanol to Freon 22 (cooled with liquid N2) and then plunged directly into liquid N2. These were cleaved with a chilled single-edged razor blade and returned to absolute ethanol prior to critical point drying, mounting, and coating with gold-palladium as described previously (Carlson et al., 1988). Coated preparations were observed and photographed in a n Hitachi S-800 field emission SEM at original magnifications of 100-70,000 diameters. RESULTS
At the level of TEM, cross sections through normal adult human retinal capillaries show they are comprised of inner continuous layers of endothelial cells Fig. 1. Low-magnification transmission electron micrograph of surrounded by discontinuous processes of intramural section through normal human retinal capillary. The vessel wall consists of continuous endothelium (EC) surrounded by an interrupted pericytes (Fig. 1).These cell types are invested by a (arrows) subendothelial basement membrane (EBM).More externally complex system of BM leaflets which together with are numerous processes of intramural pericytes (P) surrounded by PCM collectively constitutes the retinal capillary BM. pericytic basement membrane (PBM) except where pericytes and en- The system consists of: 1) a n outer Muller cell BM dothelial cells contact. The vessel wall is surrounded by Muller cells (MC) which lie subjacent to their own basement membrane (MBM). (MBM) which is uninterrupted and closely applied to Pericytic matrix (PCM) comprised of collagen fibrils and fibrillogran- basal surfaces of surrounding Muller cells, 2) a n inner ular basement-membrane-like material fills in the perivascular subendothelial BM (EBM) which is discontinuous at space. x 8,300. points where endothelial cells and pericytes are in close contact, and 3) pericytic BM (PBM) which surrounds Fig. 2. Darkfield light micrograph of isolated normal human retcytoplasmic processes of pericytes, and which may be inal microvessel basement membranes (RMBM). Following detergent treatment a n arborizing network of vessel basement membranes com- fused to MBM or EBM. In addition, a BM-like pericytic prised mainly of capillaries retains its in situ histoarchitecture. matrix (PCM) similar to glomerular mesangial matrix x 145. (MM), and numerous collagen fibrils, occupy the perivascular connective tissue space. Fig. 3. Transmission electron micrograph of detergent-treated normal human retinal capillary. All cellular elements of the vessel wall When retinas are dissociated from the underlying are solubilized leaving only the component basement membranes and retinal pigment epithelium and sequentially extracted associated extracellular matrix (compare with Fig. 1). Detail of area similar to that shown in rectangle is shown in Figure 5. EBM, suben- with detergents and endonuclease, all cellular elements are solubilized leaving only the internal limitdothelial basement membrane; PBM, pericyte basement membrane; MBM, Muller cell basement membrane; PCM, pericytic matrix; (PI, ing membrane (which usually can be removed a s a sin“pockets” occupied by pericytes in vivo. X 9,700. gle acellular sheet) and a network of translucent “tubes” clearly demonstrated by darkfield microscopy Fig. 4. Transmission electron micrograph of detergent-treated hu(Fig. 2). The network consists primarily of acellular man retinal capillary basement membrane in diabetic retinopathy. The layer of fibrillar collagen (COL) at left is related to a large adja- capillary-sized vessels which freely anastomose and cent vessel. Individual BM leaflets including subendothelial (EBM) branch from larger arteriolar and venular structures. and Muller (MBM) are recognizable. A massive increase in pericytic Cross sections show that these microvessel “ghosts” do matrix (PCM) is obvious (compare with Fig. 3). Detail of area similar not collapse and retain their in situ cylindrical shapes to that shown in rectangle is shown in Figure 6. x 9,400.
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Figs. 5, 6.
RETINAL MICROVESSEL BASEMENT MEMBRANES IN DIABETES
(Fig. 3 ) . Moreover, they maintain their morphological integrity and all subcomponents of the retinal capillary BM are recognizable in their in vivo histoarchitectural arrangements (compare Figs. 1 and 3). However, in these specimens cells are conspicuously absent and discontinuous EBMs border vessel lumens. Furthermore, spaces previously occupied by pericytes are represented by empty “pockets” surrounded by PBM. When similar preparations are made from retinas derived from patients with clinically confirmed diabetic retinopathy, some of the retinal capillary BMs are dramatically altered (Fig. 4). Although it is difficult to show micrographs representative of all acellular diabetic vessels, most show increased thickness of one or more BM subcomponents while many exhibit considerable PCM redundancy, often in concentric layers. Increased electron density of the PCM is also a feature that is common to nearly all. At increased magnifications, normal acellular walls show interrupted EBMs while MBMs are continuous and more compact with collagen fibrils frequently inserted into their adventitial surfaces (Fig. 5). PBMs surround empty pericytic “pockets” while PCM fills in much of the perivascular space and surrounds “nests” of collagen fibrils which usually are located adjacent to the MBM and rarely intermingle with PCM. Thicknesses of entire capillary BM complexes are highly variable and are related to the number and distribution of cellular elements in various portions of the vessel wall. Our data indicate, however, that when measurements are taken a t the thinnest points from circular samples (i.e., specimens least likely to be sectioned obliquely) they range from 750-900 nm (Table 1). When acellular capillary BMs in diabetic retinopathy are carefully examined, at least two types are evident. One (type A) shows obvious EBM and PBM thickening (up to 300 nm) and concentric layers of electron-dense BM-like material which occupy most of the vessel wall (Figs. 4, 6A). In a second type, (type B) EBMs and PBMs are poorly recognizable and the majority of the capillary wall is replaced by electron-dense PCM (Fig. 6B). The latter is not distinctly layered, however, and may represent a massively thickened
Fig. 5. Transmission electron micrograph of portion of acellular normal human retinal capillary basement membrane in area similar to that shown in rectangle in Figure 3. The capillary BM complex is comprised of three morphologically distinct basement membranes including a discontinuous subendothelial (EBM), a more sharply delineated Muller (MBM), and a pericytic (PBM) which surround “pockets” (P) previously occupied by pericytes. The pericytic matrix (PCM) is loosely distributed in the perivascular space. COL, collagen fibrils. x 32,300. Fig.6. Transmission electron micrographs of portions of acellular human retinal capillary basement membranes taken from patients with advanced diabetic retinopathy. The areas shown are similar to that indicated by rectangle in Figure 4. A Type A vessel in which individual EBM and PBM leaflets are variously thickened. Bracket indicates area of increased pericytic matrix (PCM) electron density (compare with Fig. 5). COL, collagen fibrils; (P), pericytic pocket. B Type B vessel with indistinct subendothelial basement membrane. Miiller (MBM) and pericyte (PBM) basement membranes are recognizable. Bracket shows area where pericytic matrix (PCM) appears greatly increased over normal counterparts (compare with Fig. 5). (P), pericytic pocket; COL, collagen fibrils. A, x 20,500. B, X 26,600.
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EBM which extends up to, but does not replace, the sub-MBM collagen layer. In both types MBMs seem relatively unchanged from their normal counterparts, averaging about 100 nm in thickness. As with normal specimens, thickness measurements of diabetic retinal BM complexes are only marginally accurate because sampling is difficult. Nevertheless, points taken randomly from the thinnest areas of more than 100 cross sections indicate that overall thicknesses of retinal capillary BM complexes ranged from 1,200-3,000 nm in diabetic retinopathy (Table 1). Although TEM sections demonstrate that human retinal capillary BMs from both normal and diabetics maintain their cylindrical shapes following cell removal, it may be argued that such intrinsic structural rigidity is related to epoxy support following fixation. To test this hypothesis, fixed acellular vessels were cryofractured and then prepared for SEM observation. Despite the absence of liquids or resins these samples did not collapse but remained intact, and they demonstrated tubular shapes in which surfaces of external MBMs and internal EBMs could be identified clearly (Fig. 7). Higher magnifications demonstrated that at least three distinct layers (MBM, EBM, and PCM) could be delineated a t the cut edge of normal capillary BMs (Fig. 8). All layers were comprised of closely packed (20-120 nm) spheroidal particles. Compared to their normal counterparts, cross sections through acellular retinal capillaries from persons with diabetic retinopathy exhibited remarkably thick walls and narrowed lumens (Figs. 9-11). Although increased wall thickness was a common feature among these vessels, their substructural features differed. For example, some were comprised mainly of fibrillar material (Fig. 9) which, a t higher magnifications, was identified as collagen fibrils with attached solitary and aggregate spheroids (Fig. 10). Others demonstrated a single, thick, and remarkably regular BM (Fig. 11A) or poorly defined lamellae of highly dense BM-like material (Fig. 11B) with both types displaying occasional pericytic pockets. The morphological features of these vessels corresponded closely to their TEM counterparts (compare Fig. 9 with Fig. 6A and Fig. 11A and 11B with Fig. 6B). When acellular retinal vessels fortuitously fractured longitudinally were viewed by SEM, their threedimensional shapes and the distributions of their subcomponent BMs were appreciated. Normal vessels showed internal EBM surfaces thrown into circumferential folds (Fig. 12). Moreover, these surfaces exhibited numerous fenestrations (Carlson, in pressb) which corresponded to areas where endothelial cells and pericytes formed close junctions in sitd. Higher-magnification views of longitudinally fractured edges (Fig. 13) demonstrate clearly their tripartite wall structure with irregular “pocket-laden” PCM interposed between distinct MBM and EBM layers. In contrast, similarly fractured acellular retinal vessel walls from diabetics often were massively thickened. This accumulation of ECM was not focal but extended for considerable distances parallel to the long axis of the vessel. At low magnifications PCM accumulations appeared dense and homogeneous (Fig. 14). High-resolution SEM showed, however, that they were comprised of densely packed spheroidal subunits which
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RETINAL MICROVESSEL BASEMENT MEMBRANES IN DIABETES
TABLE 1. Morphological features of extracellular matrix components of normal and diabetic human retinal capillaries
Thickness (nm) Diabetic
ECM component Entire capillary BM complex Miiller BM
Pericyte BM Subendothelial BM PCM Collagen fibril location
Normal 750-900
n
>100 >loo >loo >loo
90 (80-100) 90 (80-100) 70 (60-90) Loosely organized
BM-like material Sub-MBM
averaged 80 nm in diameter and extended between poorly distinguishable EBM and MBM layers (Fig. 15). Although total wall thickness was greatly increased in diabetics, spheroidal subunits closely resembled those seen in normal acellular vessels. High-resolution SEM studies of normal external MBM and internal EBM surfaces showed t h a t both were composed of spheroidal particles, the size and density of which were apparently specific for individual BM leaflets (Fig. 16A,B). MBM external surfaces showed large 50-120 nm particles and were tightly packed with little interparticulate material (Fig. 16A), while internal EBM surface particles appeared less distinct (Fig. 16B). It is possible, however, that this latter feature may be related to specimen coating or sampling error since in other areas spheroids were more crisply demarcated (data not shown). A common observation in these specimens was circular to oval fenestrations some of which were shallow (Fig. 16B) while others
Fig. 7. Scanning electron micrograph showing cryofractured normal human acellular retinal capillary basement membrane similar to that shown in Figure 3. MBM,, external surface of Miiller basement membrane; EBM,, internal surface of subendothelial basement membrane. Detail of area similar to that in rectangle is shown in Figure 8. x 17,000. Fig. 8. Transmission electron micrograph of area similar to that shown in rectangle in Figure 7. Subendothelial (EBM) and Miiller (MBM) basement membranes are comprised of layers of spheroids. Similar materials comprise the pericytic matrix (PCM) which is loosely arranged with spaces previously occupied by pericytes. x 50,000. Fig. 9. Scanning electron micrograph showing cryofractured type A human acellular retinal capillary basement membrane in diabetic retinopathy. The vessel wall is thickened (compare with Fig. 7) and is comprised of loosely arranged fibrillar material. Detail of area in rectangle is shown in Figure 10. EBMi, internal surface of subendothelial basement membrane. x 7,000. Fig. 10. Higher magnification of area shown in rectangle in Figure 9. Numerous collagen fibrils (COL) and associated spheroids are loosely arranged between more compact layers of subendothelial (EBM) and Miiller cell (MBM) basement membranes. x 50,000. Fig. 1 1 , Scanning electron micrographs of cryofractured type B human acellular retinal capillary basement membranes in diabetic retinopathy. A Vessel wall is massively thickened and is comprised of basement membrane material (bracket) similar to that shown by TEM in Figure 6A. B Vessel wall is comprised mainly of homogeneous, densely packed basement-membrane-like material (bracket). (P), space occupied by pericyte in vivo. A, x 15,000. B, X 17,000.
Type A 1,200-3,000
Type B 1,200-3,000
100
