Scanning Electron Microscopy of Human Drusen. Robert J. Ulshafer, Clark D. Allen, Bjorn Nicolaissen, Jr.,* and Melvin L. Rubin. Drusen are small, yellowish ...
Scanning Electron Microscopy of Human Drusen Robert J. Ulshafer, Clark D. Allen, Bjorn Nicolaissen, Jr.,* and Melvin L. Rubin Drusen are small, yellowish deposits that form under the retinal pigment epithelium (RPE) with senescence or under certain pathological conditions. The present study examined these structures under the scanning electron microscope. Tissue came from four eyes of 66- and 75-year-old donors who demonstrated widespread drusen of the posterior fundus noted on postmortem examination. Specimens were prepared by either detaching the RPE from Bruch's membrane, or by cryofracturing the tissue for cross-sectional views. Drusen appeared to be composed of irregularly-shaped globular masses and of distinct spherical entities. These particles varied greatly in size, and were situated between the RPE's basement membrane and the outer collagenous zone of Bruch's membrane. Surface views showed drusen components to be embedded in the collagenous zone of Bruch's membrane. Pits corresponding to the sizes of the globular and spherical masses imply that some particles were lost during tissue processing. Fractured cross sections of the irregularly-shaped globular masses revealed a homogeneous, granular matrix with no distinct ultrastructural features, while some of the fractured spherical components demonstrated an internal core. Transmission electron microscopic analysis on the same specimens that were subjected to SEM corroborated these observations. Analytical x-ray microanalysis (Kevex, Foster City, CA) in the SEM revealed major peaks for calcium and phosphorous in the crystalline spherical components, and primarily potassium and chloride in the globular structures. Invest Ophthalmol Vis Sci 28:683689,1987
studies have found cytoplasmic bags2 or buds6 containing mitochondria, coated vesicles, and microtubules associated with the deposits. These latter studies implied that the membrane-bound structures are processes of RPE cells, and that these processes eventually fragment, their contents contributing to the drusenoid deposit. Drusenoid material is usually kept from extending into the choriocapillaris by the elastic lamina of Bruch's membrane, although it has been found in the outer collagenous layer of Bruch's membrane and within the choroid. When drusen accumulation is extreme, the overlying RPE cells become separated from Bruch's membrane and the choriocapillaris, and eventually degenerate. The degenerating RPE cells may also become incorporated into drusen.5 The chemical composition of drusen appears to be primarily lipid (cerebrosides), mucopolysaccharide (sialomucin),7 and sometimes mineral salts.2'8 The present study was designed to investigate drusen morphology with the scanning electron microscope (SEM), both in surface and fractured views, and to determine its elemental composition using energy dispersed x-ray microanalysis (Kevex).
As part of the normal aging process, and under certain pathological conditions, small, yellowish, hyperreflective lesions appear under the retina when viewed by ophthalmoscopy. The spots are called drusen, and are believed to represent an accumulation of hyaline masses associated with degeneration of the retinal pigment epithelium (RPE) and/or Bruch's membrane. Although the exact origin of the debris comprising drusen is unknown, several studies have shown that abnormalities in Bruch's membrane are the first stage in druse formation, and that these abnormalities begin near the RPE basal surface. Small masses composed of membraneous and granular material begin accumulating outside the basement membrane of RPE cells, and eventually coalesce to form globular dome-like structures filling the inner collagenous zone of Bruch's membrane.1'2'3'4 Some authors have reported that the masses are derived from mitochondria, lysosomes, pigment granules, residual bodies, photoreceptor remnants and various other cytoplasmic debris,5 although this composition has not been confirmed.24 Several
From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, and the *Departments of Ophthalmology and Pathology, Ulleval Hospital University of Oslo, Oslo, Norway. Supported in part by a non-restricted University of Florida Departmental Grant from Research to Prevent Blindness, Inc. Submitted for publication: March 26, 1986. Reprint requests: Dr. Robert J. Ulshafer, Department of Ophthalmology, Box J-284, J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610.
Materials and Methods Four eyes were used in this study, two from a 75year-old donor, and two from a 66-year-old donor. On postmortem examination, all had widespread deposits of drusen in the posterior pole. None had the unrelated type of drusen that are sometimes seen in the optic
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Fig. 1. Low and high magnifications of Bruch's membrane containing drusenoid material, a) Pigment epithelium (PE) has been peeled back to reveal a deposit of drusenoid material (D) on Bruch's membrane (BM). Choroid, C, lies underneath, b) An enlargement of the area containing drusenoid material is shown. The deposit is composed of sphericle and globular structures of various sizes.
nerve head. Since none had any gross pathological conditions, the drusen were believed to be age-related. Each globe was slit and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 10-12 hr. The vitreous and sensory retina were carefully removed, and the remaining globe was cut into small pieces, with intact RPE-choroid-sclera. The pieces were returned to the fixative for at least 24 hr. Prior to processing, the tissues were washed in buffer, and those pieces not destined for x-ray analysis were postfixed in 1% osmium tetroxide for 1 hr. Dehydration of all tissues was accomplished with 5 min changes of acidified 2,2dimethoxypropane (50% followed by three changes in 100%). Specimens were then transferred to 100% ethanol. All tissues were then dried in a Balzers (Hudson, NH) BU101 Critical Point Dryer, employing liquid carbon dioxide as the transition fluid. Following drying, specimens were mounted sclera side down on SEM stubs. In some specimens that were not cryofractured, the intact RPE was removed by gently pressing doublesided tape on the specimen, and either folding the tape over onto itself, or else lifting it off the specimen and mounting it on the stub next to the remaining specimen. This allowed examination of the surface of Bruch's membrane (now facing up on the specimen), as well as the complementary surface of the RPE base-
ment membrane that adhered to the double-sided tape. All specimens were then coated with 60-70 nm goldpalladium in a Techniques (Anatech, Ltd., Alexandria, VA) Hummer X Sputter Coater. To aid interpretation of structures seen in the SEM, several specimens were returned to 100% ethanol following scanning, cleared in propylene oxide, and embedded in Epon/araldite for examination in the TEM. Sections were cut at 700 nm on an LKB Ultratome III and viewed in a Zeiss (Thornwood, NY) 9S TEM.
Results Ophthalmoscopically-visible drusen were found in many fundus regions, both macular and nonmacular. However, apparently only the most extensive deposits were clinically identified, as drusenoid deposits were noted under the SEM that were not visible under an ophthalmoscope or dissecting microscope. Deposits were randomly dispersed, and generally formed discrete accumulations or mounds of small spherical and pleomorphic particles. In regions having extensive drusen, the overlying RPE cells appeared degenerated. Figure 1 shows a specimen in which the RPE has
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been partially peeled back from Bruch's membrane. RPE cells were hexagonally shaped, and appeared morphologically comparable to those seen in specimens or regions not having drusen. Immediately under the RPE was Bruch's membrane, under which can be seen the choriocapillaris. Resting on Bruch's membrane was a region containing many small spherical structures, 0.5-5 urn in size (Fig. lb). The adjacent area of Bruch's membrane appeared smooth and particle-free. The border between the two areas was not distinct. Viewing drusen head-on in one of these regions (Figs. 2a, b), showed that the particles appeared to be embedded in a fine granular matrix. Many pits or depressions occurred in the matrix that corresponded in size to the particles. These pits probably represent particles that were lost during specimen preparation. Some of the spherical particles that were fractured during tissue processing revealed that they were composed of an inner core and several concentric layers under an outer cortex (Fig. 2b). Larger, pleomorphic masses were also seen in drusenoid deposits. Analytical x-ray microanalysis (Kevex) of the particles composing drusen revealed that those spherical bodies with a core had high levels of calcium and phosphorus (Fig. 2c). The most prevalent ions in the pleomorphic masses were potassium and chloride, important ions in RPE cytoplasm. Transmission electron microscopy performed on specimens embedded in resin following SEM demonstrated that drusen occurred in the middle layers of Bruch's membrane, between the basement membranes of the RPE and choriocapillaris (Fig. 3). TEM also confirmed that the deposits were composed of spherical and pleomorphic bodies embedded in an amorphous ground substance that also contained collagen and elastin fibrils (not shown). As in the SEM, the spherical bodies had an inner core and several concentric layers, composed of small, crystalline spicules, and a dense outer shell (Fig. 2d). Frequently, the interior of the spherical particles was lost during specimen sectioning. The pleomorphic masses were composed of a fine granular substance. In regions where the drusen buildup was greatest, thinning and other pathological changes were noted in overlying RPE. When present, RPE cells lacked extensive apical microvillae, and were thinner and more rounded than normal. Furthermore, melanin granules occurred throughout the RPE cell cytoplasm, instead of being limited primarily to the apical end (Fig. 3). Sometimes the deposit of drusenoid matrial was very thick, forming a mound under the RPE. In regions most severely involved, drusen were similar to that shown'in Figure 4. Over the center of the mound, RPE cells were missing, and only the impressions left by the network of cell junctions remained. Cells present on
the periphery of the mound were rounded. In the background of Figure 4 are distinct clumps or nodes of RPE cells that also appear to be rounded. This clumping and rounding of cells is characteristic of a degenerating RPE, 1011 and may represent another druse. A fractured view through the mound revealed that it was 60-70 jtim thick in its central point. Particles within the deposit were greater in size (up to 10 pm in diameter for spherical bodies) than those in regions containing fewer particles (Figs. 1, 2a, b).
Discussion To date, this is the first report to describe drusen in detail with the SEM. Analysis of specimens with the TEM showed similar pathological changes to those previously reported.2'4'56 The present ultrastructural description of drusen, however, differed considerably from that noted by Farkas et al.5 In that study, no globular or crystalline particles were regularly noted in the drusen deposits. However, rounded acid phosphatase reactive bodies (lysosomes) were shown in their preparations. Those bodies were considerably smaller than the particles noted in the current study. It is possible that the drusen reported here were of a different etiology than those in the previous study, or alternatively represent an earlier or later pathological stage. Other studies have proposed that at least some drusen arise from budding of RPE cells into the sub-RPE space. 26 The budding processes eventually fragment, and their contents form a heterogeneous material in Bruch's membrane, which the latter authors call drusenoid. The material described in the current study is at one of the late stages described by Ishibashi et al.6 The pleomorphic structures noted in our micrographs may be RPE processes budding through the basement membrane, similar to those previously reported. Although the authors6 did not report crystalline inclusions in the drusenoid deposits, structures similar to those described in the current report can be seen in their micrographs (see Fig. 14, p. 1926). Further, Burns and Feeney-Burns2 demonstrated mineral crystals with regularity only in drusen of eyes from older (ca. 60 yr) individuals. In a recent review on the aging macula, Marshall and Laties3 described drusen as either dome-shaped and granular, or hyalinized and globular structures. Hyalinized globules in one of their electronmicrographs (Fig. lc, p. 392, corresponding to Fig. legend 3c, p. 3973) resemble ultrastructurally the spherical, crystalline bodies that we found to contain a core having high levels of calcium and phosphorus. They and others2 also showed the presence of large, inclusion-laden macrophages in Bruch's membrane. Sarks classified
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Fig. 2. Head-on view of drusenoid material, a) Particles of various sizes appear to be embedded in a granular matrix. Several of the spherical structures have been artefactually fractured open (arrowheads), revealing that some are composed of core separated from a shell or cortex. One pleomorphic mass (*) is present near the top of the micrograph, b) Enlargement of spheroid in upper right of Figure 2a. The spherical body appears to be composed of several concentric layers including a dense core and a thin shell, c) X-ray energy spectra (Kevex) recorded from pleomorphic masses (top, similar to (*) in 2a) and spherical bodies (bottom, similar to the one in 2b). The pleomorphic masses are high in potassium and chloride, while the crystalline bodies are composed primarily of phosphorus and calcium, d) Transmission electron micrograph of spherical and pleomorphic components in drusenoid deposit. Note that the spherical elements are very electron dense, and are composed of a core (lost during sectioning in the larger crystal) and several concentric layers. The inner layers appear to be composed of crystalline spicules. The pleomorphic masses (*) are less electron dense and composed of a fine, granular substance.
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Fig. 3. Corresponding scanning (a) and transmission (b) electron micrographs of the same specimen. Drusenoid material is seen under a span of approximately eight cells of the pigment epithelium (PE). The material lies on Bruch's membrane (BM); choriocapillaris (C); a red blood cell, R, is present in the capillary on the right. The overlying RPE cells appear to be in a state of degeneration: they are rounded and lack extensive apical microvillae. The drusenoid material is composed of small spherical and larger pleomorphic masses. The larger masses (*) in the drusen are composed of amorphous or finely granular material. Several of the smaller, spherical bodies (arrowheads) are more electron dense than the larger masses and some appear to have been fractured or lost during processing. Several of these bodies are seen in the choroid. The thin, electron-dense layer covering RPE cells in 3b is the gold-palladium coat used to visualize this specimen in SEM.
drusen as being either hard or soft.1 The former are small (less than 50 jum), of hyaline origin and benign. The latter are formed from confluent mounds of amorphous material, vesicles, membraneous debris and sometimes fluid (serous) deposits. Soft drusen are believed to lead to senile macular degeneration. Using Sarks' classification, the deposits described in the current study would correspond to the soft type, based on size and composition. Intracellular crystalline inclusion bodies resembling those noted in the current study have been reported in other tissues of the body, including nephrocalcinosis 1213 and various dystrophic calcifications and calcific metaplasias.14 The electron-dense bodies noted in the former studies are approximately the same size, shape, structure, and calcium phosphate composition as reported here in drusen. The presence of the bodies in both choroid and the drusen, but not in the RPE, offers the possibility the they may originate from the choroid. Inclusions, similar in size and staining to those in
the drusen, were seen in both overlying RPE and in the choriocapillaris. However, we do not believe that melanin pigment granules are present in drusen, since they have not been noted with the TEM. Degenerative changes were noted in RPE overlying heavy deposits of drusen (Fig. 4): cells appeared to be rounding up; melanin granules occurred throughout the cells' cytoplasm rather than primarily in the apical end; apical microvillae were missing; and there appeared to be a reduction in number of mitochondria and basal infoldings. Over the most advanced drusenoid formations, RPE cells appeared to be totally degenerated or missing. The structures resembling junctional bands may represent the basal plasma membranes that remain after RPE cells die or fragment; alternatively the bands may be thickenings in RPE basal membrane. It has been previously reported that the normally apicallylocated tight junctions of RPE cells are translocated to the basal borders of the cells over severe drusen.2 Tissue preparation for the SEM is particularly harsh,
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Fig. 4. Two views of the same druse, a) Top view showing degenerating pigment epithelium overlying druse. The mound of epithelium is viewed from the surface normally exposed to the subretinal space. Some RPE cells are intact but rounded. Other cells (*) are disrupted, degenerated or missing. The hexagonal outlines, probably corresponding to the displaced junctional bars at the RPE cells' basal surface, remain. A nodule of several rounded, possibly degenerating cells appears in the upper right of the micrograph (arrow), b) The specimen has been tilted 90° to expose the inner structure of the druse. The large deposit rests on Bruch's membrane and is composed of small spherical structures and several large, smooth, pleomorphic structures (arrowheads), which may be cells or parts of cells. The same group of RPE cells pointed out in (a) is also denoted by an arrow in (b).
and is known to produce artifacts, primarily due to critical point drying and gold coating.15 However, when tissue was reembedded in epoxy following SEM and sectioned/viewed in the TEM (Fig. 3), morphology was largely similar to specimens that were not processed for SEM. The exact cause of drusen formation is not known. There is evidence that they may arise in response to primary dysfunction or degeneration in the RPE, Bruch's membrane or vessels of the choriocapillaris.16 Since disruption of any of these structures would create changes in the other two, it is difficult to assess which is the primary site of the lesion. Furthermore, it is probable that dysfunction of any of those structures would result in the same pathological end point: reduction in visual acuity leading to blindness. The majority of structures found in drusen are probably primarily derived from the pigment epithelial cells, either from fragmentation of cytoplasmic processes2'6 or from excretion of undigested phagosomes, lysosomes, mitochondria, and lipofuscin granules.35 Wandering cells, clearly of vascular origin, have also been reported as
being associated with drusen.2'5 Their presence may be an attempt by the body to rid Bruch's membrane of debris, thereby preventing a blockage of metabolic transfer between RPE and the choriocapillaris. In a recent study on age-related changes in Bruch's membrane, Feeney-Burns and Ellersieck found that accumulation of debris in Bruch's membrane is probably part of the normal aging process.14 In that study, every eye examined from donors over the age of 60 had changes in Bruch's membrane. Frequently, the earliest pathological sign at the ultrastructural level was seen in those as young as 20 years old. Similarly, Sarks et al1 found hard drusen in about 50% of all age-matched normal eyes. The current study adds a third dimension in analysing drusen accumulation. We have shown that various drusenoid formations, even in the same eye, may have different morphological characteristics, and may be composed of structures of different origin and composition. Using Kevex x-ray analysis interfaced to the SEM, we were able to differentiate between some of the structures comprising drusen. This study does not
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offer documentation to determine the origin of drusenoid material, but as an adjunct to classical histology and transmission EM, we believe the SEM may increase our knowledge on pathology and pathogenesis of this manifestation of eye disease and aging.
7. 8. 9.
Key words: drusen, retinal pigment epithelium, Bruch's membrane, aging, calcium phosphate, retinal disease 10.
References 1. Sarks SH, VanDriel D, Maxwell L, and Killingsworth M: Softening of Drusen and subretinal neovascularization. Trans Ophthalmol Soc U K 100:414, 1980. 2. Burns RP and Feeney-Bums L: Clinico-morphologic correlations of drusen of Bruch's membrane. Tr Am Ophthalmol Soc LXXCIII:206, 1980. 3. Marshall J and Laties A: The special pathology of the aging macula. In Retinal Degeneration: Experimental and Clinical Studies, La Vail MM, Hollyfield JG, and Anderson RE, editors. New York, Alan R. Liss, Inc., 1985, pp. 389-400. 4. Feeney-Bums L and Ellersieck MR: Age related changes in the ultrastructure of Bruch's membrane. Am J Ophthalmol 100:686, 1985. 5. Farkas TG, Sylvester V, and Archer D: The ultrastructure of drusen. Am J Ophthalmol 71:1196, 1971. 6. Ishibashi T, Sorgente N, Patterson R, and Ryan SJ: Pathogenesis
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of drusen in the primate. Invest Ophthalmol Vis Sci 27:184, 1986. Farkas TG, Sylvester V, Archer D, and Altona M: The histochemistry of drusen. Am J Ophthalmol 71:1206, 1971. Sarks SH: Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol 60:324, 1976. Humphreys WJ, Spurlock BO, and Johnson JS: Critical point drying of ethanol-infiltrated cryofractured biological specimens for scanning electron microscopy. Scan Electr Microsc 1974: 275, 1974. Ulshafer RJ and Allen CB: Scanning electron microscopy of the retina in an animal model of hereditary blindness. Scan Electr Microsc 1984:841, 1984. Nicolaissen B, Jr, Davanger M, and Arnesen K: Surface morphology of explants from the human retinal pigment epithelium in culture. A scanning electron microscopic study. Acta Ophthalmol 60:881, 1982. Nguyen HT and Woodard JC: Intranephronic calculosis in rats. Am J Pathol 100:39, 1980. Khan SR, Finlayson B, and Hackett R: Renal papillary changes in patient with calcium oxalate lithiasis. Urology 23:194, 1984. Cheville NF: In Cell Pathology, Ames, IO, The Iowa State University Press, 1983, pp. 159-163. Postek MT, Howard KS, Johnson AH, and McMichael KL: Scanning Electron Microscopy. Burlington, VT, Ladd Research Industries, Inc., 1980, pp. 144-145, 165. Kornzweig AL: Aging of the retinal pigment epithelium. In The Retinal Pigment Epithelium, Zinn KF and Marmor MF, editors. Cambridge, MA, Harvard University Press, 1979, pp. 478-495.
