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Pathogenesis of Drusen in the Primate Tatsuro Ishibashi, Nino Sorgenre, Randi Patterson, and Stephen J. Ryan Two monkey eyes that showed clinical evidence of drusen were studied by light and electron microscopy. The drusen-like spots had several different morphological patterns: the appearance of typical drusen, budding retinal pigment epithelial (RPE) cells, and vacuolation of retinal pigment epithelial cells. Several stages of budding were seen. In some lesions, part of the RPE cell protruded into the sub-RPE space. The upper portion of the budding cell was connected to the cytoplasm of the parent RPE cell and was surrounded by basement membrane of the RPE cell. These budding cells had plasma membranes, cytoplasm that contained organelles, and a nucleus. Disconnected buds, separate from the parent RPE cell, were also seen; these showed degeneration. Finally, an accumulation of vesicular, granular, tubular and linear material was found in the nodular space beneath the RPE cell. It is suggested that this budding of RPE cells is the initial event in drusen-formation. Invest Ophthalmol Vis Sci 27:184-193, 1986 Drusen are excrescences formed beneath the retinal pigment epithelium (RPE); they are extremely common in persons over age 40. Histopathologically, drusen are usually periodic acid-Schiff (PAS) positive. By electron microscopy, they are composed of polymorphous material of vesicular, granular, and filamentous appearance.1"4 Drusen may be present for many years without any complications; however, eyes with drusen are predisposed to the development of subretinal neovascularization and geographic atrophy of the RPE.5'6 Drusen were first fully described by Donders7 in 1854. Since then, numerous theories have been proposed concerning their pathogenesis; however, there is still no general agreement on how drusen are formed. Donders7 believed that the RPE cells were directly converted into drusen material (transformation theory), but Miiller8 proposed that drusen were deposits secreted by an otherwise intact RPE (deposition theory). Friedman et al9 suggested that drusen originated from constituents of blood (vascular theory), while Spencer10 hypothesized that the drusenoid material initially accumulated within the RPE cells and later filled Bruch's membrane (deposition and transformation theory); this latter theory agrees with the results of studies by Hogan11 and Farkas et al.12 Fine and Yanoff13 proposed that drusen were composed of aggregates of abnormally formed basement membrane material (basement membrane theory); more recently, Burns and FeeneyBurns14 suggested that drusen were composed of material resulting from the cytoplasmic fragmentation of RPE (apoptosis theory). To elucidate the pathogenesis of drusen, we studied with light and electron microscopy two monkey eyes that showed drusen-like spots in the fundi. Materials and Methods One eye from each of two male Macaca speciosa monkeys, conforming to the ARVO Resolution on the From the Department of Ophthalmology, University of Southern California School of Medicine, and the Estelle Doheny Eye Foundation, Los Angeles, California. Supported in part by grants EYO1545 and EY03040 from the National Institutes of Health, National Eye Institute, Bethesda, MD. Submitted for publication: February 6, 1985. Reprint requests: Stephen J. Ryan, MD, Department of Ophthalmology, University of Southern California, 1355 San Pablo Street, Los Angeles, CA 90033. *ig. l. Muorescein angiogram snowing nomeatcing nypeniuoreseem spots in the posterior pole, 184 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 No. 2 PATHOGENE5I5 OF DRUSEN / Ishiboshi er ol. Fig. 2. Histological section of typical drusen, showing the dome-shaped granular structure (arrow) (PAS, XI200). Fig. 3. Histological section of drusenlike spot showing nodular accumulation of cellular components in the sub-RPE space (arrow) (PAS, XI200). Use of Animals in Research, were used in this study. From acquisition records, dental evaluations and information obtained from the keepers, the animals were determined to be at least 20 yr old. Fundus photographs and fluorescein angiographs revealed drusen-like spots in the fundi. The animals were anesthetized with an intramuscular injection of ketamine hydrochloride (10 mg/kg) and the eyes were enucleated. Eyes were opened by cutting a corneoscleral window and fixed by immersion in 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 hr. The globe was dissected into several blocks approximately 2 X 1 mm in size, each of which contained drusen-like spots. After washing in 0.1 M phosphate buffer and post-fixation for 2 hr in 2% phosphate-buffered osmium te- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 166 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1986 Vol. 27 troxide, the blocks were dehydrated in a series of graded alcohols and propylene oxide, then embedded in an epoxy resin. Sections at 1 fim were cut for light microscopy and stained with Richardson's reagent; thin sections were cut on an ultramicrotome, stained with uranyl acetate and lead citrate, and examined with an electron microscope. Some blocks were dehydrated in graded alcohol and embedded in glycol methacrylate. Sections at 3 /im were cut and examined with the light microscope following staining with PAS. Results Fig. 4. Ultrastructural view of drusen-like spot consisting of cell cytoplasm that is in continuity with RPE cytoplasm (R) and surrounded by RPE basement membrane. The budding RPE cytoplasm contains some organelles. L, secondary lysosome (X2O,3OO). Ophthalmoscopic examination of the fundi revealed the presence of myriad yellow-white spots; these were concentrated in the posterior pole of one eye but were scattered throughout the fundus of the other eye. Clinically the spots were considered to represent drusen. rig. 3. inset snows a ngnt micrograpn section ot arusen-iiKe spot (arrow). [Kicnardson s, XbUU). Large ngure shows ultrastructural view ot the same spot. A large part of the RPE cells evaginate into the sub-RPE space. The RPE cells show an increase in residual bodies and secondary lysosomes, and a decrease in melanin granules. The evaginated portion of the RPE cells also has many lipofuscin granules. R, RPE cell (X11,000). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 No. 2 PATHO6ENE5I5 OF DRU5EN / Ishiboshi er ol. 187 Fig. 6. Inset shows a light micrograph section of drusen-like spot showing nodular accumulation of cellular components (arrow). (Richardson's, X600). Large figure shows ultrastructural view of the same spot. A large part of the RPE cell evaginates into the sub-RPE space. The residual RPE cytoplasm (R) is thin and the basal infoldings and mitochondria are barely discernible. .Functional complexes at the apical portions are present (arrows) (X7000). v%s.-». n 16 iivi maguinkaiiuii ui-iwLuiisnuwii m i iguicu. m e upper puruun oi me nouuie lsconneciea to me K f t cytoplasm and is surrounded by RPE basement membrane. The portion separated from the parent RPE cell shows degeneration; however, portions of basement membrane are still present (arrows). The budding cytoplasm has many organelles, including a pyknotic nucleus (N), large complex lysosomes (L) and a melanin granule (M) (X 13,000). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 188 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / Februory 1986 Fig. 8. Ultrastructural view of drusen-like nodule that is completely separate from the cytoplasm of its parent RPE cell (R). The nodule is surrounded by plasma membrane and basement membrane and contains organelles (XI 7,200). Fluorescein angiography showed nonleaking hyperfluorescence and characteristically demonstrated more drusen-like spots than were visible on ophthalmoscopy (Fig. 1). Light Microscopic Findings Light microscopic examination revealed several morphologic patterns in the drusen-like spots. One was of typical drusen, showing the dome-shaped granular structure with PAS positive staining (Fig. 2). A second pattern was that of a nodular accumulation of cellular components in the sub-RPE space. This type of nodule was occasionally PAS positive (Fig. 3). A third pattern was that of vacuolation of RPE cells, which were in their normal position within the RPE layer. It was not possible, however, to correlate the individual clinical drusen with distinct morphologic patterns. Electron Microscopic Findings The second type of lesion, ie, the nodular accumulation of cellular components, were located where drusen are typically seen but histologically did not have the appearance of drusen. To determine the relation to typical drusen, we examined this type of lesion ultrastructurally. Electron microscopy revealed several types of subRPE nodules, which seemed to represent a progression of stages in drusen-formation. Some small sub-RPE nodules were not detectable at the light microscopic level. A nodule of cell cytoplasm was evident in the Vol. 27 sub-RPE space; this was in continuity with the RPE cytoplasm and surrounded by RPE basement membrane (Fig. 4). In some cases a large part of the RPE cell evaginated into the sub-RPE space, but the upper portion of the nodule was connected to the RPE cytoplasm (Figs. 5-7). These portions of the budding RPE cells had plasma membranes and cytoplasm that contained organelles, including melanin granules, mitochondria, vesicles, endoplasmic reticulum, ribosomes, secondary lysosomes, residual bodies and occasionally a pyknotic nucleus (Figs. 4-7). Another type of nodule was completely separate from the cytoplasm of its parent RPE cell; it was surrounded by plasma membrane and basement membrane and contained organelles (Fig. 8). Various stages of degeneration were evident in the cell portions separated from the parent RPE cell as well as in the tightly packed organelles of the budding RPE cells (Figs. 79A, B). Some separated nodules showing degeneration demonstrated the disruption of basement membrane and plasma membrane (Figs. 9A, B). A large complex lysosome, the result of fusion of a number of residual bodies containing lipofuscin granules, was frequently observed (Fig. 7). Still another type of nodule appeared to have further degenerated and fragmented, dispersing vesicular, granular, tubular, and linear material into the sub-RPE space and the inner layer of Bruch's membrane (Figs. 10,11). Finally, an accumulation of vesicular, granular, and filamentous material, so-called drusenoid material, was found in the nodular space beneath the RPE cell (Figs. 12, 13). The parent RPE cells showing budding (Fig. 5) or drusen formation (Figs. 13, 14) showed an increase in residual bodies and secondary lysosomes, and a decrease in melanin granules; the residual bodies represented lipofuscin granules, and the secondary lysosomes contained melanolysosomes, melanolipofuscin granules, and phagolysosomes. In RPE cells that had shed a major portion of their cytoplasm, the residual cytoplasm was thin, and the basal infoldings and mitochondria were barely discernible. However, the cells remained attached to adjacent RPE cells with junctional complexes at the apical portions (Fig. 6). In contrast, other cells overlying large sub-RPE nodules still contained many mitochondria and basal infoldings (Fig. 11), suggesting that these may represent regenerating cells. Discussion There are only a few reports of drusen-like lesions in non-human primates. Drusen have been described Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 No. 2 PATHOGENESI5 OF DRUSEN / Ishibashi er ol. Fig. 9. Ultrastructural view of drusen-like nodule showing degeneration. A, The upper cell fragment (a) is surrounded by basement membrane (arrow). The lower cell fragment (b) is also surrounded by basement membrane (arrows), which shows partial disruption (arrowheads) (X21,000). B, some cell fragments show disruption of the basement membrane (arrows) and plasma membrane (arrowheads). The edges of the disrupted portion of plasma membrane have a curled up appearance (X26,000). in a young female baboon,11 in which the lesions were discrete, round, eosinophilic structures lying on Bruch's membrane. Stafford12 observed a drusen-like alteration in an aged female rhesus monkey, but did not study the eye morphologically. Fine and Kwapien13 observed yellow-white hyperfluorescent dots in the fundi of a female rhesus monkey, but on histologic examination these were seen to be caused by a vacuolation of individual RPE cells, and not by drusen or by loss of RPE cells. The vacuolation of the RPE cells was considered to be a form of lipoidal degeneration.14 FeeneyBurns et al15 suggested that hyperfluorescent non-leaking window defects seen in cynomolgus monkey eyes represented RPE filled with lipid vacuoles, or with more Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 190 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Februory 1986 t t Mg. JU. Ultrastructural view ol drusen-iike spot that appears to have degenerated and fragmented, dispersing vesicular, granular, tubular and linear material into the sub-RPE space and the inner layer of Bruch's membrane (X25,400). lipofuscin and less melanin. Recently, Stafford et al16 reported the clinical and pathological correlation of typical drusen in rhesus monkey eyes. Vol. 27 In this study of Macaca speciosa eyes, drusen-like spots were found to show three different morphological patterns, ie, vacuolated RPE cells, budding RPE cells, and typical drusen. The vacuolated RPE cells and typical drusen correspond with findings previously described. 131416 Ultrastructural findings of the budding of RPE cells and their degeneration and fragmentation are similar to the earliest defined drusen of young human eyes.10 It is suggested that this budding of RPE cells is the initial event in drusen formation. Although several theories have been proposed concerning the origin and pathogenesis of drusen, most observers believe that drusen-formation is via secretion of undigested materials from the RPE into the inner layer of Bruch's membrane. Farkas et al8 proposed an additional theory, ie, that drusen are the result of a pathologic autolysis of the RPE caused by a breakdown of lysosomes. In our study, lysosomal changes were found in both the parent and the budding RPE cells, but were also seen in RPE cells that did not show budding. However, we could not find a clear relationship between the lysosomal changes of RPE cells and the budding. Our findings suggest an alternate explanation to the theory that the mechanism of drusen formation is "secretion" of undigested material from the RPE cells; rather, it is due to budding of the RPE cells. Table 1 summarizes •!^7.^:- rig. i i . insei snows a ngni micrograpn section oi arusen-iiKe noauie larrowj (Kicnarason s, xouu;. Large ngure snows uitrastructurai view of the same nodule showing degeneration. RPE cells (R) overlying the large sub-RPE nodule have mitochondria and basal infoldings (X6800). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 No. 2 PATHOGENESIS OF DRUSEN/ Ishibashi er al. a postulated sequence of events leading to the formation of drusen. Only one previous study10 proposed this budding mechanism in drusen formation—and likened it to the process called apoptosis. Since a similarity was noted in younger eyes between RPE cytoplasm and drusen contents, it was hypothesized that the membranebound portion of the basal cytoplasm of an RPE cell protrudes into Bruch's membrane through a break in the basement membrane of the RPE cell. Our findings revealed that nodules surrounded by basement membrane were obviously derived from RPE cells, because of the direct connection between the nodules and the RPE cytoplasm. It is suggested that the drusenoid material could be composed not only of cytoplasm containing organelles, plasma membrane and the nucleus of the RPE cell, but also of the basement membrane of the RPE cell. The term apoptosis was first proposed by Kerr et al.17 Apoptosis is a mechanism for controlled cellular deletion in tissue involution, atrophy, remodeling and tumor regression; shrinkage necrosis on morphological terms is synonymous with apoptosis.18 Electron microscopy showed that the structural changes in apoptosis took place in two discrete stages: the first com- 191 Fig. 12. Ultrastructural view of drusen-like spot showing an accumulation of vesicular, granular, and filamentous materials beneath the RPE cell (X26,500). prised the formation of apoptotic bodies; the second, their phagocytosis and degradation by other cells. The process began with the separation of a cell from its neighbors and condensation of its cytoplasm. This was I Fig. 13. Inset shows a light micrograph showing globular deposition of granular material in the sub-RPE space (arrow) {Richardson's, X600). Large figure shows ultrastructural view of the same spot showing a large accumulation of drusenoid material in the sub-RPE space and in the inner layer of Bruch's membrane (X8900), Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 192 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1986 Vol. 27 rig. i i . uurasiruciuraj view 01 an r t r t <.K.J over sman accumulation or arusenoia material; mere is an increase in residual oodies and secondary lysosomes, and a decrease in melanin granules. A small amount of drusenoid material is seen in the sub-RPE space (X 10,600). followed by prolific budding with the production of small membrane-bound cellular fragments, which often occurred in clusters and which did or did not contain nuclear remnants. They were eventually ingested by macrophages, to be degraded by lysosomal enzymes. The processes of drusen-formation seen in our study Table 1. Pathogenesis of Drusen Stage I: Degenerative changes of RPE cell Stage U: Budding of RPE cell in the sub-RPE space Stage III: Separation of budding RPE cell from parent RPE cell Stage IV: Degeneration and fragmentation of RPE cell bud Stage V: Formation of drusenoid material seem to be similar to the processes described in apoptosis. However, we did not observe separation of the plasma membranes from those of adjacent RPE cells or condensation of the basal cytoplasm of the RPE cell before the onset of budding. As a result, we do not use the term apoptosis in this paper. Although we could not detect total deletion of the RPE cell, it seems reasonable that this process does occur if the original RPE cell lacks a nucleus and sufficient organelles to remain viable. Based on the present results, it seems reasonable to postulate that the initial event in drusen-formation is budding of RPE cells. The degeneration and fragmentation of the budding RPE cells then release the drusenoid material into the sub-RPE space and inner layer of Bruch's membrane. Additional studies are needed to elucidate the precise mechanism of the budding. Key words: drusen, monkey, Bruch's membrane, retinal pigment epithelium, electron microscopy Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017 PATHO6ENESI5 OF DRUSEN / Ishiboshi er ol. No. 2 Acknowledgments The animals used in this study were maintained in facilities fully accredited by the American Association of Laboratory Animal Science. We thank Thomas E. Ogden, MD, PhD, for his invaluable advice and Rosario Espinoza for her technical assistance. The editorial assistance of Ann Dawson was greatly appreciated. References 1. Hogan MJ: Bruch's membrane and disease of the macula: Role of elastic tissue and collagen. Trans Ophthalmol Soc UK 87: 113, 1967. 2. Farkas TG, Sylvester V, and Archer D: The ultrastructure of drusen. Am J Ophthalmol 71:1196, 1971. 3. Fine BS and Yanoff M: Ocular Histology: A Text and Atlas. Hagerstown, MD, Harper & Row, 1979, pp. 219-223. 4. Burns RP and Feeney-Burns L: Clinico-morphologic correlations of drusen of Bruch's membrane. Trans Am Ophthalmol Soc 78: 206, 1980. 5. Gass JDM: Drusen and disciform macular detachment and degeneration. Arch Ophthalmol 90:206, 1973. 6. Green WR and Key SN: Senile macular degeneration: A histopathologic study. Trans Am Ophthalmol Soc 75:180, 1977. 7. Donders FC: Beitrage zur pathologischen Anatomie des Auges. Graefes Arch Clin Exp Ophthalmol 1:106, 1854. 193 8. Muller H: Untersuchungen uber die Glashaute des Auges, insbesondere die Glaslamelle der Choroidea und ihre senilen Veranderungen. Graefes Arch Clin Exp Ophthalmol 2:1, 1856. 9. Friedman E, Smith TR, and Kuwabara T: Senile choroidal vascular patterns and drusen. Arch Ophthalmol 69:220, 1963. 10. Spencer WH: Pathogenesis of macular degeneration: light microscopy. Trans Am Acad Ophthalmol Otolaryngol 69:662, 1965. 11. Barnett KC, Heywood R, and Hague PH: Colloid degeneration of the retina in a baboon. J Comp Pathol 82:117, 1972. 12. Stafford TJ: Maculopathy in an elderly sub-human primate. Modern Problems of Ophthalmology 12:214, 1974. 13. Fine BS and Kwapien RP: Pigment epithelial windows and drusen: an animal model. Invest Ophthalmol Vis Sci 17:1059,1978. 14. Fine BS: Lipoidal degeneration of the retinal pigment epithelium. Am J Ophthalmol 91:469, 1981. 15. Feeney-Burns L, Malinow MR, Klein ML, and Neuringer M: Maculopathy in cynomolgus monkeys: a correlated fluorescein angiographic and ultrastructural study. Arch Ophthalmol 99: 664, 1981. 16. Stafford TJ, Anness SH, and Fine BS: Spontaneous degenerative maculopathy in the monkey. Ophthalmology 91:513, 1984. 17. Kerr JFR, Wyllie AH, and Currie AR: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239, 1972. 18. Kerr JFR: Shrinkage necrosis: A distinct mode of cellular death. J Pathol 105:13, 1971. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933358/ on 06/17/2017