Download Pathogenesis of drusen in the primate.

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,
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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-
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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).
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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).
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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
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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
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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
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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.
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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).
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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),
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1986
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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
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PATHO6ENESI5 OF DRUSEN / Ishiboshi er ol.
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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.
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