Download Epithelial characteristics of the endothelium in Chandler`s

Epithelial Characteristics of the Endothelium
in Chandler's Syndrome
Lawrence W. Hirst, W. Richard Green, Martha Luckenbach, Zenaida de la Cruz, and Walter J. Stark
The cells lining the posterior surface of a corneal button obtained from a patient with Chandler's
syndrome were examined by transmission and scanning electron microscopy, and by keratin staining.
The ultrastructural appearance with intracytoplasmic 8 nm filaments, desmosomes, microvillous projections, and positive keratin staining suggests that these may be epithelial cells. Clinical specular
microscopy of this cornea prior to transplantation revealed characteristic changes that were seen to
be related to the density of microvillous projections on the cell membrane. Invest Ophthalmol Vis
Sci 24:603-611, 1983
Since the original description1 of Chandler's syndrome in 1955, it has generally become accepted that
the early corneal edema, later glaucoma, and peripheral anterior synechia formation with iris distortion
are due to a migration of abnormal cells from the
posterior corneal surface on over the anterior angle
and onto the anterior surface of the iris.2"8 The origin
and exact nature of the cells, and the reason for their
migration, remain unknown. These cells have sometimes been called fibroblast-like,3 but generally they
have been considered to be endothelial in nature.
More recently, it has been suggested that these cells
have some characteristics of epithelial cells.9 Bron and
Tripathi10 suggested that they were undifferentiated
mesothelial cells.
We present a case of Chandler's syndrome, as confirmed by clinical examination, specular microscopy,
and histologic study of a corneal transplant button.
Keratin staining of the posterior corneal cell layer and
scanning and transmission electron microscopy of the
same specimen gave evidence that the corneal endothelium in Chandler's syndrome has characteristics
of corneal epithelial cells.
Case Report
A 68-year-old white man was first seen at the Wilmer Institute
in November 1975 because of a superficial corneal foreign body
of the left eye. He had a past history of spontaneous corneal edema
of the left eye in 1970, which resolved during 5 days without treatment, and during which time his intraocular pressure remained 17
mmHg. In 1974 he had a similar corneal edema of the left eye,
with a pressure of 12 mmHg. Between these episodes he was followed up regularly and was apparently found to have normal intraocular pressure (IOP).
On examination in 1975, his vision was 20/25 in the right eye
and 20/25 in the left. He had a clear right cornea with no endothelial abnormalities noted by slit-lamp examination and a clear
anterior chamber. The right lens showed 1 + nuclear sclerosis. In
the left eye he had a very early band keratopathy, and some mapdot-fingerprint changes in the peripheral corneal epithelium. He
was noted at this time to have an abnormal endothelial layer by
slit-lamp examination, and an IOP of 21 mmHg in the right and
27 mmHg in the left by applanation. The anterior chambers were
deep, iris details were normal, and the cup/disc ratio was 0.2 in
both eyes.
He was next seen in 1977 with acute onset of pain in his left eye
and visual blurring, an IOP of 50 mmHg, and corneal edema diagnosed as a result of acute angle-closure glaucoma. A peripheral
iridectomy was performed, and his IOP remained normal after
surgery with topical antiglaucoma medications. When re-examined
in October 1981, because of decreasing vision in his left eye, the
vision was 20/25 in the right eye and light perception with accurate
projection in the left eye. The right eye had a clear cornea (Fig.
1) with a grade 3 open angle in all areas and an IOP of 18 mmHg
by application. In the left eye, band keratopathy was increased,
and it obscured the view of the central corneal endothelium (Fig.
1). Peripheral corneal endothelium by slit-lamp examination had
a guttate appearance. The anterior chamber was deep, and a peripheral iridectomy was present at the 1-o'clock position. There
was ectropion of the iris pigment layer at the 12-o'clock position,
and inferiorly at the 5:30-position in the midperiphery there appeared to be an iris nevus (Fig. 1). Gonioscopy revealed peripheral
anterior synechia (PAS) for almost 270°, extending anteriorly in
front of Schwalbe's line. The left pupil dilated fully, and he was
noted to have a dense nuclear sclerotic cataract. Ophthalmoscopic
examination was not possible because of the media, but ultrasound
From the Wilmer Ophthalmological Institute, the Johns Hopkins Hospital, Baltimore, Maryland.
Supported in part by U.S. Public Health Service Research Grant
EY 02476, Computer Core Grant #2P30 EY 01765, and EM Core
Grant EY 01765.
Presented in part at the Annual Meeting of the Association for
Research in Vision and Ophthalmology, Sarasota, Florida, May
1982.
Submitted for publication June 10, 1982.
Reprint requests: Lawrence W. Hirst, MD, Wilmer Institute,
Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21205.
After July 1, 1983: Bethesda Eye Institute, 3655 Vista Avenue, St.
Louis, MO 63110.
0146-0404/83/0500/603/$ 1.25 © Association for Research in Vision and Ophthalmology
603
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INVESTIGATIVE OPHTHALMOLOGY b VI5UAL 5CIENCE / May 1983
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Fig. 1, Right eye of patient, with a normal cornea
and iris. Left eye of patient
showing central band keratopathy, ectropion of the iris
pigment layer at the 12o'clock position, and one
iris nevus at the 5:30-position.
suggested no evidence of gross retinal pathology. Examination was
also performed with a small-field specular microscope and with a
wide-field specular microscope.
He had a left penetrating keratoplasty and extracapsular cataract
extraction performed on the left eye in December 1981. Three
quarters of the corneal button, the broad iridectomy specimen, and
a conjunctival biopsy specimen werefixedin glutaraldehyde-formaldehyde solution for light microscopy and ultrastructural examination. A rim of the donor corneal tissue and the remaining
quarter of the patient's corneal button were frozen and sectioned
for antikeratin antibody staining" and monoclonal antikeratin
antibody staining (by methods from personal communication with
Tung Tien Sun, PhD.
Of the patient's 23 living relatives, 20 were given a slit-lamp
examination of the anterior segment.
Results
Clinical Examinations
Family examination: Of the 20 family members
examined there was no history of glaucoma in any,
and slit-lamp examination disclosed the corneas, endothelial surfaces, and iris details in all members to
be normal. One relative had map-dot-fingerprint corneal changes and cataracts, and in another, some very
fine pupillary remnants associated with an anterior
polar cataract. Their ages ranged from 5 years to 78
years. One daughter and her two children, who had
no history of ocular disease, were not available for
examination. No evidence of minor posterior polymorphous endothelial dystrophic changes was seen
on the endothelial surface of any of the family members.
Specular microscopy ofthe patient: Both small-field
and wide-field specular microscopy of the patient's
unaffected right eye revealed a normal endothelial
surface, with perhaps some increased pleomorphism
of endothelial cells (Figs. 2A, B). The entire central
8 to 10 mm of corneal endothelium was scanned with
the wide-field specular microscope to confirm the
absence of any discrete abnormalities.
In the left eye, only the areas of corneal endothelium above and below and to the sides of the band
keratopathy could be viewed, and in all areas that
were visible by the wide-field unit there was no evidence of normal endothelium (Figs. 3 A, B). Reversal
of cell outlines and variations of cell shape were
prominent, with a diffuse uniform involvement in all
areas observed. Small-field specular microscopy at
high magnification disclosed some cell boundary outlines, but mainly a reversal of cell outlines, the center
of the cell being dark and the periphery of the cell
being light, with some highlighted refractile edges
(Fig. 3B).
Histopathology
Light microscopy: Cornea. The corneal button
showed slight irregularity of the epithelial surface, and
the basement membrane wasfibrillated,with a minor
degree offibrouspannus formation between this and
Bowman's layer. The stroma was normal. Descemet's
membrane was very thin, and the endothelial cell
layer was mildly attenuated. In the center of the button, 19 nuclei of endothelial cells were counted in the
view of a single high-power field (X400).
Iris: Light microscopic examination of 1-micron
thick sections in epoxy resin (Epon) disclosed a small
fragment of iris with a single layer of cells with an
apparent basement membrane on the anterior surface. Occasional pigment granules were present in
these cells.
Transmission electron microscopy: Cornea. Descemet's membrane was thin throughout and measured about 2.5 microns in thickness. The cells lining
the posterior surface were mostly in a single layer
{Fig. 4), but showed some overlapping in areas, and
in occasional areas were arranged in two layers. No
distinct basement membrane was present where the
cells were apposed to Descemet's membrane. The internal portion of Descemet's membrane was, however, composed of a thin fibrillar basement membrane-like material. The endothelial cells had some
characteristics of epithelial cells (Fig. 4). These consisted of numerous filaments (keratofibrils) that averaged 8 nm in diameter (Fig. 4). These cells adjacent
to Descemet's membrane contained numerous free
ribosomes and glycogen particles. A moderate number of mitochondria and pinocytotic vesicles were
present. In some cells the pinocytotic vesicles were
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CHANDLER'S SYNDROME / Hirsr er ol.
Fig. 2A. Bio-Optics specular micrograph of right eye of patient, showing a fairly normal endothelial mosaic with some pleomorphism
of cells (bar = 50 microns). B, Pocklington wide-field specular micrograph of normal right eye of patient, showing absence of any discrete
abnormalities in endothelial layer (bar = 100 microns).
quite numerous. The cells composing the layer facing
the anterior chamber contained sparse, but well-developed, microvilli. There were well-developed desmosomal attachments in some areas (Fig. 4). Other
attachments were composed of a placoid density of
the cellular membranes and a space between the cells.
The innermost cells also contained a few keratohyaline granules and had intracytoplasmic features that
were similar to the cells next to Descemet's mem-
brane. The cell processes were serpiginous and well
approximated.
Iris. The anterior surface of the iris was covered by
a monolayer of cells that showed polarity with a moderate number of surface microvilli and well-defined
single or multiple layers of basal basement membrane
measuring from 0.1 to 0.5 micron in thickness (Fig.
5). The cells had numerous, well-developed desmosomal attachments near the apical borders (Fig. 5).
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Moy 1983
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tig. JA. Bio-Uptics specular micrograph otleft eye, showing grade III abnormalities with "reversal" of cell outlines and only occasional
normal cell boundaries (bar = 50 microns). B, Pocklington wide-field specular micrograph of left eye, showing diffuse endothelia! changes
and no normal mosaic visible (bar = 100 microns).
These cells contained numerous filaments that measured 8 to 9 nm in diameter, numerous free ribosomes and glycogen particles, and rarely mitochondria (Fig. 5). No distinct keratohyaline granules were
seen.
Scanning electron microscopy: Cornea. The cell
layer lining the posterior corneal surface showed cell
pleomorphism with no regular hexagonal array (Fig.
6). Most noticeable were the microvillous projections
on most cells, with some cells showing a predominance of microvillous projections around their periphery near the intercellular boundaries and others
showing this predominance centrally (Fig, 6). The
cells with mainly peripheral microvillous projections
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CHANDLER'S SYNDROME / Hirsr er ol.
Fig. 4, Posterior surface
of the thin Descemet's
membrane (arrowheads) is
lined by one (upper inset) or
two layers (upper photograph) of cells that are:
joined by desmosomes (circle and lower photograph),
contain cytoplasmic filaments (arrows, upper and
lower photos) that measured 8 nm in diameter and
are arranged in fusiform aggregates, and have microvillous projections (asterisk)
(upper, X21,000; lower,
X 104,000; inset, paraphenylenediamine X480).
appeared to have a nuclear-like mound that was microvillous-free centrally.
Anlikeratin antibody staining: Rabbit preimmune
serum revealed no staining of either surface of the
control cornea. Using rabbit antikeratin antibodies
at a dilution of 1 to 50, the control cornea (from the
corneal rim of the donor button) was noted to fluoresce strongly on the epithelium with no fluorescence on the endothelial layer. With rabbit preimmune serum, the patient's cornea showed no fluorescent staining of either surface. With the rabbit
antikeratin antibody, however, strong fluorescence
was noted on both the surface epithelium and the
posterior cellular layer (Fig. 7). There were no areas
of endothelial cells that did not take up the fluorescent stain strongly.
Monoclonal antibody staining to keratin showed
positive fluorescence with antibody AE1, AE3,
and AE4.
Discussion
The clinical features in our case included: two documented episodes of unilateral corneal edema with
normal IOP and spontaneous resolution of the edema;
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1983
L
evidence of unilateral IOP rise, and abnormal endothelial surface by slit-lamp examination, in 1975;
increased IOP requiring iridectomy and further medical glaucoma therapy in 1977; broad PAS formation
for 270°, anterior to Schwalbe's line; ectropion of iris
pigment layer and iris nevus formation; characteristic
features on specular microscopy12 of the involved eye
and normal endothelium in the uninvolved eye; and,
lack of corneal and iris abnormalities and glaucoma
in 20 out of 23 of the patient's living relatives.
All of these features corroborate the clinical diagnosis of Chandler's syndrome1 of the left eye. The
presence of a surgical iridectomy in 1977 raises the
possibility that this patient has an epithelial ingrowth.
J
Vol. 24
Fig. 5. Anterior surface of
iris is covered by a single
layer of cells (main figure
and upper inset, arrowhead)
that have: numerous apical
microvillous processes, a
single layer (bracket and arrowhead, left lower inset) or
multiple layers (arrow) of
basement- membrane, are
joined by apically located
desmosomes (circles and
lower middle inset), and
have numerous cytoplasmic
filaments (bracket and asterisks, left and right lower
insets) (X8800; upper inset paraphenylenediamine,
X480; lower left inset,
X35,000; lower middle inset, X 50,000; lower right inset, X55,000).
The existence of corneal disease, including recurrent
corneal edema and corneal endothelial abnormalities,
prior to the surgical iridectomy makes this unlikely.
Also, the absence of abnormality of corneal endothelium in other family members, together with the
normal endothelial layer in the patient's other eye,
makes the diagnosis of posterior polymorphous endothelial dystrophy highly improbable.13
Since the early descriptions of Chandler's syndrome, ' and the clinical identification of a spectrum
of corneal and iris abnormalities in the iridocorneal
endothelial (ICE) syndrome, the etiology of the endothelial layer abnormalities and iris changes has
been the subject of much contention. Recent evi-
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Fig. 6. Scanning electron micrograph of posterior
corneal cell layer, showing prominent microvillous
projections at cell boundaries and over the surface
of paler cells, except for a "nuclear" bare area. A
second cell-type, apparently darker, has far fewer
microvillous projections (X1000).
dence suggests that the primary defect in Chandler's
syndrome (and in the ICE syndrome) is an abnormal
posterior corneal cell layer, which migrates across the
trabecular meshwork and iris, causing the iris defects
and glaucoma.2"8 Although the site of origin of these
migrating cells appears definitely to be the posterior
corneal surface, their phylogenetic origin is still conjectural. It has been suggested that these may, in fact,
be mesothelially undifferentiated cells,10 or they may
resemble fibroblasts with an increased rough-surface
intracytoplasmic reticulum and junctional complexes
with microvillous projections.3 The possibility that
these cells resemble epithelial cells has been suggested
by Richardson.9 In fact, the ultrastructural descriptions of the posterior cornea of a number of the reported cases include intracytoplasmic filaments7'9 and
microvillous projections,7-9 although the morphology
of these cells as epithelial-like has not been substantiated. In fact, in contradistinction to the cells lining
the posterior corneal surface in posterior polymorphous endothelial dystrophy, Rodrigues et al8 make
the point that the cells lining the posterior cornea in
Chandler's syndrome have no epithelial-like characteristics.
The presence of ultrastructural features of increased intercellular digitations, microvillous surface
projections, bundles of 10-nm intracytoplasmic filaments, desmosomal formation, and production of
basement membrane and collagen could indicate that
these cells have an epithelial origin or nature. Unlike
the cells in posterior polymorphous endothelial dystrophy or of epithelial ingrowth, in the present patient
they did not form more than two layers on the cornea
or more than a monolayer on the iris. The prominent
and unequivocal staining, with rabbit antikeratin an-
tibody, of the cells lining the endothelial surface as
compared to the nonstaining of the control cornea
supports the contention that these cells are epithelial
in nature." Why this staining of corneas from other
Chandler's syndrome cases proved negative for keratin14 remains an enigma. This method has been effectively used to demonstrate the presence of keratin
in posterior polymorphous dystrophy.15
The results of specular microscopy, as described
previously in grade 3 of the ICE syndrome,12 are similar to the results in the present case, with prominent
light-dark "reversal" of cell outlines and large central
areas that are devoid of detail. The specular microscopic morphology is mainly related to the cell
boundary interface with the aqueous humor, and this
is affected by changes in refraction at this surface.
Scanning electron microscopy of this same posterior
corneal surface disclosed that the most evident abnormality (as compared to a normal endothelial
layer) was the varying density of the microvillous
projections, which were prominent in this patient.
The major density was around the periphery of the
cell, with nuclear-like projections present centrally,
which had only a few microvillous projections. This
correlates very well with the specular microscopic
appearance, with the cell outlines being demarcated,
and with the central area (nearly devoid of microvillous projections) being dark.
Our antikeratin antibody staining results confirmed the epithelial-like characteristics previously
suspected by some ultrastructural examinations.79
We also correlated the scanning electron microscopy
and specular microscopy of the endothelial layer of
this cornea of a patient with Chandler's syndrome.
The specular microscopic morphology and appear-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1983
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Fig. 7. Rabbit antikeratin antibody
staining of control cornea (A, B, C)and
of Chandler's syndrome cornea (D, E,
F). A and D reveal no fluorescence
with staining by rabbit preimmune
serum (X80). B shows epithelial fluorescence with no endothelial fluorescence (double arrowheads) in the control cornea, with rabbit antikeratin antibody staining. E shows strong
epithelial and endothelial fluorescence
(double arrowheads) in the Chandler's
syndrome cornea with rabbit antikeratin antibody staining. C and F show
the difference in endothelial fluorescence of control cornea vs Chandler's
syndrome corneas, at higher magnification (X22O).
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CHANDLER'S SYNDROME / Hirsr er ol.
No. 5
ance can now be understood in terms of the microvillous projections present on the aqueous-humor border of the cell membrane.
No explanation has been forthcoming as to why
human endothelial cells, which are noteworthy for
their inability to mitose, should migrate off of the
cornea.
Whether the epithelial characteristics of the "endothelium" in Chandler's syndrome represent a congenital variant (as in posterior polymorphous dystrophy) or are an acquired change in the endothelium
is unknown. The fact that corneal endothelium can
sometimes develop characteristics of fibrocytes and
can produce collagen seems well established.1617 Also,
the fact that cells on the posterior corneal surface
were epithelial-like in the present case of Chandler's
syndrome may offer an explanation of this unusual
in vivo behavior of these cells lining the posterior
corneal surface, and also the relative impermeability
of these corneas to fluorescein.18 These findings
should prompt a closer examination of other cases
of Chandler's syndrome, to confirm whether or not
this condition is an epithelialization disease.
Key words: Chandler's syndrome, specular microscopy, corneal endothelium, ultrastructure, epithelial cells, keratin
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Acknowledgments
14.
The authors are grateful to Tung-Tien Sun, PhD, for
keratin staining of the corneal specimens, and thank David
Andrews, MS, and Charlotte Chalmers for assistance during
preparation of the manuscript.
15.
16.
References
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2. Shields MB, Campbell DG, and Simmons RJ: The essential
iris atrophies. Am J Ophthalmol 85:749, 1978.
3. Rodrigues MM, Streeten BW, and Spaeth GL: Chandler's syn-
17.
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Eagle RC, Jr, Font RL, Yanoff M, and Fine BS: Proliferative
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