Download Electron Microscopy in Cases of Marginal Degeneration of

Electron microscopy in cases of marginal
degeneration of the cornea
Takeo Iwamoto, A. Gerard DeVoe, and R. Linsy Farris
Marginal degeneration of the cornea was studied with the electron microscope on specimens
obtained either by lamellar keratoplasty or superficial keratectomy from four patients. Half of
each specimen was used for light microscopy and histochemical studies. Two clinical types
could be distinguished in these patients: an "inflammatory type" (Cases 1 to 3) characterized
by marked vascularization in and around the corneal lesions and congestion of the corneal
and surrounding conjunctival vessels, and a "quiescent type" (Case 4) characterized by less
marked vascularization with little or no vascular injection. Electron microscopy of the peripheral
corenal lesions of these patients revealed in the inflammatory type: (1) vascularization of the
superficial stroma; (2) marked dilation of the vessel lumen in the corneal and adjacent limbal
blood vessels, with thrombosis, swelling of the endothelial cells and pericytes, and occasional
disintegration of the vessel wall; (3) diffuse as well as localized perivascular and subepithelial
cell infiltrations, which were either predominantly lymphocytic (Case 2) or neutrophilic (Cases
1 and 3); (4) typical fibrinoid degeneration or necrosis, as indicated by intensive stainings
with eosin, period acid—Schiff (PAS), and Mallory's phosphotungstic acid hematoxylin
(FTAH), of perivascular and subepithelial regions (Case 2) and a similar change in localized
areas of the stroma (Case 1). In the quiescent type of marginal degeneration (Case 4), marked
intra- and extracellular vacuolations were noted. The changes in the inflammatory type suggested a hypersensitivity, such as seen in collagen diseases, and those in the quiescent type a
fatty change or degeneration. Besides these distinctive ultrastructural features of each clinical
type, there were also other alterations in the peripheral cornea, which appeared common to
both types. They included: (1) extensive degenerative changes of the basal epithelial cells;
(2) abnormally thickened subepithelial "basement membrane-like layer" (BML) which
contained numerous "unusual fibrils" (similar but less prominent BML was seen in the normal
adult human); (3) invasion of connective tissue, with or toithout blood vessels, into the subepithelial region (some of the connective tissue cells contained vacuoles, and adjacent
keratocytes were highly activated). The ultrastructure of these alterations are described and
its pathologic significance discussed.
Key words: marginal degeneration, cornea, limbus, degeneration, election microscopy,
pathology, collagen disease, vasculitis, hypersensitivity, basement membrane
From the Department of Ophthalmology, Columbia
University, 635 West 165th Street, New York,
JLVJL arginal degeneration of the cornea
(Terrien's marginal degeneration) 1 is a
rare, usually bilateral, condition starting
,
. , ,. ,
•••
..i j .
£ .
™ * d l S C r e e f * i n t Subepithelial opacities
of t h e
peripheral cornea, usually the upper,
whi
c h progress centrally and circumferentially and finally result in stromal degenera-
Supported by National Institutes or Health Research Grant No. EY-00190-16 from the National Eye Institute.
Manuscript submitted Dec. 20, 1971; revised
manuscript accepted Feb. 18, 1972.
241
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Investigative Ophthalmology
April 1972
242 huamoto, DeVoe, and Farris
tion, with peripheral furrow formation,
ectasia, and profuse vascularization. The
disease has been called by various names,
e.g., chronic peripheral furrow-keratitis,2
symmetric marginal dystrophy,3 marginal
sclerosis and atrophy,4 peripheral corneal
ectasia/1 senile marginal atrophy,0 keratoleptynsis marginale/ marginal degeneration of
the cornea,s and marginal keratectasia.9
Various age groups are affected,1'10 apparently with a predilection for aged
males.10 Earlier histopathologic studies have
shown occasional thickness variations and
some cellular modifications in the otherwise
normal epithelium,912 splitting, disorganization, and loss of the Bowman's membrane
and stromal lamellae, and a conversion of
these tissues into or their replacement by
a connective tissue often accompanied by
blood vessels.4-(i> s> ° Fatty deposits in the
pathologic stroma11 and changes of Descemet's membrane9'13> 14 have also been
seen.1'15 The true etiology and pathogenesis
are unknown: Among innumerable suggestions,1' 10 there have been two main theories
concerning the nature of this disease9-1G> 17
—an inflammation5' 1S>10 and a degeneration theory,4'"> 14>20 the latter being considered in close relationship with arcus
senilis. While many investigators supported
the degeneration theory partly based on the
earlier histopathologic studies which showed
little or no signs of inflammation,1'Gi °
some light microscopic studies have reported inflammatory signs as well.12'13 Recently, several authors have suggested a
"collagen disease" as a possible basis for
this disordered condition of the peripheral
cornea.21'22
To our knowledge, no electron microscopy has been reported to date. In our
own clinical experiences, there seem to be
two different clinical manifestations of marginal degeneration of the cornea, which
could be called tentatively: (1) an inflammatory type, and (2) a quiescent type (see
Discussion section). We have studied both
types with the electron microscope. The
purpose of this paper is to describe the ultrastructural alterations of the peripheral
corneal lesions in these patients and to discuss their pathologic significance.
Case reports
Case 1. This 35-year-old Spanish man was seen
in September, 1969, with a chief complaint of a
spot on the right eye. The patient could not speak
English; therefore, only a limited history was obtained. Corrected vision was hand movements in
the right eye and 20/30 in the left. The right eye
was moderately injected, but the left eye was
without injection. Slit lamp examination revealed
a diffuse opacification of the right cornea. There
was a superficial vascularization of the periphery
superiorly, and the lower two thirds of the cornea
was thinned to approximately one half normal
thickness, and contained deep and superficial vessels extending from the limbus (Fig. 1, A). The
left cornea revealed early thinning in the superior
and inferior periphery, accompanied by superficial
opacification and vascularization. No other ocular
abnormalities were detected. Bilateral marginal
degeneration was diagnosed, and on September
23, 1969, a 12 mm. lamellar keratoplasty was
done on the right eye; tissues from the inferior
cornea and limbus were used for this study. Postoperatively, the lamellar graft healed well, some
vascularization of the graft took place, and the
eye quieted after treatment with steroid drops and
0.2M ethylenediaminetetraacetic acid (EDTA)
solution.
Case 2. This 59-year-old Caucasian man was
first seen on May 12, 1969, with a chief complaint
of burning of both eyes. There was a history of
occlusion of a branch of the left retinal artery,
angina, tiredness, and periodic pain in the shoulder
and knee joints; there was no history of diabetes.
Corrected vision was 20/25 and Jl in the right
eye and 20/40 and J5 in the left eye. There was
mild injection and edema in both conjunctiva,
with intact extraocular movements and normal
intraocular pressure. The fundi showed normal
disc, sclerotic retinal vessels, and slight hyperpigmentation of both the maculas. Slit lamp examination revealed tortuous1 conjunctival vessels
bilaterally. The left cornea showed superficial
whitish opacification in the periphery circumferentially; this was most marked superiorly with
a 3.5 mm. extension centrally from the limbus.
Conjunctival vessels extended over the opacification which also showed thinning to approximately
two thirds normal thickness (Fig. 1, B). Similar
but less intense corneal lesions were seen in the
right eye, with prominent conjunctival bleeder vessels at the limbus. There were early cataractous
changes in the left eye, and clear anterior chamber in both eyes. A diagnosis of bilateral marginal
degeneration was made. In June, 1969, a work-up
for collagen disorder was done with the only ab-
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Electron microscopy in corneal degeneration 243
1
Fig. 1. Clinical aspects of marginal degeneration. A, Case 1, right eye. B, Case 2, left eye.
C, Case 3, right eye. D, Case 4, left eye. A, B, and C belong to an inflammatory type, D a
quiescent type.
normal result being a two times normal elevation
of the gamma-1 serum poteins. Also in June, 1969,
a superficial keratectomy was done on the superior
corneal le.sion of the left eye; parts of the tissue
were used for this study. Postoperatively, the
corneal conditions remained active with the opacification enlarging in the right cornea and linear
fhiorescein staining developing at the margin of
the left superior corneal lesion. Following treatment with Decadron drops once daily, the corneal
opacifications stopped progressing and all fluorescein staining disappeared. Under this regimen,
there has been no further activity of the corneal
lesions during a follow-up of two years.
Case 3. This 76-year-old Caucasian man was
first seen on November 21, 1969, with a chief
complaint of irritation and tearing of the right
eye for several weeks. There was- a history of a
cerebrovascular attack in 1965, resulting in lefthanded paralysis, and also of cataract extractions
on both eyes; there was no history of diabetes.
Corrected vision was 20/400 and J18 in both
eyes. The right eye was moderately injected, and
the left eye was without injection. The fundus was
normal in the right eye; the left fundus could not
be seen, however, because of severe bullous
keratopathy. The slit lamp examination showed the
right cornea to have peripheral thinning, superficial
opacification and vascularization superotemporally
and superonasally, with fluorescein staining at
the margin of the peripheral lesions. In the left
eye there was superficial vascularization and
edema with stromal opacification and no evidence
of corenal thinning or peripheral localization of
the lesion. The anterior chamber was clear in the
right eye, and the intraocular pressure was normal
in both eyes. Marginal degeneration in the right
eye and bullous keratopathy in the left eye were
diagnosed. Despite treatment in the right eye with
subconjunctival heparin and topical gamma globulin, die corneal lesion continued to progress, and
by April 2, 1970, the corneal thinning and vascularization had extended to a point that only a
small island of normal thickness cornea remained
inferiorly and centrally (Fig. 1, C). In April,
1970, a superficial keratectomy and scleral graft
were done on the right eye; tissues from the lower
cornea and limbus were taken for this study, Following the operation, the eye quieted; however,
glaucoma developed and three cyclodiathermy
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Investigatioe Ophthalmology
April 1972
244 Iwamoto, DeVoe, and Farris
procedures were necessary to control the ocular
pressure before inserting a keratoprosthesis on
May 4, 1971; postoperatively vision has improved
to 20/50 and J2.
Case 4. This 30-year-old Caucasian man was
first seen on November 3, 1969, with a chief complaint of vision worsening in the left eye since
seven years of age. Thinning of the peripheral
cornea in both eyes had been noted since ten
years of age. There was a history of hay fever and
also allergies to penicillin and ragweed. There was
no history of joint problems, dermatitis, or diabetes. Corrected vision was 20/20 Jl in the right
and 20/200 J10 in the left eye. External examination revealed quiet eyes without injection. Extraocular movements were intact, and the fundi were
normal in both eyes. Slit lamp examination revealed an inferior temporal opacity of the right
cornea with ingrowth of a few small blood vessels
from the conjunctiva. There was also a peripheral
corneal opacity inferiorly, extending nasally. The
left cornea showed a peripheral opacity superiorly from 9:30 to 3:30, extending approximately
3 mm. into the cornea. There was marked thinning
of the cornea within the opacification to approximately one fifth the normal thickness. Although
limbal vessels appeared to have increased near
the lesions, there was no evidence of their ingrowth into the opacity (Fig. 1, D). Fluorescein
did not stain the corneal lesions. The anterior
chamber was clear and the intraocular pressure
was normal in both eyes. A diagnosis of bilateral
marginal degeneration was made, and on July 28,
1970, a ring lamellar graft was done on the left
eye, tissues from the superior portions being used
for this study. During a follow-up of two years,
there has been occasional injection in the right
eye relieved by one or two drops of Decadron eye
drops. Two deep stromal vessels have appeared
also in the right cornea superiorly; these vessels
have not been enlarged and do not appear progressive. Vision in the right eye has remained
stable, and vision in the left eye has1 been improved to 20/20 and Jl with a scleral contact
lens.
Material and methods
Pathologic tissues were obtained either by lamellar keratoplasty or superficial keratectomy from
the patients reported. Half of the specimens, except that from Case 4, were fixed with ten per
cent formalin solution, embedded in paraffin,
and used for light microscopy and histochemical
studies; the staining included hematoxylin-eosin,
periodic acid-Schiff (PAS), Congo red, and phosphotungstic acid hematoxylin (PTAH). The other
half of the specimens were prepared for electron
microscopy with the same methods reported elsewhere23; i.e., they were fixed with three to four per
cent glutaraldehyde in 0.05M phosphate buffer (pH
7.4) for three to ten days, postfixed with one per
cent OsOi in Caulfield buffer for two hours, dehydrated with graded alcohol, and embedded in
Araldite (Durcupan ACM). First, thick (1 to 2 fi)
sections were made, stained with Giemsa, and
used for a preliminary light microscopy, which included a gross estimation of the pathologic changes
and location of the areas to bs studied with the
electron microscope. Areas thus selected were
trimmed, thin-sectioned, and doubly stained with
uranyl acetate and lead citrate. Normal human
corneal tissues obtained by enucleation necessary
because of malignant melanomas of the choroid
of retinoblastomas were prepared by the same
methods and used as controls. A Porter-Blum
microtome was used for sectioning and a Zeiss
EM-9S for election microscopy.
When necessary, the results of the light microscopy are also stated in the Results section, which
is otherwise confined to the electron microscopic
findings; those light microscopic findings which
seemed to duplicate those of the electron microscopy are omitted.
Results
The pathologic tissues studied were confined to the superficial layers of the peripheral corneas, in which there were clinical
lesions of marginal degeneration. Blood
vessels in the limbal areas were studied in
some cases. We have seen frequently an abnormal thickening of the subepithelial basement membrane zone in these patients, and
therefore a brief description of this structure in the normal human being seems warranted. When compared with the central
corneal region, the normal peripheral cornea shows a thickening of the subepithelial
basement membrane zone and increased
basal indentations of the epithelium2'1'23
(Fig. 2, A). The thickened basement membrane zone is composed of two main components: basement membrane-like material
and "unusual fibrils" with peculiar periodicity (Fig. 2, B). Some normal collagen fibrils
and thin fibrils are also often included in
this zone. The unusual fibrils have a dense
structure and closely resemble the "special
fibrils" observed elsewhere in animal tissues,20 as pointed out by McTigue and
Fine.24 In our observation of the normal
human control subjects (ages, 51, 52, and
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Electron microscopy in corneal degeneration 245
Fig. 2. Figs. 2 to 8 are electron micrographs; the line on the bottom in each figure represents
Ifi unless otherwise indicated. Normal adult human subject. A, Peripheral cornea, showing a
normally thick subepithelial "basement membrane-like layer" or "BML" (bm). ep = Basal
epithelial cell; Bl = Bowman's layer. Epithelial base shows indentations (large arrow). Some
cytoplasmic projections (small arrow) are seen in the intercellular space (^11,000.) B, Higher
power of the BML (from limbal area). It consists mainly of basement membrane-like material
(b) and many "unusual fibrils" (arrow). (x90,000.)
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Investigative Ophthalmology
April 1972
246 hoamoto, DeVoe, and Farris
55), the average thickness of the basement
membrane zone was approximately I/A
(rarely up to 2/A) in the peripheral cornea.
In the limbus, in which there is a loose
subepithelial connective tissue instead of
the Bowman's membrane, the basal indentations of the epithelium were usually more
marked, and the basement membrane zone
showed varying thickness with essentially
the same structural components as those
in the peripheral cornea. The basement
membrane zone of the central cornea consisted only of basement membrane material
(up to 500A) in the young human (age 6);
however, in the adult (ages 40 and 55) a
slight thickening (0.2 to 0.6^) was seen,
and its fine structure was similar to that of
the peripheral cornea. Although we have
often seen an abnormal thickening of the
subepithelial basement membrane zones in
our patients with marginal degeneration as
mentioned, the fine structure appeared the
same essentially as that of the normal control subjects. However, since these zones
contained elements other than basement
membrane-like material, we use the term
"basement membrane-like layer" or its abbreviation "BML" to describe such a structure in both normal and pathologic tissues
in this paper.
Case 1. Extensive degenerative changes
were seen in the basal epithelial cells of
the peripheral cornea (Figs. 3, A and 4, C).
Some of these cells appeared edematous,
but the majority showed flattened dense
cell bodies with condensed tonofilaments
and pyknotic nuclei. Some of the latter cells
seemed to be disintegrated and eventually
incorporated into the subepithelial BML
(Fig. 3, B). The BML in this patient was
enormously thick, particularly in vascularized regions of the peripheral cornea.
Here, it amounted to as much as 10/A in
thickness and appeared to have involved
the Bowman's layer (or membrane) or in
some areas a subepithelial connective tissue which had replaced the Bowman's layer
(Fig. 3, A). The BML consisted of basement membrane-like material, normal collagen fibrils, thin fibrils, some cellular de-
bris, and numerous "unusual fibrils" similar
to those seen in the BML of the normal
peripheral cornea (Fig. 3, C). Vascularization was seen in the superficial stroma of
the peripheral cornea. Many of these corneal and limbal blood vessels were dilated
and filled with blood cells, and some vessels
appeared thrombotic (Fig. 4, A). Endothelial cells and pericytes often seemed to
be swollen, and fairly numerous neutrophils
and a few plasma cells infiltrated around
some vessels (Fig. 3, A). Connective tissue changes similar to those seen in the
fibrinoid degeneration area in Case 2 were
found in localized stromal regions, particularly around blood vessels (Fig. 4, B). In a
nonvascularized corneal region, which was
slightly more central than the vascularized
area, the subepithelial BML was up to 5/x
thick. However, a place was found where
a 5 to 6/x thick layer closely resembling the
subepithelial BML was located deeper in
the stroma, as if an original thicker subepithelial BML was separated into two distant layers by the interposition of a newly
invaded connective tissue (Fig. 4, C).
Some fibroblasts in this connective tissue
were full of vacuoles with or without intravacuolar substances (Fig. 4, D). The
keratocytes in deeper stroma near this
region were activated with a structure
similar to that shown in Fig. 7, D.
Case 2. Epithelium over the peripheral
corneal regions studied was similar to that
of conjunctiva as seen by light and electron microscopy. These epithelial cells in
the middle and basal layers often appeared
edematous, with lucent cell bodies (Fig.
5, B). Vascularization was marked in the
superficial stroma, and numerous cell infiltrations were found in wide areas involving the subepithelial region and the
superficial stroma. The cells were predominantly lymphocytic (Fig. 5, A) with some
blast cells,27 but fairly many macrophages
(Fig. 5, C) and a few plasma cells were
also found. These cells also invaded the
intercellular spaces of the epithelium, and
in portions the basal epithelial cells seemed
to have been destroyed, causing a thin-
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Electron microscopy in corneal degeneration 247
Fig. 3, Case 1. A, Peripheral cornea, showing degenerative basal epithelial cells (ep), markedly
thickened (up to 10M) subepithelial BML (bin), and a small blood vessel in the superficial
stroma. The vessel endothelial cells (en) appear swollen, vl — Vessel lumen with erythrocytes;
n = a neutrophil. (The higher magnification is omitted, because of the well-known structure.42)
(x2,700.) B, Higher power of the portion (ep) in A, showing a degenerative epithelial cell
(ep,); the flattened cytoplasm is dense with condensed tonofilaments, and the nucleus (n)
appears pyknotic. Below it, degenerated epithelial cells (ep:) seem to have further disintegrated, to be incorporated into the subepithelial BML (bm). (*26,600.) C, Higher power of
the area (bm) in A, showing the ultrastructure of the abnormally thickened BML. It is composed mainly of basement membrane-like material (b) and many "unusual fibrils" (arrows);
some normal collagen fibrils (c) and thinfibrils1(t) are also seen. (xl08,000.)
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248 Iwamoto, DeVoe, and Farris
Investigative Ophthalmology
April 1972
Fig. 4. Case 1. A, A thrombotic blood vessel (en — endothelium, vl = vessel lumen) in vascularized stroma of peripheral cornea. Apparently, portions of the vessel wall (arrows) have been
disrupted, releasing aggregates of numerous platelets (pi) outside the vessel wall. A similar
thrombosis has been seen elsewhere.43 (Original magnification x3,800.) B, Stroma of vascularized peripheral cornea, showing a localized area composed of normal collagen fibrils (c)
and a dense granular substance (g); this is similar to the structure of "fibrinoid degeneration"
(Fig. 6, A). The possibilitj' that the granular substance may be formed by disintegration of
some collagen fibrils is suggested (armies). A region with irregular pattern of 500 A banding
is seen at b; this banding may occur when thin fibrils1 are arranged in close apposition,23
(Original magnification x58,500.) C, A nonvascularized corneal region more central than that
in Fig, 3. ep = Basal layer of the epithelium with changes similar to those in Fig. 3, B; bin,
= thickened subepithelial BML; bin. = deeply located BML layer (its fine structure closely
resembles the subepithelial BML). It appears as if a connective tissue (ct) has invaded an
abnormally thickened subepithelial BML, thereby separating the BML into two distant layers.
(Original magnification x2,700.) D, Higher power of the connective tissue cell marked by
arrow in C. It has many vacuoles (v) with or without substance. (Original magnification
x37,200.)
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Electron microscopy in corneal degeneration 249
Fig. 5. Case 2. A, Peripheral corneal region. Here the epithelium (ep) is thinned3 seemingly as
a result of the destruction of basal epithelial cells. Many lymphocytes (ly) infiltrate in the intercellular space of the epithelium and in the superficial srroma. The connective tissue area (ct)
here is particularly electron dense and corresponds to the region of "fibrinoid degeneration" as
indicated by histochemical stainings in the other half specimen (inset); the fine structure is
shown in Fig. 6. (Original magnification x3,960.) Inset, light micrograph of the same area as
in A, showing a strongly positive staining by phosphotiingstic acid hematoxylin at the subepithelial and superficial stroma regions (middle zone), ep = Epithelium; bv = blood vessel.
(Original magnification x300.) B, Higher power of the middle layer of the epithelium in A,
showing edematous epithelial cells (ep) with lucent cytoplasm and nucleoplasm (n). Note that
these cells have the structure similar to conjunctiva] epithelial cells with well-developed cytoplasmic projections (arrow) and some bundled tonofilaments (t). ly = Lymphocyte located in
intercellular space. (Original magnification xl^OOO.) C, A macrophage (m) in the stromal
region. As shown, it often attached to lymphocytes (ly). (Original magnification xl.6,800.)
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250 Iwamoto, DeVoe, and Farris
ning of the epithelium (Fig. 5, A). Stromal blood vessels often showed swelling of
the endothelial cells (Fig. 6, D), and a
partial disintegration of the vessel wall
was also seen occasionally (Fig. 6, C).
Most striking changes were observed in
wide areas of the connective tissue, including the subepithelial region and superficial stroma. As shown by histochemical
stainings on the other half specimen, these
connective tissue areas were strongly eosinophilic, PAS positive, and stained intensively blue with PTAH, indicating the
state of "fibrinoid degeneration" or necrosis (Fig. 5, A). Electron microscopy of
these regions revealed a wide area of
dense granular substance in which relatively few normal collagen fibrils of various
densities and many thin fibrils were embedded (Fig. 6, A and B).
Case 3. Basal epithelial cells were often
edematous at the peripheral cornea, and
the subepithelial BML was generally thicker (2 to 3 /*) than normal (Fig. 7, A);
this was partially much thicker as mentioned below. Occasionally, a loose connective tissue accompanied with blood
vessels was seen to intervene in the subepithelial region, thereby placing the Bowman's layer down into the deeper stromal
region; the tip of this connective tissue
seemed to be directed to the thickened
BML (Fig. 7, B). Blood vessels were
also located between the intervening connective tissue and an elevated base of
the epithelium, and some of them appeared thrombotic, filled with numerous
platelets and other blood cells (Fig. 7,
C). Near these vessels, neutrophils were
often interposed between the epithelial
base and the BML. Deeper stromal collagen lamellae near the invading connective tissue were wavy, and many keratocytes in this region were activated with
increased cisternae of rough surfaced endoplasmic reticulum and an enlarged Golgi
complex (Fig. 7, D).
Most blood vessels in the limbal stroma
were greatly dilated, filled mainly with
Investigative Ophthalmology
April 1972
red cells, and some of the endothelial cells
appeared swollen. In one localized area,
a massive cell infiltration was found involving these vessels as well as an adjacent
subepithelial area of the peripheral cornea.
In the latter region, some basal epithelial
cells seemed to have been destroyed, and
the BML here was markedly thickened
(up to 8 /A). These infiltrated cells were
mostly neutrophils, but other leukocytes,
plasma cells, and mast cells were also
mixed; some of these mast cells seemed
to be degranulating.
Case 4. Marked degenerative changes
were seen in the basal epithelial cells of
the peripheral cornea. These cells had
a dense cytoplasm and a pyknotic nucleus,
and many of them possessed bizzare cytoplasmic projections, sometimes resembling
spider's legs. Those cytoplasmic projections
formed irregular infoldings of the epithelial
base, and the BML filling these infoldings
exhibited a layer of extremely irregular
thickness (up to 3 /A) (Fig. 8, A). Normal
Bowman's layer was lost and replaced by
a compact connective tissue which continued to the deeper stromal region. The
diameter of the collagen fibrils were various
(100 to 600 A), with the thicker fibrils
predominating. There was often abnormally little space between these fibrils (Fig.
8, E), and some portions appeared almost
diffusely homogeneous, composed of fibrils
without interfibrillar spaces. Patches of
dense granular substance were scattered
over the connective tissue area (Fig. 8,
B). Most cells in the stroma seemed to
be degenerating keratocytes or fibroblasts
with a dense cytoplasm and a pyknotic
nucleus (Fig. 8, B and C). In some portions, however, these cells appeared to
be more intact and often contained many
vacuoles with or without intravacuolar substance (Fig. 8, B). These portions with
vacuolated cells gradually made transition
into a wide peripheral conieal region, in
which numerous vacuoles of various size
occupied almost the entire thickness of
the stroma which we studied (Fig. 8,
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Electron microscopy in corneal degeneration 251
Fig. 6. Case 2. Area of "fibrinoid degeneration" shown in Fig. 5, A. A, Superficial stroma,
showing connective tissue changes of fibrinoid degeneration. It consists of relatively sparsed
collagen fibrils of various densities (d, c:) and wide areas of dense granular substance (g).
Ct and & — Cross-sections of collagen fibrils. (x37,000.) B, Higher power of a portion of the
dense granular area of fibrinoid degeneration, showing the presence of many thin fibrils
(arrows); corresponding areas of the other half specimen failed to stain positive with Congo
red. There is only one normal collagen fibril (c) in this field. (x84,000.) C, A portion of a
small blood vessel located in the subepithelial region, showing a disintegrated endothelial wall
(de). vl = Vessel lumen. (*24}000.) D, A swollen endothelium (en) of another blood vessel
within the fibrinoid region (fd). er = Erythrocyte in the vessel lumen. (xl2,800.)
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Fig. 7. Case 3. A, A nonvascularized corneal region slightly more central than vascularized
areas, showing still an abnormally thickened BML (bin) which contains small vesicles. Basal
epithelial cells (ep) here seem edematous. Bl = Bowman's layer. (Original magnification
xl2,000.) B, A portion of peripheral cornea, where a triangular connective tissue (ct) accompanied with blood vessels (arrow) invades the subepithelial region, thereby placing the Bowman's layer (Bl) down into the deeper stromal region. The tip of the connective tissue seems
to be directed to the BML (bm). ep = Epithelium. (Original magnification x2,250.) C, Portion of a thrombotic blood vessel (en = endothelium, pi = numerous platelets filling the vessel
lumen) which was located in the subepithelial region of the peripheral cornea; this vessel was
accompanied with an invaded connective tissue such as shown in B. A similar platelet thrombus
has been seen elsewhere.43' d<1 (Original magnification xj.0,500.) D, A keratocyte in the superficial stroma near the region of the invading connective tissue shown in B. It has a highly activated structure, with many cisterns of rough-surfaced endoplasmic reticulum (er) and a large
Golgi complex (g), (Marked changes are not seen in the stromal collagen fibrils here.) (Original magnificatnon xl0,500.)
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Electron microscopy in corneal degeneration 253
C and D). Some of these vacuoles were
intracellular, but many others were extracellular. Here, accumulations of larger
vacuoles could be seen in deeper stroma.
A compact arrangement of the collagen
fibrils was most marked in this region.
Some apparently intact lymphocytes (Fig.
8, D) and a few blood vessels with a
thick laminated basal lamina (Fig. 8, F)
were found in the vacuolated area at deep
stroma near limbus.
Discussion
The etiology and pathogenesis of marginal degeneration are obscure. There
have been two main theories concerning
the nature of this disease: one theory regarded the disease as an inflammation, and
the other as a degenerative process. Many
recent investigators1'G> ° supported the latter
view partly based on the earlier histopathologic studies which showed little or
no signs of inflammation. However, inflammatory signs also have been noted in some
light microscopic studies.12'13 In addition,
recent reports on this disease suggested
the possibility of "collagen disease,"21'22
which may possibly be concerned with
a type of inflammation. As earlier authors0
admitted and also in our own experiences,
there seem to be two clinical manifestations in marginal degeneration of the
cornea. The one type is, as seen in Cases
1 to 3, characterized by a marked vascularization in and around the corneal lesions
and also frequently by a congested status
of both the corneal lesions and the surrounding conjunctival blood vessels. Not
infrequently, fluorescein stains borders of
the corneal lesion. Topical steroid therapy
causes the fluorescein staining to disappear
and the eye becomes quieter with less
congestion; however, the cornea continues
to lose its transparency and melt in a central direction. In the other type, such as
in Case 4, the vascularization of the lesions
is less marked, usually with little or no
surrounding vessel injection, and the clinical picture is without evidence of active
inflammation. We tentatively call the former
group an inflammatory type and the latter
a quiescent type. Although there are some
similarities between our quiescent type
(Case 4) and "pellucide marginal degeneration (Schlaeppi),"2S the presence of a
few blood vessels in the right corneal lesions and a superior location of the left
corneal lesions in Case 4 preclude their
exact identity.
To our knowledge, there have been no
ultrastructural reports on this disease. We
have studied the two clinical types with
the electron microscope. Although the
studies were confined to the superficial
layers of the pathologic peripheral corneas,
the results were consistent with our tentative clinical classification. In the "inflammatory type" most blood vessels were hyperemic with a dilated vessel lumen filled
with blood cells, and frequently there
were marked inflammatory cell infiltrations,
whereas in the quiescent type the most
notable changes were suggestive of fatty
change, to be discussed below. Some pathologic manifestations found in the inflammatory type, such as the specific cell types
which predominated in the infiltrates
(lymphocytes in Case 2, neutrophils in
Cases 1 and 3), vascular changes (Cases
1 to 3), and particularly fibrinoid degeneration (Case 2), are known to occur in
hypersensitivity reactions in experimental
animals as well as in various collagen diseases29' 30: For example, lymphocytic infiltrations can be seen typically in delayed
hypersensitivity. While neutrophilic infiltrations are seen in anaphylactic and
Arthus reactions, these may also be found
in the early stages of delayed hypersensitivity.31' 32 Vasculitis, accompanied with
endothelial alterations (including swelling33'
31
), disruption or necrosis of the vessel
wall and thrombosis, is often observed in
Arthus reaction and some collagen diseases. In addition, edema and lymphocytic infiltrations of the limbal epithelium32' 3i and a close association of
lymphocytes and macrophages,32 such as
found in Case 2, have been seen in experimentally induced biphasic and delayed
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254 Iwamoto, DeVoe, and Farris
frivestigative Ophthalmology
April 1972
8
Fig. 8. Case 4. A, Spider-like epithelial basal cell (ep) in the peripheral cornea, bm = Irregularly thickened BML. Normal Bowman's layer is lost and replaced by stromal connective tissue (ct). (xj.7,600.) B, A stromal cell filled with intracytoplasmic vacuoles (v) with or without
substance; the nucleus (n) appears pyknotic. cl = Dense patches in stromal connective tissue.
(xl7,600.) C, A superficial stromal region filled with intra- and extra-cellular vacuoles (arrows);
larger vacuoles were present in deeper stromal regions. Most stromal cells (k) are dense and
degenerating. (x3,960.) D, Deeper stroma of the same region as in C, showing numerous large
vaculoes (v). A relatively intact lymphocyte (ly) is seen among the vacuoles. (x33960.) E, Compactly arranged collagen fibrils in the vacuolated stromal region. There are no spaces between
individual collagen fibrils (arrow). (x70,800.) F, A small blood vessel (en — endothelium,
m = mitochondria, p = pericyte) seen in the deep stromal region, showing a thick laminated
basal lamina (bm). (x40,000.)
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Electron microscopy in corneal degeneration 255
hypersensitivities of the cornea. Fibrinoid
degeneration may not be specific in hypersensitivity but is still considered to be
an important manifestation in collagen diseases*0; the ultrastructure of the fibrinoid
region in Case 2 appeared similar to that
of the fibrinoid observed in an experimental
antigen-antibody reaction in the rabbit cornea,35 although the latter was modified by
labeled ferritin particles. On these grounds,
it would seem possible to speculate that
those changes seen in the inflammatory
type of marginal degeneration are the
manifestations of hypersensitivity, the different pathologic pictures in individual
cases reflecting different stages. If this
were true, one conceivable antigenic factor
could be the abnormally thickened BML
which contained numerous "unusual fibrils."
Recently, the "subepithelial fibrils" in mice,
which closely resembled the "special fibrils"
in other animals-0 as well as our "unusual
fibrils," have been suggested to be modified collagen.30 It may be that the constituents of the BML, including the unusual
fibrils and other elements possibly released
from degenerated basal epithelial cells, become antigenic in this disease and produce
a hypersensitivity of an autoimmune type.
Collagenous proteins, either by themselves
or as a carrier for a hapten, could become
antigenic, as suggested by others.37 As
speculated by Teng38 in keratoconus, the
thickened BML in our patients seem to
have been initiated by an increased production of basement membrane-like material, which was released from degenerated
basal epithelial cells and which may contain some proteolytic enzymes.39 It would
then be conceivable that the numerous unusual fibrils found in the BML are collagenous fibrils, so modified under the influence
of the substances contained in the basement membrane-like material which has
infiltrated the Bowman's layer as well as
a replaced connective tissue. It is noteworthy that a much less prominent but
similar BML can be seen in normal human
corneas as well, particularly in the peripheral cornea and limbus. Although at present
the structural differences between our
pathologic tissues and normal control tissues appeared mainly quantitative, it is
possible that some qualitative changes may
also be present.
On the other hand, a characteristic ultrastructure in the quiescent type (Case 4)
was marked intra- and extracellular vacuolations in wide stromal regions of the
peripheral cornea. These vacuoles resembled those observed in lipid keratopathy,40 suggesting that the main alteration
in Case 4 was a fatty change or degeneration; a fatty change in this disease has
been noted by earlier investigators.11'1G> 20
The collagen fibrils in these areas showed
an abnormally compact arrangement, suggesting a possibility of tissue anoxia. As
to the question whether these changes
occurred after a prior vascularization41 or
not, we have seen only a few blood vessels
in deep stroma near the limbus. While
a possibility that some vessels might have
regressed in these regions could not be
excluded, our clinical impression was in
favor of the interpretation that the changes
preceeded the vascularization; actually no
vessels could be detected clinically to enter
the lesion in this eye.
In addition to the prevailing changes
discussed above, there were some other
which appeared to be common to both
types. These included: (1) degeneration of
basal epithelial cells; (2) abnormal thickening of the BML, which may be secondary to the first change, as discussed; (3)
invasion of a connective tissue into the
subepithelial region. In Cases 1 and 4,
superficial layers of the epithelium were
partially missing; however, we interpreted
these to have occurred during the prolonged surgical procedure. The third mentioned change could be seen most clearly
in the inner areas of the corneal lesions
in the inflammatory type (Cases 1 and
3), while it seemed to have had taken
place all over in the quiescent type (Case
4); some cells within such an invading
connective tissue in the former type (Case
1) possessed many vacuoles which often
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256
Investigative Ophthalmology
April 1972
Iioamoto, DeVoe, and Farris
contained intravacuolar substance, and
these appeared similar to some cells found
in the latter type (Case 4). In Case 3,
the tip of the invading connective tissue
seemed to be directed to the BML, and
in Case 1 such a connective tissue appeared to have separated the BML into
two distant layers. Another noteworthy finding was markedly activated deeper stromal cells near the invading connective
tissue (Cases 1 and 3). Although highly
speculative, summarizing our findings it
appeared as if the following processes
were taken place: For unknown reasons,
basal epithelial cells of the peripheral
cornea extensively degenerated, causing an
abnormally thickened BML. The thickened
BML motivated an invasion of connective
tissue either of corneal or conjunctival
origin, thereby replacing the Bowman's
membrane (or layer). When there was
no accompanying vascularization in the
connective tissue (corneal origin?), it
ended in a presumed condition of fatty
change. When vascularized, allowing the
migration of leukocytes, a presumed hypersensitivity occurred, possibly with the thickened BML acting as antigenic factors.
In our tissues obtained by lamellar
keratoplasty or superficial keratectomy, a
so-called "furrow" or gutter could not be
clearly visualized. However, at least the
corneal changes described above are those
of the peripheral corneal lesions in which
there were certain degrees of corneal thinning clinically. Among these, the areas
observed in Case 2, where most epithelial cells appeared to be conjunctival
in nature, may possibly represent the region in which a vascularized connective
tissue extended from the limbal conjunctiva
to replace the tissue defect of a previously
formed furrow. The direct mechanism
causing the corneal thinning could not be
determined in our study. However, there
were several factors which might be concerned with it. They were: (1) elements
contained in the BML; (2) fibrinoid degeneration (Case 2) or a similar change
(Case 1); (3) infiltrating cells; (4) pre-
sumed fatty change and its related factors,
including altered fibroblasts.
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