Electron Microscopy in Cases of Marginal Degeneration of the Cornea

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 JLVJL arginal degeneration of the cornea University, 635 West 165th Street, New York, (Terrien's marginal degeneration)1 is a rare, usually bilateral, condition starting

Supported by National Institutes or Health Re- ..i j. £ . , . , ,. , ••• search Grant No. EY-00190-16 from the Na- ™* dlSCre e f*int Subepithelial opacities tional Eye Institute. of the peripheral cornea, usually the upper, Manuscript submitted Dec. 20, 1971; revised which progress centrally and circumferenti- manuscript accepted Feb. 18, 1972. ally and finally result in stromal degenera- 241

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tion, with peripheral furrow formation, corneal lesions in these patients and to dis- ectasia, and profuse vascularization. The cuss their pathologic significance. disease has been called by various names, e.g., chronic peripheral furrow-keratitis,2 Case reports 3 symmetric marginal dystrophy, marginal Case 1. This 35-year-old Spanish man was seen sclerosis and atrophy,4 peripheral corneal in September, 1969, with a chief complaint of a ectasia/1 senile marginal atrophy,0 keratolep- spot on the right eye. The patient could not speak tynsis marginale/ marginal degeneration of English; therefore, only a limited history was ob- s 9 tained. Corrected vision was hand movements in the cornea, and marginal keratectasia. the right eye and 20/30 in the left. The right eye 1 10 Various age groups are affected, ' ap- was moderately injected, but the left eye was parently with a predilection for aged without injection. Slit lamp examination revealed males.10 Earlier histopathologic studies have a diffuse opacification of the right cornea. There was a superficial vascularization of the periphery shown occasional thickness variations and superiorly, and the lower two thirds of the cornea some cellular modifications in the otherwise was thinned to approximately one half normal 912 normal epithelium, splitting, disorganiza- thickness, and contained deep and superficial ves- tion, and loss of the Bowman's membrane sels extending from the limbus (Fig. 1, A). The and stromal lamellae, and a conversion of left cornea revealed early thinning in the superior and inferior periphery, accompanied by superficial these tissues into or their replacement by opacification and vascularization. No other ocular a connective tissue often accompanied by abnormalities were detected. Bilateral marginal 4 (i> s> blood vessels. - ° Fatty deposits in the degeneration was diagnosed, and on September pathologic stroma11 and changes of Desce- 23, 1969, a 12 mm. lamellar keratoplasty was met's membrane9'13> 14 have also been done on the right eye; tissues from the inferior seen.1'15 The true etiology and pathogenesis cornea and limbus were used for this study. Post- operatively, the lamellar graft healed well, some are unknown: Among innumerable sugges- vascularization of the graft took place, and the 1 10 tions, ' there have been two main theories eye quieted after treatment with steroid drops and concerning the nature of this disease9-1G> 17 0.2M ethylenediaminetetraacetic acid (EDTA) —an inflammation5' 1S>10 and a degenera- solution. tion theory,4'"> 14>20 the latter being con- Case 2. This 59-year-old Caucasian man was sidered in close relationship with arcus first seen on May 12, 1969, with a chief complaint of burning of both eyes. There was a history of senilis. While many investigators supported occlusion of a branch of the left retinal artery, the degeneration theory partly based on the angina, tiredness, and periodic pain in the shoulder earlier histopathologic studies which showed and knee joints; there was no history of diabetes. little or no signs of inflammation,1'Gi ° Corrected vision was 20/25 and Jl in the right some light microscopic studies have re- eye and 20/40 and J5 in the left eye. There was 12 13 mild injection and edema in both conjunctiva, ported inflammatory signs as well. ' Re- with intact extraocular movements and normal cently, several authors have suggested a intraocular pressure. The fundi showed normal "collagen disease" as a possible basis for disc, sclerotic retinal vessels, and slight hyper- pigmentation of both the maculas. Slit lamp ex- this disordered condition of the peripheral 1 cornea.21'22 amination revealed tortuous conjunctival vessels bilaterally. The left cornea showed superficial To our knowledge, no electron micros- whitish opacification in the periphery circum- copy has been reported to date. In our ferentially; this was most marked superiorly with own clinical experiences, there seem to be a 3.5 mm. extension centrally from the limbus. two different clinical manifestations of mar- Conjunctival vessels extended over the opacification which also showed thinning to approximately ginal degeneration of the cornea, which two thirds normal thickness (Fig. 1, B). Similar could be called tentatively: (1) an inflam- but less intense corneal lesions were seen in the matory type, and (2) a quiescent type (see right eye, with prominent conjunctival bleeder ves- Discussion section). We have studied both sels at the limbus. There were early cataractous types with the electron microscope. The changes in the left eye, and clear anterior chamber in both eyes. A diagnosis of bilateral marginal purpose of this paper is to describe the ul- degeneration was made. In June, 1969, a work-up trastructural alterations of the peripheral for collagen disorder was done with the only ab-

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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 be seen, however, because of severe bullous of the gamma-1 serum poteins. Also in June, 1969, keratopathy. The slit lamp examination showed the a superficial keratectomy was done on the superior right cornea to have peripheral thinning, superficial corneal le.sion of the left eye; parts of the tissue opacification and vascularization superotemporally were used for this study. Postoperatively, the and superonasally, with fluorescein staining at corneal conditions remained active with the opaci- the margin of the peripheral lesions. In the left fication enlarging in the right cornea and linear eye there was superficial vascularization and fhiorescein staining developing at the margin of edema with stromal opacification and no evidence the left superior corneal lesion. Following treat- of corenal thinning or peripheral localization of ment with Decadron drops once daily, the corneal the lesion. The anterior chamber was clear in the opacifications stopped progressing and all fluores- right eye, and the intraocular pressure was normal cein staining disappeared. Under this regimen, in both eyes. Marginal degeneration in the right there has been no further activity of the corneal eye and bullous keratopathy in the left eye were lesions during a follow-up of two years. diagnosed. Despite treatment in the right eye with Case 3. This 76-year-old Caucasian man was subconjunctival heparin and topical gamma glob- first seen on November 21, 1969, with a chief ulin, die corneal lesion continued to progress, and complaint of irritation and tearing of the right by April 2, 1970, the corneal thinning and vas- eye for several weeks. There was- a history of a cularization had extended to a point that only a cerebrovascular attack in 1965, resulting in left- small island of normal thickness cornea remained handed paralysis, and also of cataract extractions inferiorly and centrally (Fig. 1, C). In April, on both eyes; there was no history of diabetes. 1970, a superficial keratectomy and scleral graft Corrected vision was 20/400 and J18 in both were done on the right eye; tissues from the lower eyes. The right eye was moderately injected, and cornea and limbus were taken for this study, Fol- the left eye was without injection. The fundus was lowing the operation, the eye quieted; however, normal in the right eye; the left fundus could not glaucoma developed and three cyclodiathermy

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procedures were necessary to control the ocular cent glutaraldehyde in 0.05M phosphate buffer (pH pressure before inserting a keratoprosthesis on 7.4) for three to ten days, postfixed with one per May 4, 1971; postoperatively vision has improved cent OsOi in Caulfield buffer for two hours, de- to 20/50 and J2. hydrated with graded alcohol, and embedded in Case 4. This 30-year-old Caucasian man was Araldite (Durcupan ACM). First, thick (1 to 2 fi) first seen on November 3, 1969, with a chief com- sections were made, stained with Giemsa, and plaint of vision worsening in the left eye since used for a preliminary light microscopy, which in- seven years of age. Thinning of the peripheral cluded a gross estimation of the pathologic changes cornea in both eyes had been noted since ten and location of the areas to bs studied with the years of age. There was a history of hay fever and electron microscope. Areas thus selected were also allergies to penicillin and ragweed. There was trimmed, thin-sectioned, and doubly stained with no history of joint problems, dermatitis, or dia- uranyl acetate and lead citrate. Normal human betes. Corrected vision was 20/20 Jl in the right corneal tissues obtained by enucleation necessary and 20/200 J10 in the left eye. External examina- because of malignant melanomas of the choroid tion revealed quiet eyes without injection. Extra- of retinoblastomas were prepared by the same ocular movements were intact, and the fundi were methods and used as controls. A Porter-Blum normal in both eyes. Slit lamp examination re- microtome was used for sectioning and a Zeiss vealed an inferior temporal opacity of the right EM-9S for election microscopy. cornea with ingrowth of a few small blood vessels When necessary, the results of the light micros- from the conjunctiva. There was also a peripheral copy are also stated in the Results section, which corneal opacity inferiorly, extending nasally. The is otherwise confined to the electron microscopic left cornea showed a peripheral opacity supe- findings; those light microscopic findings which riorly from 9:30 to 3:30, extending approximately seemed to duplicate those of the electron micros- 3 mm. into the cornea. There was marked thinning copy are omitted. of the cornea within the opacification to approximately one fifth the normal thickness. Although limbal vessels appeared to have increased near Results the lesions, there was no evidence of their in- The pathologic tissues studied were con- growth into the opacity (Fig. 1, D). Fluorescein did not stain the corneal lesions. The anterior fined to the superficial layers of the periph- chamber was clear and the intraocular pressure eral corneas, in which there were clinical was normal in both eyes. A diagnosis of bilateral lesions of marginal degeneration. Blood marginal degeneration was made, and on July 28, vessels in the limbal areas were studied in 1970, a ring lamellar graft was done on the left some cases. We have seen frequently an ab- eye, tissues from the superior portions being used for this study. During a follow-up of two years, normal thickening of the subepithelial base- there has been occasional injection in the right ment membrane zone in these patients, and eye relieved by one or two drops of Decadron eye therefore a brief description of this struc- drops. Two deep stromal vessels have appeared ture in the normal human being seems war- also in the right cornea superiorly; these vessels ranted. When compared with the central have not been enlarged and do not appear pro- gressive. Vision in the right eye has remained corneal region, the normal peripheral cor- stable, and vision in the left eye has1 been im- nea shows a thickening of the subepithelial proved to 20/20 and Jl with a scleral contact basement membrane zone and increased lens. basal indentations of the epithelium2'1'23 (Fig. 2, A). The thickened basement mem- Material and methods brane zone is composed of two main com- Pathologic tissues were obtained either by lamel- ponents: basement membrane-like material lar keratoplasty or superficial keratectomy from and "unusual fibrils" with peculiar periodic- the patients reported. Half of the specimens, ex- cept that from Case 4, were fixed with ten per ity (Fig. 2, B). Some normal collagen fibrils cent formalin solution, embedded in paraffin, and thin fibrils are also often included in and used for light microscopy and histochemical this zone. The unusual fibrils have a dense studies; the staining included hematoxylin-eosin, structure and closely resemble the "special periodic acid-Schiff (PAS), Congo red, and phos- fibrils" observed elsewhere in animal tis- photungstic acid hematoxylin (PTAH). The other sues,20 as pointed out by McTigue and half of the specimens were prepared for electron 24 microscopy with the same methods reported else- Fine. In our observation of the normal where23; i.e., they were fixed with three to four per human control subjects (ages, 51, 52, and

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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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55), the average thickness of the basement bris, and numerous "unusual fibrils" similar membrane zone was approximately I/A to those seen in the BML of the normal (rarely up to 2/A) in the peripheral cornea. peripheral cornea (Fig. 3, C). Vasculariza- In the limbus, in which there is a loose tion was seen in the superficial stroma of subepithelial connective tissue instead of the peripheral cornea. Many of these cor- the Bowman's membrane, the basal inden- neal and limbal blood vessels were dilated tations of the epithelium were usually more and filled with blood cells, and some vessels marked, and the basement membrane zone appeared thrombotic (Fig. 4, A). Endo- showed varying thickness with essentially thelial cells and pericytes often seemed to the same structural components as those be swollen, and fairly numerous neutrophils in the peripheral cornea. The basement and a few plasma cells infiltrated around membrane zone of the central cornea con- some vessels (Fig. 3, A). Connective tis- sisted only of basement membrane material sue changes similar to those seen in the (up to 500A) in the young human (age 6); fibrinoid degeneration area in Case 2 were however, in the adult (ages 40 and 55) a found in localized stromal regions, particu- slight thickening (0.2 to 0.6^) was seen, larly around blood vessels (Fig. 4, B). In a and its fine structure was similar to that of nonvascularized corneal region, which was the peripheral cornea. Although we have slightly more central than the vascularized often seen an abnormal thickening of the area, the subepithelial BML was up to 5/x subepithelial basement membrane zones in thick. However, a place was found where our patients with marginal degeneration as a 5 to 6/x thick layer closely resembling the mentioned, the fine structure appeared the subepithelial BML was located deeper in same essentially as that of the normal con- the stroma, as if an original thicker sub- trol subjects. However, since these zones epithelial BML was separated into two dis- contained elements other than basement tant layers by the interposition of a newly membrane-like material, we use the term invaded connective tissue (Fig. 4, C). "basement membrane-like layer" or its ab- Some fibroblasts in this connective tissue breviation "BML" to describe such a struc- were full of vacuoles with or without in- ture in both normal and pathologic tissues travacuolar substances (Fig. 4, D). The in this paper. keratocytes in deeper stroma near this Case 1. Extensive degenerative changes region were activated with a structure were seen in the basal epithelial cells of similar to that shown in Fig. 7, D. the peripheral cornea (Figs. 3, A and 4, C). Case 2. Epithelium over the peripheral Some of these cells appeared edematous, corneal regions studied was similar to that but the majority showed flattened dense of conjunctiva as seen by light and elec- cell bodies with condensed tonofilaments tron microscopy. These epithelial cells in and pyknotic nuclei. Some of the latter cells the middle and basal layers often appeared seemed to be disintegrated and eventually edematous, with lucent cell bodies (Fig. incorporated into the subepithelial BML 5, B). Vascularization was marked in the (Fig. 3, B). The BML in this patient was superficial stroma, and numerous cell in- enormously thick, particularly in vascu- filtrations were found in wide areas in- larized regions of the peripheral cornea. volving the subepithelial region and the Here, it amounted to as much as 10/A in superficial stroma. The cells were predomi- thickness and appeared to have involved nantly lymphocytic (Fig. 5, A) with some the Bowman's layer (or membrane) or in blast cells,27 but fairly many macrophages some areas a subepithelial connective tis- (Fig. 5, C) and a few plasma cells were sue which had replaced the Bowman's layer also found. These cells also invaded the (Fig. 3, A). The BML consisted of base- intercellular spaces of the epithelium, and ment membrane-like material, normal col- in portions the basal epithelial cells seemed lagen fibrils, thin fibrils, some cellular de- to have been destroyed, causing a thin-

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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 thin fibrils1 (t) are also seen. (xl08,000.)

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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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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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ning of the epithelium (Fig. 5, A). Stro- red cells, and some of the endothelial cells mal blood vessels often showed swelling of appeared swollen. In one localized area, the endothelial cells (Fig. 6, D), and a a massive cell infiltration was found in- partial disintegration of the vessel wall volving these vessels as well as an adjacent was also seen occasionally (Fig. 6, C). subepithelial area of the peripheral cornea. Most striking changes were observed in In the latter region, some basal epithelial wide areas of the connective tissue, in- cells seemed to have been destroyed, and cluding the subepithelial region and super- the BML here was markedly thickened ficial stroma. As shown by histochemical (up to 8 /A). These infiltrated cells were stainings on the other half specimen, these mostly neutrophils, but other leukocytes, connective tissue areas were strongly eosin- plasma cells, and mast cells were also ophilic, PAS positive, and stained inten- mixed; some of these mast cells seemed sively blue with PTAH, indicating the to be degranulating. state of "fibrinoid degeneration" or necro- Case 4. Marked degenerative changes sis (Fig. 5, A). Electron microscopy of were seen in the basal epithelial cells of these regions revealed a wide area of the peripheral cornea. These cells had dense granular substance in which rela- a dense cytoplasm and a pyknotic nucleus, tively few normal collagen fibrils of various and many of them possessed bizzare cyto- densities and many thin fibrils were em- plasmic projections, sometimes resembling bedded (Fig. 6, A and B). spider's legs. Those cytoplasmic projections Case 3. Basal epithelial cells were often formed irregular infoldings of the epithelial edematous at the peripheral cornea, and base, and the BML filling these infoldings the subepithelial BML was generally thick- exhibited a layer of extremely irregular er (2 to 3 /*) than normal (Fig. 7, A); thickness (up to 3 /A) (Fig. 8, A). Normal this was partially much thicker as men- Bowman's layer was lost and replaced by tioned below. Occasionally, a loose con- a compact connective tissue which connective tissue accompanied with blood tinued to the deeper stromal region. The vessels was seen to intervene in the sub- diameter of the collagen fibrils were various epithelial region, thereby placing the Bow- (100 to 600 A), with the thicker fibrils man's layer down into the deeper stromal predominating. There was often abnormal- region; the tip of this connective tissue ly little space between these fibrils (Fig. seemed to be directed to the thickened 8, E), and some portions appeared almost BML (Fig. 7, B). Blood vessels were diffusely homogeneous, composed of fibrils also located between the intervening con- without interfibrillar spaces. Patches of nective tissue and an elevated base of dense granular substance were scattered the epithelium, and some of them ap- over the connective tissue area (Fig. 8, peared thrombotic, filled with numerous B). Most cells in the stroma seemed to platelets and other blood cells (Fig. 7, be degenerating keratocytes or fibroblasts C). Near these vessels, neutrophils were with a dense cytoplasm and a pyknotic often interposed between the epithelial nucleus (Fig. 8, B and C). In some por- base and the BML. Deeper stromal colla- tions, however, these cells appeared to gen lamellae near the invading connec- be more intact and often contained many tive tissue were wavy, and many kerato- vacuoles with or without intravacuolar sub- cytes in this region were activated with stance (Fig. 8, B). These portions with increased cisternae of rough surfaced endo- vacuolated cells gradually made transition plasmic reticulum and an enlarged Golgi into a wide peripheral conieal region, in complex (Fig. 7, D). which numerous vacuoles of various size Most blood vessels in the limbal stroma occupied almost the entire thickness of were greatly dilated, filled mainly with the stroma which we studied (Fig. 8,

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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.)

Downloaded from iovs.arvojournals.org on 09/28/2021 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 Bow- man'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, Por- tion 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.) (Orig- inal magnificatnon xl0,500.)

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C and D). Some of these vacuoles were group an inflammatory type and the latter intracellular, but many others were extra- a quiescent type. Although there are some cellular. Here, accumulations of larger similarities between our quiescent type vacuoles could be seen in deeper stroma. (Case 4) and "pellucide marginal degen- A compact arrangement of the collagen eration (Schlaeppi),"2S the presence of a fibrils was most marked in this region. few blood vessels in the right corneal le- Some apparently intact lymphocytes (Fig. sions and a superior location of the left 8, D) and a few blood vessels with a corneal lesions in Case 4 preclude their thick laminated basal lamina (Fig. 8, F) exact identity. were found in the vacuolated area at deep To our knowledge, there have been no stroma near limbus. ultrastructural reports on this disease. We have studied the two clinical types with Discussion the electron microscope. Although the The etiology and pathogenesis of mar- studies were confined to the superficial ginal degeneration are obscure. There layers of the pathologic peripheral corneas, have been two main theories concerning the results were consistent with our tenta- the nature of this disease: one theory re- tive clinical classification. In the "inflam- garded the disease as an inflammation, and matory type" most blood vessels were hy- the other as a degenerative process. Many peremic with a dilated vessel lumen filled recent investigators1'G> ° supported the latter with blood cells, and frequently there view partly based on the earlier histo- were marked inflammatory cell infiltrations, pathologic studies which showed little or whereas in the quiescent type the most no signs of inflammation. However, inflam- notable changes were suggestive of fatty matory signs also have been noted in some change, to be discussed below. Some patho- light microscopic studies.12'13 In addition, logic manifestations found in the inflam- recent reports on this disease suggested matory type, such as the specific cell types the possibility of "collagen disease,"21'22 which predominated in the infiltrates which may possibly be concerned with (lymphocytes in Case 2, neutrophils in a type of inflammation. As earlier authors0 Cases 1 and 3), vascular changes (Cases admitted and also in our own experiences, 1 to 3), and particularly fibrinoid degenera- there seem to be two clinical manifestation (Case 2), are known to occur in tions in marginal degeneration of the hypersensitivity reactions in experimental cornea. The one type is, as seen in Cases animals as well as in various collagen dis- 1 to 3, characterized by a marked vasculari- eases29' 30: For example, lymphocytic infil- zation in and around the corneal lesions trations can be seen typically in delayed and also frequently by a congested status hypersensitivity. While neutrophilic infil- of both the corneal lesions and the sur- trations are seen in anaphylactic and rounding conjunctival blood vessels. Not Arthus reactions, these may also be found infrequently, fluorescein stains borders of in the early stages of delayed hypersensi- the corneal lesion. Topical steroid therapy tivity.31' 32 Vasculitis, accompanied with causes the fluorescein staining to disappear endothelial alterations (including swelling33' and the eye becomes quieter with less 31), disruption or necrosis of the vessel congestion; however, the cornea continues wall and thrombosis, is often observed in to lose its transparency and melt in a cen- Arthus reaction and some collagen dis- tral direction. In the other type, such as eases. In addition, edema and lympho- in Case 4, the vascularization of the lesions cytic infiltrations of the limbal epi- is less marked, usually with little or no thelium32' 3i and a close association of surrounding vessel injection, and the clini- lymphocytes and macrophages,32 such as cal picture is without evidence of active found in Case 2, have been seen in experi- inflammation. We tentatively call the former mentally induced biphasic and delayed

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8 Fig. 8. Case 4. A, Spider-like epithelial basal cell (ep) in the peripheral cornea, bm = Irreg- ularly 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, Com- pactly 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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hypersensitivities of the cornea. Fibrinoid the structural differences between our degeneration may not be specific in hyper- pathologic tissues and normal control tis- sensitivity but is still considered to be sues appeared mainly quantitative, it is an important manifestation in collagen dis- possible that some qualitative changes may eases*0; the ultrastructure of the fibrinoid also be present. region in Case 2 appeared similar to that On the other hand, a characteristic ultra- of the fibrinoid observed in an experimental structure in the quiescent type (Case 4) antigen-antibody reaction in the rabbit cor- was marked intra- and extracellular vacuo- 35 nea, although the latter was modified by lations in wide stromal regions of the labeled ferritin particles. On these grounds, peripheral cornea. These vacuoles re- it would seem possible to speculate that sembled those observed in lipid keratop- those changes seen in the inflammatory athy,40 suggesting that the main alteration type of marginal degeneration are the in Case 4 was a fatty change or degenera- manifestations of hypersensitivity, the dif- tion; a fatty change in this disease has ferent pathologic pictures in individual been noted by earlier investigators.11'1G> 20 cases reflecting different stages. If this The collagen fibrils in these areas showed were true, one conceivable antigenic factor an abnormally compact arrangement, sug- could be the abnormally thickened BML gesting a possibility of tissue anoxia. As which contained numerous "unusual fibrils." to the question whether these changes Recently, the "subepithelial fibrils" in mice, occurred after a prior vascularization41 or which closely resembled the "special fibrils" not, we have seen only a few blood vessels 0 in other animals- as well as our "unusual in deep stroma near the limbus. While fibrils," have been suggested to be modi- a possibility that some vessels might have 30 fied collagen. It may be that the con- regressed in these regions could not be stituents of the BML, including the unusual excluded, our clinical impression was in fibrils and other elements possibly released favor of the interpretation that the changes from degenerated basal epithelial cells, be- preceeded the vascularization; actually no come antigenic in this disease and produce vessels could be detected clinically to enter a hypersensitivity of an autoimmune type. the lesion in this eye. Collagenous proteins, either by themselves In addition to the prevailing changes or as a carrier for a hapten, could become 37 discussed above, there were some other antigenic, as suggested by others. As 38 which appeared to be common to both speculated by Teng in keratoconus, the types. These included: (1) degeneration of thickened BML in our patients seem to basal epithelial cells; (2) abnormal thick- have been initiated by an increased pro- ening of the BML, which may be second- duction of basement membrane-like ma- ary to the first change, as discussed; (3) terial, which was released from degenerated invasion of a connective tissue into the basal epithelial cells and which may con- 39 subepithelial region. In Cases 1 and 4, tain some proteolytic enzymes. It would superficial layers of the epithelium were then be conceivable that the numerous un- partially missing; however, we interpreted usual fibrils found in the BML are collage- these to have occurred during the pro- nous fibrils, so modified under the influence longed surgical procedure. The third men- of the substances contained in the base- tioned change could be seen most clearly ment membrane-like material which has in the inner areas of the corneal lesions infiltrated the Bowman's layer as well as in the inflammatory type (Cases 1 and a replaced connective tissue. It is note- 3), while it seemed to have had taken worthy that a much less prominent but place all over in the quiescent type (Case similar BML can be seen in normal human 4); some cells within such an invading corneas as well, particularly in the peripher- connective tissue in the former type (Case al cornea and limbus. Although at present 1) possessed many vacuoles which often

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contained intravacuolar substance, and sumed fatty change and its related factors, these appeared similar to some cells found including altered fibroblasts. in the latter type (Case 4). In Case 3, the tip of the invading connective tissue seemed to be directed to the BML, and REFERENCES in Case 1 such a connective tissue ap- 1. Duke-Elder, S., and Leigh, A. G.: Diseases of peared to have separated the BML into the outer eye, in Duke-Elder, S., editor: Sys- tem of ophthalmology, vol. 8, part 2, St. two distant layers. Another noteworthy find- Louis, 1965, The C. V. Mosby Company, p. ing was markedly activated deeper stro- 909. mal cells near the invading connective 2. Schmidt-Rimpler, H.: Augenheilkunde und tissue (Cases 1 and 3). Although highly Ophthalmoskopie, Berlin, 1889, Friedrich speculative, summarizing our findings it Wreden, p. 486. appeared as if the following processes 3. Terrien, F.: Dystrophie marginale syme'trique des deux cornees avec astigmatisme r^gulier were taken place: For unknown reasons, consecutif et guerison par la cauterisation basal epithelial cells of the peripheral ignee, Arch. Ophthalmol. (Paris) 20: 12, cornea extensively degenerated, causing an 1900. abnormally thickened BML. The thickened 4. Fuchs, E.: Ueber Randsklerose und Randa- BML motivated an invasion of connective trophie der Hornhaut, Albrecht von Graefe's Arch. Klin. Ophthalmol. 52: 317, 1901. tissue either of corneal or conjunctival 5. Lauber, H.: Ueber periphere Hornhautektasie, origin, thereby replacing the Bowman's Klin. Monatsbl. Augenheilkd. 43: 382, 1905. membrane (or layer). When there was 6. Fuchs, E.: XJber senile Randatrophie der no accompanying vascularization in the Hornhaut, Albrecht von Graefe's Arch. Klin. connective tissue (corneal origin?), it Ophthalmol. 89: 386, 1915. ended in a presumed condition of fatty 7. Trantas, A.: Ectasie peripherique de la cornee de Terrien (Keratoleptynsis marginale), Clin. change. When vascularized, allowing the Ophthalmol. 14: 621, 1925. migration of leukocytes, a presumed hyper- 8. Seefelder, R.: Weiterer Beitrag zur patho- sensitivity occurred, possibly with the thick- logischen Anatomie der Randdegeneration der ened BML acting as antigenic factors. Hornhaut, Klin. Monatsbl. Augenheilkd. 48: 321, 1910. In our tissues obtained by lamellar 9. Coats, G.: Pathological examination of the keratoplasty or superficial keratectomy, a specimen from a case of marginal keratectasia so-called "furrow" or gutter could not be previously shown by Mr. J. Herbert Fisher, clearly visualized. However, at least the Trans. Ophthalmol. Soc. U.K. 31: 5, 1911. corneal changes described above are those 10. Stucchi, C. A.: La maladie de Terrien (De- generescence marginale de la cornee). Kerato- of the peripheral corneal lesions in which plastie transfixiante-histopathologie, Ann. there were certain degrees of corneal thin- Ocul. (Paris) 201: 720, 1968. ning clinically. Among these, the areas 11. Seefelder, R.: Klinisches und Anatomisches observed in Case 2, where most epi- iiber periphere Rinnenbildung und periphere thelial cells appeared to be conjunctival Ektasie der Hornhaut, Klin. Monatsbl. Augen- heilkd. 45: 475, 1907. in nature, may possibly represent the re- 12. Legrand, J., and Hervouet, F.: Ectasie mar- gion in which a vascularized connective ginale de la cornee (maladie de Terrien). tissue extended from the limbal conjunctiva Keratoplastie-etude anatomo-pathologique, to replace the tissue defect of a previously Ann. Ocul. 186: 97, 1953. formed furrow. The direct mechanism 13. Rupprecht, J.: Pathologisch-anatomischer Bei- trag zur Kenntnis der peripheren Hornhautek- causing the corneal thinning could not be tasie, Klin. Monatsbl. Augenheilkd. 45: 34, determined in our study. However, there 1907. were several factors which might be con- 14. Schieck, F.: t)ber die periphere Hornhautek- cerned with it. They were: (1) elements tasie, ihre Pathogenese und operative Be- contained in the BML; (2) fibrinoid de- handlung, Dtsch. Ophthalmol. Ges. 47: 283, 1928. generation (Case 2) or a similar change 15. Hogan, M. J., and Zimmerman, L. E., editors: (Case 1); (3) infiltrating cells; (4) pre- Ophthalmic pathology. An atlas and textbook,

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ed. 2, Philadelphia and London, 1962, W. 31. Jones, J. V.: Cell-mediated immunity and de- B. Saunders Company, p. 320. layed hypersensitivity, in Gold, E. R., and 16. Francois, J.: La d6g6nerescence marginale de Peacock, D. B., editors: Basic immunology, la cornee, Arch. Ophthalmol. (Paris) 53: 616, Bristol, 1970, John Wright & Sons, Ltd., p. 1936. 285. 17. Asayama, R., Sakaue, E., and Sawamoto, Y.: 32. Howes, E. L.: Cellular hypersensitivity in the A case of the marginal keratectasia, Rinsho cornea. An analysis of the limbus and limbal Canka 18: 17, 1964. cellular infiltration by light and electron 18. Seefelder, K. S.: Zur Entstehung der periph- microscopy, Arch. Ophthalmol. 83: 475, 1970. eren Hornhautektasie, Klin. Monatsbl. Augen- 33. Faith, G. C, and Trump, B. F.: The glomer- heilkd. 44: 61, 1906. ular capillary wall in human kidney disease: 19. Ischreyt, C: Ein Fall von peripherer Horn- Acute glomerulonephritis, systemic lupus ery- hautektasie infolge von Trachom, Klin. Mon- thematosus, and preeclampsia-eclampsia. Com- atsbl. Augenheilkd. 45: 197, 1907. parative electron microscopic observations and 20. Thamm, W.: Ueber Randfurchenbildung und a review, Lab. Invest. 15: 1682, 1966. Randektasie der Hornhaut, Klin. Monatsbl. 34. Elliot, J. H., Flax, M. H., and Leibowitz, Augenheilkd. 87: 44, 1931. H. M.: The limbal cellular infiltrate in ex- 21. Frasca, G.: Marginal degeneration of the perimental corneal hypersensitivity. I. Mor- cornea and its possible relationship to collagen phologic studies after primary sensitization, diseases, Rass. Ital. Ottalmol. 27: 255, 1958. Arch. Ophthalmol. 76: 104, 1966. (Cited in Ophthalmic Literature 12: 2801, 35. Wagner, B. M.: Hyalin and fibrinoid: Cur- 1958.) rent status, in Wagner, B. M., and Smith, D. 22. Scialdone, D.: Sistrofia marginale simmetrica E., editors: The connective tissue, Baltimore, della cornea di Fuchs-Terrien, Ann. Ottalmol. 1967, The William & Wilkins Company, p. Clin. Ocul. 92: 491, 1966. 68. 23. Iwamoto, T., and DeVoe, A.G.: Electron 36. Rowlatt, C: Subepithelial fibrils associated microscopic studies on Fuchs' combined dys- with the basal lamina under simple epithelia trophy. I. Posterior portion of the cornea, in mouse uterus: Possible tropocollagen ag- INVEST. OPHTHALMOL. 10: 9, 1971. gregates, J. Ultrastruct. Res. 26: 44, 1969. 24. McTigue, J. W., and Fine, B. S.: The base- 37. O'Dell, D. S.: Immunology of collagen and ment membrane of the corneal epithelium, in related materials, in Gould, B. S., editor: Uyeda, R. editor: Electron microscopy. 6th Treatise on collagen, vol. 2: Biology of colla- International Congress, Vol. 2, Tokyo, 1966, gen, part A, London and New York, 1968, Maruzen Co. Ltd., p. 775. Academic Press, Inc., p. 311. 25. McTigue, J. W.: The human cornea: A light 38. Teng, C. C: Electron microscope study of and electron microscopic study of the normal the pathology of keratoconus. Part I, Am. cornea and its alterations in various dystro- J. Ophthalmol. 55: 18, 1963. phies, Trans. Am. Ophthalmol. Soc. 65: 591, 39. Dohlman, C. H.: The function of the corneal 1967. epithelium in health and disease, INVEST. 26. Palade, G. E., and Farquhar, M. G.: A special OPHTHALMOL. 10: 383, 1971. fibril of the dermis, J. Cell Biol. 27: 215, 40. Jack, R. L., and Luse, S. A.: Lipid keratop- 1965. athy, An electron microscopic study, Arch. 27. Movat, H. Z., and Fernando, N. V. P.: The Ophthalmol. 83:678, 1970. fine structure of the lymphoid tissue during 41. Cogan, D. G., and Kuwabara, T.: Lipid kera- antibody formation, Exp. Mol. Pathol. 4: 155, topathy and atheroma, Circulation 18: 519, 1965. 1958. 28. Schlaeppi, V.: La dystrophie marginale in- 42. Iwamoto, T., and Witmer, R.: Aqueous humor ferieure pellucide de la cornee, Mod. Probl. cytology with the election microscope, Al- Ophthalmol. 1: 672, 1957. brecht von Graefe's Arch. Klin. Ophthalmol. 29. Hopps, H. C: Hypersensitivity diseases, in 174: 110, 1967. Anderson, W. A. D., editor: Pathology, vol. 1, 43. Schulz, H.: Thrombocyten und Thrombose im Saint Louis, 1966, The C. V. Mosby Com- elektronenmikroskopischen Bild, Berlin, Hei- pany, p. 359. delberg, New York, 1968, Springer-Verlag, 30. Vazquez, J. J., and McCarter, J. H.: Im- p. 83. munological aspects of connective tissue dis- 44. King, D. W., editor: Ultrastructural aspects eases, in Wagner, B. M., and Smith, D. E., of disease, New York, Evanston, and London, editors: The connective tissue, Baltimore, 1966, Hoeber Medical Division, Harper & 1967, The Williams and Wilkins1 Company, Row, publishers, p. 112. p. 161.

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