Descemet's membrane in the iridocorneal-endothelial syndrome: Morphology and composition

Exp. Eye Res. (199i) 61, 323-333

Descemet's Membrane in the IridocorneaI-Endothelial Syndrome" Morphology and Composition S. G. L E V Y a,b*, A. C. E. M c C A R T N E y b ' [ ", H. S A W A D A c, P. J. C. D O P P I N G - H E P E N S T A L R.A. ALEXANDER

d,

b A N D J. M O S S ~

Department of Histopathology, Charing Cross and Westminster Medical School London, U.K. bDepartment of Pathology, Institute of Ophthalmology, London, U.K. °Department of Anatomy, Yokohama City University School of Medicine, Yokohama, Japan and dSt Johns Institute of Dermatology, St Thomas" Hospital London, U.K. (Received Lund 26 September 1994 and accepted in revised form 14 March 1995) The iridocorneal-endothelial syndrome is a disease of the ocular anterior segment characterized by corneal failure, glaucoma and iris destruction. Specular photomicroscopical and histological studies suggest the disorder is caused by a population of abnormal corneal endothelial cells. In other corneal endotheliopathies Descemet's membrane, the basement membrane underlying the endothelial cells, is disfigured by the presence of an abnormal region of extracellular matrix termed a posterior collagenous layer, which is laid down by the diseased endothelial cells. In this study we sought to establish the typical morphology and composition of Descemet's membrane in the iridocorneal-endothelial syndrome. Ultrastructural examination of Descemet's membrane in 27 keratoplasty specimens identified three morphologic patterns. In the majority there was a posterior collagenous layer which in all cases consisted of an anterior layer of wide-spaced collagen and a posterior layer of microfibrils embedded in an amorphous matrix. In four specimens which did not possess a posterior collagenous layer the anterior banded zone of Descemet's membrane was absent. In five corneas Descemet's membrane was normal. The composition of the posterior collagenous layer was examined by immunoelectron microscopy (five corneas) and histochemistry (six corneas). Collagen Types I, III. V. VI and VIII. fibronectin, tenascin and oxytalan were microfibrillar components, collagen Type VIII formed wide-spaced collagen whilst laminin was present in the amorphous matrix. The stereotyped derangements of structure and composition identified in the endothelial basement membrane may significantly influence the pathobiology of this disorder. © 1995 Academic Press Limited Key words: corneal endothelium ; iridocorneal-endothelial syndrome : basement membrane : Descemet's membrane; posterior collagenous layer; wide-spaced collagen ; microfibrils; immunoelectron microscopy.

1. Introduction The iridocorneal-endothelial s y n d r o m e (ICE syndrome) is characterized clinically by a ' h a m m e r e d silver' appearance of the corneal endothelium, corneal failure, g l a u c o m a and florid iris atrophy (Campbell, Shields and Smith, 1 9 7 8 ; Shields, Campbell and Simmons, 1 9 7 8 ; Eagle et al., 1 9 7 9 ; Shields et al., 1 9 7 9 ; Frangoulis et al., 1 9 8 5 : Wilson and Shields, 1989). Specular photomicroscopy of the corneal endothelium in this disease shows an a b n o r m a l cell type with features of pleomorphism, indistinct intercellular borders and light-dark reversal of the specular reflex (Setala and Vannas, 1 9 7 9 ; Bourne, 1982 ; Neubauer, Lund and Leibowitz, 1 9 8 3 : Sherrard et al., 1 9 8 5 ; Laganowski et al., 1 9 9 1 ; Sherrard, Frangoulis and Kerr Muir, 1 9 9 1 ; Bourne and Brubaker, 1 9 9 2 : Laganowski, Kerr Muir and Hitchings, 1992). The term 'ICE-cell' has been introduced to describe this cell (Sherrard et al., 1985). * For correspondence at : The Bristol Eye Hospital. Lower Maudlin Street, Bristol BS1 2LX, U.K. "~ Current address : Department ofHistopathology, United Medical and Dental Schools, St Thomas' Hospital. London. U.K. 0 0 1 4 4 8 3 5 / 9 5 / 0 9 0 3 2 3 + 11 $08.00/0

In some instances both ICE-cells and n o r m a l endothelial cells are seen, an appearance designated as 'subtotal-ICE' (Sherrard et al.. 1985). Ultrastructural examination of the endothelium of ICE s y n d r o m e keratoplasty specimens (Levy et al., in press; Sherrard et al., 1991 : Kramer et al., 1992) demonstrates cells with epithelial features w h i c h are the histological correlate of the ICE-cell. Cells w h i c h resemble those of n o r m a l corneal endothelium are the equivalent of the n o r m a l cells seen by specular photomicroscopy in cases of subtotal-ICE. Smaller n u m b e r s of inflammatory cells, mostly macrophages, and fibroblast-like cells are also present. In some endstage corneas, endothelial cell denudation is the p r e d o m i n a n t appearance. Descemet's m e m b r a n e (DM) is the specialized basem e n t m e m b r a n e of the corneal endothelium (Jakus, 1956), a posterior epithelium derived from neural crest rather t h a n surface ectoderm (Johnston et al., 1979). Ultrastructural examination of normal h u m a n DM demonstrates two zones, the anterior banded zone (ABZ) and the posterior n o n - b a n d e d zone (PNBZ) (]akus, 1 9 5 6 ; Murphy, Alvarado and luster, 1 9 8 4 ; Marshall, Konstas and Lee, 1991a). The ABZ is © 1995 Academic Press Limited

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adjacent to the corneal stroma and is about 3"5 # m thick. It is defined by the presence of a form of extracellular matrix known as wide-spaced collagen. Wide-spaced collagen is composed of an approximately hexagonal array of broad 'nodes' connected to each other at a characteristic spacing of 100 nm by thinner 'rods' (]akus, 1956; Sawada, 1982). Descemet's membrane is unique amongst basement membranes in containing large quantities of this material. The PNBZ lies between the ABZ and the basal surface of the corneal endothelial cells. Its width increases with age, reaching 20/~m or more in elderly subjects (Murphy et al., 1984); by comparison, basement membranes elsewhere are only l O 0 - 3 5 0 n m thick (MartinezHernandez and Amenta, 1983: Leblond and Inoue, 1989; Hay, 1991). Normal h u m a n DM contains collagens Type IV (Newsome et al., 1981; Ben-Zvi et al,, 1986: Tsuchiya et al., 1986; Marshall et al., 1991a) and Type VIII (Tamura et al,, 1991), laminin (Ben-Zvi et al., 1986: Kohno et al., 1987; Horikoshi, Koide and Shirai. 1988; Morton et al., 1989; Marshall et al., 1991a) and fibronectin (Ben-Zvi et al., 1986; Tervo et al.. 1986; Kohno et al., 1987). Any process injurious to the corneal endothelium may be associated with morphological abnormalities of DM (Waring III, 1982). In most instances there is an additional region containing microfibrils, widespaced collagen, amorphous material, striated collagen fibrils and sometimes fibroblastic cells. These elements appear in various configurations, for example in Fuchs' endothelial dystrophy they form focal excrescences called guttata which may be obvious clinically (Fuchs, 1910; Hogan, Wood and Fine, 1974), The term 'posterior collagenous layer' (PCL) was adopted (Waring III, 1982) to describe this abnormal region which lies between the endothelial cells and DM proper. Occasionally DM is abnormal in being completely absent rather than by formation of a PCL. This occurs following breaks in DM due to penetrating injury (Alexander and Rahi, 1986), birth trauma, buphthalmos and keratoconus when the free edges of the basement membrane retract (Cogan and Kuwabara, 1971), in posterior polymorphous dystrophy (de Felice et al., 1985) and in developmental diseases such as Peters' anomaly (Waring [II, 1982). Studies on the composition of the basement membrane of diseased corneal endothelium have shown collagen Types IV and VIII and fibrin/fibrinogen in Fuchs' endothelial dystrophy (Kenney et al., 1984), Collagen Types IV and VIII and ofigosaccharides reactive with lectins Ricinus communis agglutinin I and peanut agglutinin are present in pseudophakic bullous keratopathy (Kenney and Chwa, 1990). However the information obtained from these studies is limited since the techniques used did not distinguish between the components of DM proper, which include both Types IV and VIII collagen, and those of the PCL. Few data were available on the appearances of DM in the ICE syndrome ; in one report on eight specimens

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a PCL was present in seven cases, the eighth being normal (Alvarado et al., 1986). We therefore sought to define the characteristic morphology of DM in the ICE syndrome by ultrastructural examination of a large series of corneas taken from patients at the time of penetrating keratoplasty. Having shown consistent patterns of abnormality in the morphology of DM, we subsequently sought to elucidate the nature of its abnormal components using immunoelectron microscopical and histochemical methods. 2. Materials and Methods

Criteria for Diagnosis of the ICE Syndrome Patients were included in the study when they had clinical signs (Campbell et al., 1978; Shields et al., 1978; Eagle et al., 1979; Shields et al., 1979; Frangoulis et al., 1985; Wilson et al., 1989) and specular photomicroscopical appearances (Setala et al., 1979; Bourne, 1982; Neubauer et al., 1983; Sherrard et al., 1985; Laganowski et al., 1991, 1992; Sherrard et al., 1991; Bourne et al., 1992) typical of the ICE syndrome. All patients had the 'hammeredsilver' appearance of the corneal endothelium as well as various combinations of corneal oedema, often at normal or minimally elevated intraocular pressure, glaucoma and iris signs such as atrophy, synechiae and nodule formation. In addition ICE-cells were visualized by specular photomicroscopy in all patients except those with severe corneal oedema. In order to avoid inadvertant inclusion of patients with other disorders such as posterior polymorphous dystrophy (Laganowski, Sherrard and Kerr Muir, 1991) or Fuchs' endothelial dystrophy (Fuchs, 1910 ; Hogan et al., 1974) subjects were excluded if any of the following criteria applied to them: (1) onset at 10 years of age or less; (2) a family history of a similar disorder: (3) clinically evident guttate or vesicular lesions of the corneal endothelium: (4) any preceding major ocular disorder, surgery or penetrating/severe trauma (except filtering surgery necessitated by the ICE syndrome process itselfl.

Acquisition and use of corneal specimens 1. ICE syndrome specimens. Twenty seven corneas were taken from patients with the ICE syndrome at the time of transplantation and processed immediately. The methods used to obtain h u m a n tissue complied with the Declaration of Helsinki. The patients ages ranged from 21 to 58 years. Disease duration varied from 1 to 18 years. All the corneas were processed for routine transmission electron microscopical examination of DM. Five specimens with a PCL were selected for immunoelectron microscopical studies, carried out on portions which had been either frozen or embedded in Lowicryl K4M resin. Six corneas were examined by histochemical techniques, carried out on portions which

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had been processed into paraffin wax; the ICE syndrome corneas used for histochemical studies were chosen from those shown by routine transmission electron microscopy to possess a PCL.

2. Normal corneas. Seven normal corneas used for comparison with the ICE syndrome specimens were received from an eye-bank and processed within 18 hr of removal. The corneas were not used for transplantation because information on HIV and hepatitis status had not been made available to the eye-bank. They were from donors without known ocular disease and appeared normal clinically. Donor ages ranged from 20 to 60 years. All seven were examined by routine transmission electron microscopy. Three were used for immunoelectron microscopical and histochemical studies. Routine Transmission Electron Microscopy Specimens were prepared for routine electron microscopy by fixation in 3 % glutaraldehyde in O"1 M phosphate buffer and post-fixation in 2% osmium tetroxide, followed by dehydrating in ascending grades of ethanol and embedding in Spurr resin. Ultrathin sections were cut in a plane perpendicular to the corneal surface and stained with saturated uranyl acetate in 50% ethanol and Reynold's lead citrate. Ultrathin sections were cut from five regions, each separated by 150 #m, of every ICE syndrome cornea in order to examine different areas of the endothelium.

Antibodies A monoclonal antibody, clone 9H3, to collagen Type VIII was prepared and characterized as previously described (Sawada, Konomi and Hirosawa, 1990); it was used at a dilution of 1 : 2. The other antigens were examined with commercial polyclonal antibodies; anti-human collagen Type I (10111) diluted 1 : 1 0 0 0 from Institut Pasteur de Lyon (Lyon, France), antih u m a n collagen Type III (PS49) diluted 1 : 1 0 from Monosan (Uden, Netherlands), anti-human collagen Type V (PS 51) diluted 1:5 from Monosan, a n t i - h u m a n and bovine collagen Type VI (1360-01) diluted 1:2 O0 from Southern Biotechnology (Birmingham, AL, U.S.A.), anti-human fibronectin (A245) diluted 1 : 100 from Dako (High Wycombe, U.K.), anti-human laminin (AB949) diluted 1 : 1 0 0 from Chemicon (Temecula, CA, U.S.A.) and anti-human tenascin (AB1906) diluted 1 : 1 0 0 from Chemicon. Antibodies to collagen Type I, fibronectin and tenascin were applied to Lowicryl K4M-embedded tissue. Antibodies to collagen Types III, V, VI and VIII and laminin were unsuitable for tissue processed into Lowicryl K4M and were therefore applied to ultrathin frozen sections. Secondary antibodies conjugated to 10 n m colloidal gold were purchased from Biocell (Cardiff, U.K.); goat

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anti-rabbit IgG (EM. GAR 10), goat anti-mouse IgG and IgM (EM. GAF 10) and rabbit anti-goat IgG (EM. RAG 10).

Immunoelectron Microscopy 1. Lowicryl K4M resin-embedded tissue. Specimens were fixed in 4% paraformaldehyde in 0"1 M phosphate buffer pH 7.2 for 4 hr at 4°C. The samples were processed into Lowicryl K4M resin as previously described (Woodrow et al., 1989). Ultrathin sections containing the corneal endothelium and immediately adjacent stroma were collected on uncoated 700mesh nickel grids. Immunocytochemistry was performed by immersing grids in 20-/tl drops of the following solutions in a covered container: 1% ovalbumin in phosphate buffered saline (PBS) (PBS contained 0.1% bovine serum albumin (BSA), 0"02 M sodium azide and 0"05 % Tween 20) for 30 min, primary antibody diluted in PBS for 2 hr, washes in ten drops of PBS over 10 min, gold-conjugated secondary antibody diluted 1 + 1 0 with PBS for 1 hr, washes in five drops of PBS over 10 rain, 2"5% glutaraldehyde in PBS for 3 min. Grids were then jet-washed in distilled water and stained in saturated uranyl acetate in 50% ethanol for 20 min, followed by Reynold's lead citrate for 3 rain. 2. Frozen tissue Specimens were fixed in 4 % paraformaldehyde in 0.1 M phosphate buffer (1 hr at 4°C), cut into pieces approximately 3 × 1 m m in size, cryoprotected in 2.3 M sucrose in PBS (1 hr) and snap frozen in liquid nitrogen. Ultrathin sections containing the corneal endothelium and immediately adjacent stroma were cut at - 8 7 ° C in a cryo-ultramicrotome (FC-4D, Reichert-]ung). Sections were collected with a droplet of 2.3 M sucrose in PBS and placed onto 400mesh pioloform- and carbon-coated nickel grids. Grids were stored section-side down on droplets of PBS + 1% BSA for up to several hours and were then floated onto 20-/,1 droplets of the following solutions: 1% gelatin in PBS for 10 min, 0.02 M glycine in PBS for 3 m i n , P B S + I % BSA for 2 x l m i n , primary antibody diluted in PBS + 1% BSA for 1 hr, washes in PBS+ 1% BSA for 5 x 1 min, gold-conjugated secondary antibody diluted 1 + 1 0 with P B S + I % BSA for 1 hr. washes in PBS for 2 min, 2'5% glutaraldehyde in PBS for 1 min, washes in five drops of distilled water over 5 min. Grids were then stained in uranyl acetate oxalate for 5 min and embedded in 1 : 1 2 % aqueous uranyl acetate-l-5 % methyl cellulose for 3 min. Controls for Immunoelectron Microscopy Controls for the monoclonal antibody to collagen Type VIII were: omission of the primary antibody: monoclonal antibody to amyloid A (Dako M759) served as an antibody of irrelevant specificity since

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FIG. 1. Transmission electron micrograph of normal corneal endothelium. DM lies between the endothelial cell and corneal stroma and is divided into two regions: the ABZ which contains wide-spaced collagen and the amorphous PNBZ. (Basement membrane is above arrow), x 8300. Congo Red staining s h o w e d a m y l o i d to be a b s e n t from DM: similarly processed tissue k n o w n to c o n t a i n collagen Type VIII (bovine DM; S a w a d a et al., 1 9 9 0 ) was used as a positive control. Controls for the p o l y c l o n a l antibodies w e r e : omission of the p r i m a r y a n t i b o d y ; s u b s t i t u t i o n of the p r i m a r y a n t i b o d y with n o n - i m m u n e s e r u m from the s a m e species a n d use of a n i n a p p r o p r i a t e a n t i b o d y ( g r o w t h h o r m o n e , Dako L1814). Similarly processed h u m a n tissue was used for positive controls: c o r n e a l s t r o m a for collagen Types I, [II, V a n d VI (Marshall et al.. 1991 a : Marshall, Konstas a n d Lee, 199 l b ) , k i d n e y for fibronectin and l a m i n i n (Hynes. 1 9 9 0 ) a n d a biopsy from a patient with breast c a r c i n o m a for t e n a s c i n (Koukoulis et al., 1991).

FIG. 2. The arrangement of the PCL seen in ICE syndrome specimens. The ABZ and PNBZ of DM proper are of normal appearance. A bilayered PCL lies between the PNBZ and the basal surface of the endothelial cell. The anterior layer of the PCL contains wide-spaced collagen in a dense amorphous matrix. The posterior layer contains microfibrils in a looser amorphous matrix. In this instance a normal endothelial cell is seen. (Basement membrane is above arrow). Transmission electron micrograph, x 8300.

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POSTERIOR w

Microfibrils

Wide-spaced collagen Amorphous matrix [Posterior non-banded zone] Wide-spaced collagen

[Anteri~

zone]

[

Descemet's membrane ] ] Corneal stroma ] Striated collagen fibrils abnormal ABZ

ANTERIOR

PCL

Fro. 3. Drawing of the basement membrane morphology of ICE syndrome corneal endothelium. Abnormal ABZ: the widespaced collagen of the ABZ is absent or abnormal and the remaining basement membrane consists of amorphous material. PCL: a PCL is located between the PNBZ of DM proper and the endothelial cells. It consists of an anterior layer of wide-spaced collagen and a posterior layer of microfibrils.

FIG. 4. Wide-spaced collagen in ICE syndrome PCL consists of nodes interconnected by thinner rods. Transmission electron micrograph. × 110000.

Histochemical Techniques F i v e - m i c r o m e t r e - t h i c k sections of n e u t r a l formalinfixed, paraffin w a x - e m b e d d e d tissue were used for the following h i s t o c h e m i c a l tests. 1. Verhoeffs iron h a e m a t o x y l i n t e c h n i q u e for elastic tissue (Verhoeff, 1 9 0 8 ; Gomori, 1950). 2. Oxidised a l d e h y d e fuchsin t e c h n i q u e for o x y t a l a n (Edwards, 1968). 3. Congo red t e c h n i q u e for a m y l o i d (Eastwood a n d Cole. 1971). A positive c o n t r o l w a s used for e a c h test.

3. Results Basement Membrane Morpholog!] In the n o r m a l c o r n e a s DM consisted of two regions (Fig. 1): the ABZ, located b e t w e e n the corneal s t r o m a a n d the PNBZ, c o n t a i n e d w i d e - s p r e a d collagen seen as rows of u p r i g h t nodes spaced 100 n m apart. The PNBZ w a s a n a m o r p h o u s region b e t w e e n the ABZ a n d the basal surface of the c o r n e a l endothelial cells. Three distinct m o r p h o l o g i c p a t t e r n s were identified in the ICE s y n d r o m e specimens. There were no

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FIG. 5. Microfibrils in ICE syndrome PCL are fine, non-branching thread-like structures. Transmission electron micrograph. 60 000.

apparent correlations with subject age or disease duration. Comparison between basement membrane and cellular morphology did not reveal consistent associations. Pattern one: bi!a!jered PCL. This was the most common pattern, being observed in nineteen corneas. These specimens (Figs 2 and 3) displayed a uniform appearance in which the ABZ and PNBZ were morphologically normal but posterior to DM proper was a PCL which consisted of two layers. The layers of DM/PCL from anterior to posterior were: (i) and (ii) an ABZ and PNBZ of normal appearance: (iii) a layer containing wide-spaced collagen whose morphology was similar to that present in the ABZ, except that it was more electrondense (Fig. 4). Its orientation was the same in this abnormal layer as in the ABZ, i.e. the nodes generally pointed along the antero-posterior axis of the cornea and so appeared upright in ultrathin sections cut in a plane perpendicular to the corneal surface. Widespaced collagen either formed spindle-shaped aggregates (Fig. 4) or was aligned into masses of greater size, somewhat resembling the normal ABZ. The widespaced collagen was embedded in an amorphous matrix (Figs 2 and 4). There were also some microfibrils and striated collagen fibrils in this layer but they were minority components. (iv) A layer of microfibrils was located between the layer containing wide-spaced collagen and the basal surface of the endothelial ceils, iVIicrofibrils (Fig. 5) were fine, nonbranching structures up to 2 5 nm in diameter. They were without clear cross-striations in routinely processed tissue: however, in the ultrathin cryosections used for immunoelectron microscopy many microfibrils appeared cross-striated (Fig. 6). In some specimens they were arranged in loose interweaving bundles, in others they were not grouped together in

FIG. 6. Transmission electron micrograph demonstrating labelling of microfibrils in the posterior layer of the PCL by antibody to collagen Type V. Similar labelling of microfibrils was seen with antibodies to collagen Types L III, VI and VIII, fibronectin and tenascin. Cryosection. x 60000.

any discernable way. Microfibrils were embedded in an amorphous matrix which was considerably less electron-dense than that seen in the layer containing wide-spaced collagen (Fig. 2). In the most posterior region of the microfibril layer, i.e. the region closest to the endothelial cells, there was often no amorphous matrix and only micro fibrils were present. A few widespaced collagen aggregates and striated collagen fibrils were also seen in this layer. Overall basement membrane thickness was

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FIG. 8. An ICE syndrome specimen in which the widespaced collagen of the ABZ is grossly disorganised and the residual basement membrane severely vacuolated. There is no PCL. Note the ICE-cell. (Basement membrane is above arrow). Transmission electron micrograph. × 24000. The PC1 w a s acellular in all specimens except one in w h i c h it c o n t a i n e d a few fibroblast-like cells.

Pattern two: wide-spaced collagen of the ABZ absent or abnormal. In four c o r n e a s the ordered a r r a y of wide-

FIG. 7. In this ICE syndrome cornea the ABZ is completely absent and the basement membrane is amorphous except for scattered electron-lucent spaces. There is no PCL. Note the ICE-cells which demonstrate desmosomes, tonofilaments and microvilli. {Basement membrane is above arrow). Transmission electron micrograph. × 15000. increased c o m p a r e d to n o r m a l b e c a u s e of the PCL. Only t w o specimens d e m o n s t r a t e d g u t t a t a a n d in one of these only a single g u t t a t e excrescence was seen.

spaced collagen in the ABZ was either totally a b s e n t (Figs 3 a n d 7) or severely disrupted (Fig. 8). The residual b a s e m e n t m e m b r a n e consisted of a m o r p h o u s material which contained numerous electron-lucent spaces. None of these specimens possessed a PCL. In one of the four c o r n e a s DM was m o r p h o l o g i c a l l y n o r m a l in u l t r a t h i n sections from one of the five regions e x a m i n e d . This was the only case in w h i c h there was v a r i a t i o n in the p a t t e r n of b a s e m e n t m e m b r a n e m o r p h o l o g y in different regions of an ICE s y n d r o m e specimen.

Pattern three: normal morphology. In five corneas, (including the specimen in w h i c h most regions d e m o n s t r a t e d an a b n o r m a l ABZ), DM m o r p h o l o g y was normal. The corneal e p i t h e l i u m a n d s t r o m a of the ICE

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Fro. 10. Oxytalan, (seen as a dark line above arrow), is present in the endothelial basement membrane of this ICE syndrome cornea. Total endothelial cell loss has occurred in this specimen. Light micrograph, x 500. wide-spaced collagen of the ABZ and collagen Types I, III, V, and VI were present in the corneal stroma. 2. Histochemistry. Six ICE syndrome corneas in which routine transmission electron microscopy demonstrated a PCL were examined: in all cases the basement m e m b r a n e of the endothelium was strongly positive for oxytalan (Fig. 10) and negative for amyloid and elastic tissue. The three normal corneas used for comparison were negative for oxytalan, amyloid and elastic tissue. 4. Discussion

The composition and significance of the PCL FIG. 9. Laminin is present in the amorphous matrix in which wide-spaced collagen is embedded. Transmission electron micrograph of a cryosection, x 110000. syndrome specimens were unremarkable except for appearances suggestive of oedema.

Basement membrane composition 1. Immunoelectron microscopy. Five ICE syndrome corneas with a PCL were examined by immunoelectron microscopy. Results were the same in all cases. There was specific labelling of the microfibrils in the posterior layer of the PCL (Fig. 6) with antibodies to collagen Types [, III, V, VI and VIII. Similar labelling was seen using antibodies to fibronectin and tenascin. Wide-spaced collagen in the anterior layer of the PCL contained Type VIII collagen: this finding has been reported previously (Levy et al., 1993). The presence of laminin (Fig. 9i was demonstrated in both layers of the PCL in the amorphous material in which widespaced collagen and microfibrils were embedded. Immunostaining in other regions of the ICE syndrome specimens was similar to that seen in the normal corneas ; laminin and fibronectin were present in the ABZ and PNBZ, Type VIII collagen formed the

The microfibrillar layer of the PCL of ICE syndrome corneas was shown by immunoelectron microscopy to contain a complex mixture of substances, including several different collagens and glycoproteins. Oxytalan, which is a microfibril-associated substance (Fullmer and Lillie, 1958; Alexander et al., 1981: Goldfischer et al., 1983), was demonstrated histochemically at the light microscope-level and is probably located in the same layer of the PCL. The large number of microfibril components identified suggests that at least some are heterotypic, i.e. that individual microfibrils are composed of more than a single substance in a m a n n e r analogous to the collagen fibrils of the corneal stroma (Birk, Silver and Trelstad, 1991). Microfibrils were not cross-striated in tissue processed for routine transmission electron microscopy, but interestingly m a n y appeared striated in ultrathin cryosections. Striations in collagen fibrils less than 25 nm in diameter are often not resolved in routinely processed tissue (Hay, 1991; Linsenmayer, 1991). Presumably definition of the striations was greater in material which had been frozen. Collagen Type VIII was a component of microfibrils and also of the wide-spaced collagen in the anterior layer of the PCL. Our findings are compatible with

DESCEMET'S M i = M B R A N E IN THE ICE S Y N D R O M E

those of previous studies which have shown that this collagen assembles into both wide-spaced collagen and microfibrils in bovine extracellular matrix (Sawada et al., 1990; Sawada and Konomi, 1991). The PCL might be secreted by any one or a combination of the different cell types of ICE syndrome corneal endothelium. ICE-cells are one candidate, although they are associated exclusively with this syndrome whilst PCL formation occurs in all corneal endotheliopathies. Alternatively, the PCL might be a product of the normal cells which copopulate the endothelium. This would explain why PCLs are laid down in other disorders and is also consistent with data from experimental studies on the behaviour of normal c o r n e a l endothelial cells; these secrete collagen Types I, II[, IV and V, most of which are present in ICE syndrome PCL, after injury or exposure to inflammatory ceils (Kay et al., 1982; Kenney, Cheung and Smith, 1983; Kay, Nimni and Smith, 1984; Tsuchiya et al., 1986; Kay, Rivela and He, 1990). The bilayered arrangement of the PCL seen in this disease might arise by secretion of different substances at different phases of disease evolution or by aggregation of microfibrils and wide-spaced collagen into separate layers after simultaneous secretion. The fact that no specimen demonstrated either layer on its own supports the latter hypothesis. It is unclear as to whether the deposition of PCL components leads to their permanent presence within the PCL or whether they are subsequently degraded. Is the PCL important or simply an impressive epiphenomenon? There are two reasons to suggest that it m a y be of genuine significance in the pathobiology of the ICE syndrome. Firstly, the bilayered arrangement of the PCL was strikingly similar in all the ICE syndrome specimens in which a PCL was present. This suggests that PCL structure m a y be dictated by specific mechanisms operative in this disorder rather than merely reflecting a general state of endothelial disease. The absence of the guttate PCL formations characteristic of Fuchs' endothelial dystrophy lends support to this hypothesis. Secondly, the importance of the influence exerted by the components of basement m e m b r a n e s on cellular behaviour is well established (Martinez-Hernandez et al., 1983 ; McDonald, 1989; Timpl, 1989; Yurchenco and Schittny, 1990 ; Hay, 1991). Hay states ' the idea that ECM (extracellular matrix) is an inert supporting material...a mere scaffolding.., is now bygone ... The metabolism and fate of the cell, its shape and m a n y of its properties are continuously related to and dependent upon the composition and organisation of the matrix ... The matrix ... talks back to the cells that create it' (Hay, 1991). The term ' d y n a m i c reciprocity' has been proposed to describe this relationship (Sage and Bornstein, 1991). In the specific context of corneal endothelium, changes in cell morphology and behaviour have been shown in vitro in response to laminin (Gospodarowicz

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et al., 1981; Sabet and Gordon, 1987; Gordon, 1988, 1990), fibronectin (Gospodarowicz et al., 1979; Beach and Kenney, 1982; Kay et al., 1982; Hseih and Baum, 1985; Sabet et al., 1987; Gordon, 1988, 1990; Wilson and Lloyd, 1991) and type VIII collagen (Sage et al., 1984; Sage, 1985). The presence of tenascin is often associated with cellular injury although its precise functions in this respect are u n k n o w n (Tervo et al., 1990, 1991; Koukoulis et al., 1991). These substances were all demonstrated in ICE syndrome PCL where they m a y modulate the behaviour of diseased corneal endothelial cells, perhaps influencing such activities as movement, adhesion, division and differentiation. The abnormal ABZ

We found that the ICE syndrome is in some instances associated with the absence of the ABZ of DM. This abnormality has not been previously described and m a y be unique to the ICE syndrome. It differs from other disorders in which DM is occasionally absent (Cogan et al., 1971 ; Waring III, 1982 ; de Felice et al., 1985; Alexander et al., 1986), since in these cases the entire basement m e m b r a n e is missing rather than only one of its layers. It m a y be that this appearance represents abnormal turnover and removal of the wide-spaced collagen of the ABZ, perhaps resulting from the secretion of proteolytic enzymes by one of the different cell types present on the endothelium of ICE syndrome corneas. It is tempting to speculate that ICE-cells are the source of such proteolytic enzymes; further studies are required to address the subject of the turnover of basement m e m b r a n e in this disorder.

Acknowledgements The authors are grateful to the following ophthalmologists for providing specimens: R. Buckley, the late T. A. Casey, ]. Dart, L. Ficker, R.A. Hitchings, M.G. Kerr-Muir, C.M. Kirkness, A. Patterson, N. S. C. Rice, S. Ritten, E. S. Sherrard and A. McG. Steele. Specular photomicroscopy studies of ICE syndrome patients were performed by R. Buckley, M.G. Kerr-Muir, H. Laganowski, S. Ritten and E. S. Sherrard. The authors would also like to thank Ms Ruth Rahman for printing the electron micrographs and Mr Robin Howes, Mr Ian Shore and Dr Michael Barrett for technical assistance. Dr Levy was supported by the T. F. C. Frost fund.

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