ACTA O PHT HAL MO L OGICA VOL. 55 1977

Department of Ophthalmology, Arhus Kommunehospital, and the Institute of Anatomy, University of Aarhus, Denmark

POLYSACCHARIDE COATING OF HUMAN CORNEAL ENDOTHELIUM BY

HENRIK DAA SCHRPIDER and STEFFEN SPERLING

Electron microscopy revealed the presence of a 600-1500 A thick layer of polysaccharide on the surface of human corneal endothelial cells. The surface layer was visualized by combined fixation and staining in a mixture of ruthenium red and osmium tetroxide. The coating material was stable for at least 39 h post mortem and was retained on disintegrating cells.

Key words: cell coat - corneal endothelium - human cornea - mucopolysaccharide - ruthenium red - electron microscopy.

Since the demonstration of mucopolysaccharide surface coatings o n human vascular and peritoneal endothelial cells by McGovern (1956) and o n the surface of more than 50 different cell types in the r a t by Rambourg, N e u t r a & Leblond (1966), several investigators h a v e reported observations o n mammals and birds

pointing towards the presence of some substance o n the posterior corneal surface. At present very f e w d a t a a r e available concerning the physical or chemical structure of such a coating material. Abelsdorff & Wessely (1909) found a viscous material occupying the main part of the anterior chamber of various owls. Bkriny, Berggren & Vrabec (1957) found an increasing density of the material towards the owl cornea, and found that the material could be partially depolymerized by testicular hyaluronidase. Vrabec (1957, 1958) and Wolff (1968) obtained replicas of a layer on endothelial cells and observed silver Received April 24, 1977.

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Henrik Daa Schrsder and S t e f f e n Sfierling impregnation of some substance on the posterior corneal surface. Speakman (1959a,b) found a n osmium tetroxide positive layer on the posterior corneal surface in rabbit, cat and monkey, which did not stain by traditional histochemical methods for identification of acid mucopolysaccharides. Iwamoto & Smelser (1965) observed that thorotrast particles clumped a t some distance from the endothelial cell wall when injected into the anterior chamber of human eyes. Preziosi (1966) observed some substance covering the endothelial surface of Rhesus monkeys when viewed in phase contrast microscopy. Kirk & Hassard (1969) showed that the outline of endothelial cells in cat, ox and man could be brought forth under controlled osmotic and ionic conditions. They inferred that some substance coated the intercellular borders and suggested that an extension of the substance to the entire endothelial surface prevented the staining of dead feline endothelial cells by lissamine green. Hodson (1971) presented evidence of endothelia1 macromolecular synthesis by in vitro experiments with radioactively labelled glucose. During this study Harnish (1976) verified the presence of acid mucopolysaccharides on cells covering the trabeculum of human eyes by application of the ruthenium red osmium tetroxide technique introduced by Luft (1966). This study was undertaken after observations parallelling those reported by Preziosi (1966) in incident light and phase contrast microscopy on living bovine endothelial cells.

Material and Methods T h e material comprised 11 normal human corneas from patients two days to 81-yearsold, fixed a t various time intervals post mortem. Corneas were obtained after storage a t 16-22OC for 8-10 h and a t 4OC for the remaining post mortem time. One central corneal button with normal endothelial cells from a case of keratoconus and one central button from a normal cornea were prepared immediately after excision. Luft (1966) introduced a technique by which some cell coats are made visible in light and electron microscopy by the coupling of ruthenium red and osmium tetroxide to acid mucopolysaccharides in the coat. The original technique of fixation in glutaraldehyde and ruthenium red prior to treatment by osmium tetroxide and ruthenium red was discarded after repeated failures of retaining the endothelial cells in contact with the membrane of Descemet. Combined staining and fixation was carried out by immersion of the tissue in equal parts of ruthenium red, 1500 ppm. in distilled water, 5 O / o osmium tetroxide and 0.2 N cacodylate buffer (pH 7.3) for ll/z h at 25OC. After fixation, the tissue was transferred to cacodylate buffer, dehydrated in graded ethanol to propylene oxide and embedded in epon. Some ultrathin sections were grid stained by uranyl acetate and lead citrate before examination in a Jeol 100 C microscope, other sections were examined without additional staining.

ResuIts When the tissue was fixed in ruthenium red osmium tetroxide a coherent deposit of osmium tetroxide appeared on the endothelial surface (Fig. 1). In sections perpendicular to the cell wall (indicated by a distinct threelaminar cell membrane), the deposit measured between 600 and 1500 A. Extensions

Coating of Corneal Endothelium

Fig. 1. Cellular coating on endothelium from an infant aged 2 days. Fixed 14 h post mortem in ruthenium red osmium tetroxide and contrast stained. Magnification: 6600 x.

of the surface deposit reached the tight junctions. No surface material was found in the intercellular spaces basal to the tight junctions. Threadlike, branching and coiled extensions of the deposit were found as far as 8 p m from the coherent surface layer (Figs. 2 and 3). Only small amounts of electron dense material were found on the cell surfaces when ruthenium red was omitted from the fixative (Fig. 4). No time related change was noted in the density or in the configuration of the surface deposit. The coating material was still present on the fragmented outer limiting membranes of disintegrating cells with gross degenerative changes (Fig. 5 ) . Addition of ruthenium red to osmium tetroxide did not affect the preservation of intercellular ultrastructure. The ultrastructure of cells fixed in ruthenium red csmium tetroxide immediately post mortem appeared normal. Endothelial cells fixed 8 to 39 h post mortem showed vacuoles in the cytoplasm, mitochcndrial swelling, fragmentation of cristae and decreased density of the matrix material (Figs. 1-7). I n each cornea, cells with only slight mitochondria1 swelling as well as cells with clumping of nuclear chromatin, endoplasmatic disorganisation and rupture of the limiting membranes were observed. T h e relation of the surface coating to the phenomenum of vacuolization

82 I Acta ophthal. 53, 5

53

Henrik Dan Schr~derand Steflen Sperling

described by Kirk & Hassard (1969) was investigated. A cornea obtained 14 h post mortem was bisected. One half was fixed in ruthenium red osmium tetroxide (Fig. 6), while the other half was immersed in 0.9 O/O NaCl for 10 min before fixation. The vacuolization appeared by widening of the intercellular spaces between basal desmosomes and the tight junctions while the coating material was unchanged (Fig. 7 ) .

Figs. 2-4. 2 and 3. Extensions of the cellular coating on endothelium from a patient aged 50 years. Fixed 9 h post mortem in ruthenium red osmium tetroxide. Magnification: 10 000 x. 4. Endothelium from a patient aged 56 years. Fixed 10 h post mortem in buffered osmium tetroxide and contrast stained. Magnification: 10 000 x.

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Coating of Corneal Endothelium

F i g . 5. Cellular coating on a disintegrating cell from a patient aged 81 years. Fixed 15 h post mortem in ruthenium red osmium tetroxide and contrast stained. Magnification: 20 000 x.

Comments Ruthenium red is an ionic complex of ruthenium and ammonia. The molecular weight is 860 and the net charge of the complex is +6 (Fletcher et al. 1961). Ruthenium red precipitates a variety of negatively charged large polymers and fails to precipitate neutral polysaccharides and aliphatic compounds (Luft 1966). It stains pectin in plant tissue (Jensen 1962), bone matrix and interfibrillary substance in tendons (Heidenhain 1913), coating on intestinal microvilli (Luft 1964) and the cell coat of amoebae (Szubinska 1964). The amount of ruthenium red bound in precipitated polysaccharides does not increase the mass of the stained tissue components sufficiently to make it visible under the electron microscope. In presence of osmium tetroxide, a coupled reaction of acid polysaccharide, ruthenium red and osmium tetroxide occurs (Luft 1966). Mitochondria1 disorganization, cytoplasmatic vacuolization and clumping of nuclear chromatin were the only marked early degenerative changes. Identical observations were reported by Shaeffer (1963) and Schultz (1971) in post mortem materials. 823

Henrik Daa Schreder and S t e f f e n Sfierling

Figs. 6 a n d i. 6. Tightly adhering endothelial cells from an infant 2 days old. Fixed 14 h post mortem in ruthenium red osmium tetroxide and contrast stained. Magnification: 8500 x . 7 . Second half of the cornea in Fig. 6. Preparation as in Fig. 6 after 10 min in 0.9O/o NaC1. Magnification: 8500 x.

T h e photomicrographs obtained in this study shows coherent ruthenium red osmium tetroxide precipitates on the cellular surface forming a layer of 6001500 A and strings of precipitate extending as far as 8 p m from the surface. This finding confirms the existence of a macromolecular layer coating the endothelial cells. T h e photographs may represent the in uivo configuration of the coating material or they may picture a distorted and shrunken gel with an increasing density towards the surface of the cells similar to the gel found on the owl corneas (Biriny, Berggren & Vrabec 1957). The finding of similar layers of coating material on cells fixed only seconds after removal from a living patient and on dead and disintegrating cells fixed hcurs after corporal death suggests that the coating material is stable for a t least 39 h post mortem. A t present the physiological role of this coating material is entirely unknown. The presence of a presumably negatively charged layer on the surface of normal endothelial cells calls for revision of earlier estimates of ionic gradients across the endothelial cell membrane. 824

Coating of Corneal Endothelium

Acknowledgment The authors wish to express their gratitude to Mr. Bjarne Krunderup and Mrs. Ilona Bach-Ssrensen, Institute of Anatomy, Department B, for their skilled technical assistance.

References Abelsdorff G. & Wessely K. (19U9) Vergleichend-physiologische Untersuchungen iiber den Flussigkeitswechsel des Auges in der Wirbelthierreihe. Arch. Augenheilk. 64, Erganzungshef t, 65- 125. Bbrbny E., Berggren L. & Vrabec F. (1957) T h e mucincus layer covering the corneal endothelium in the owl S t r i r Aluco. Brit. /. Ophtlzal. 41, 25-30. Fletcher J. M. et al. (1961) Ruthenium Red. /. chem. Soc. 2, 2000-2006. Harnish J. P. (1976) Elektronmikroskopische Darstellung saurer Mukopolysakkaride im Trabekelwerk. Klin. Mbl. Augenheifk. 169, 90-94. Heidenhain M. (1913) Uber die Bearbeitung der Sehnen zu Kurszwecken, inbesondere iiber die Verwendung des Rutheniumrots und Malloryschen Bindgewebsfarbung. Z. wiss. Mikr. 30, 161-167. Hodson S. (1971) Makromolecular synthesis in corneal endothelium. Ex@. Eye Res. 1 1 , 15-19. Iwamoto T. & Smelser K. (1965) Electron microscopy of the human corneal endothelium with reference to transport mechanisms. Invest. Ophthal. 4, 270-284. Jensen W . A. (1962) Botanical Histoclremistry. Freeman, San Francisco 1962. Kirk A. H. & Hassard D. T. R. (1969) Supravital staining of the corneal endothelium and evidence for a membrane on its surface. Canad. /. Ophthal. 4 , 405-415. Luft J. H. (1964) Electron microscopy of cell extraneous coats as revealed by ruthenium red staining. /. Cell Biol. 23, 54A-55A. Luft J. H. (1966) Fine structure of capillary and endocapillary layer as revealed by ruthenium red. Fed. Proc. 25, 1773-1783. McGovern V. J. (1965) Mast cells and their relationship to endothelial surfaces. /. Path. Bact. 7 1 , 1-6. Prezicsi V. A. (1966) A n apparatus for phase-contrast microscopic observation of viable corneal endothelium during freezing and thawing. Amer. /. Ophthal. 62, 19-26. Rambourg A,, Neutra M. & Leblond C. P. (1966) Presence of a “cell coat” rich in carbohydrate a t the surface of cells in the rat. Anat. R e f . 154, 41-52. Schaeffer E. M. (1963) Ultrastructural changes in moist chamber corneas. Invest. Ophthal. 2, 272-282. Schultz R. 0. (1971) Laboratory evaluation of cryopreserved corneal tissue. Trans. amer. ophthal. SOC. 69, 563-608. Speakman J. (1959a) Endothelial cell vacuolation in the cornea. Brit. /. Ophthal. 43, 139-146. Speakman J. (1959b) Stain permeability and ultrastructure of the corneal endothelium. Arch. Ophthal. (Chicago) 62, 882-888. Szubinska B. (19G4) Electron microscopy of the interaction of ruthenium violet with the cell membrane complex of Amoeba proteus. /. Cell Biol. 23, 92 A.

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Henrik Daa Schreder and S t e f f e n Sperling Vrabec F. (1957) The inner surface of the trabecular meshwork studied by a replica technique. Amer. J . Oplitlral. 44, 7-12. Vrabec F. (1955) Studies on the corneal and trabecular endothelium. Brit. J . Oplitlzal. 42, 529-534. Wolff J. (19G8) Inner surface of regions in the anterior chamber. Docum. ophtlzal. 25, 113-247.

Author’s address: Steffen Sperling, Department of Ophthalmology, Arhus Kommunehospital, University of Aarhus. DK-8000 Arhus C, Denmark.

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Polysaccharide coating of human corneal endothelium.

ACTA O PHT HAL MO L OGICA VOL. 55 1977 Department of Ophthalmology, Arhus Kommunehospital, and the Institute of Anatomy, University of Aarhus, Denmar...
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