A C T A O P H T H A L M O L O G I C A VOL. 5 6 1 9 7 8
Ophtlialniology (Head: Niels Elilers), Arhus Kommrrneliospital. and 2Department of Palaeoecology (Head: Nils Spjeldnes), In.ctiticte of Ecology and Genetics, University of Arhus, Denmark IDepartment
of
SCANNING ELECTRON MICROSCOPIC OBSERVATIONS
OF A MUCOPOLYSACCHARIDE COATING ON HUMAN CORNEAL ENDOTHELIUM BY
STEEN ROJ JACOBSEN1 and STEFFEN SPERLING?
Scanning electron microscopic observations of human corneal endothelium revealed a binding of ruthenium red-osmium tetroxide to a material on the inner surface of the endothelial cells on the cornea and the trabecular meshwork. The coating material was partially digested by proteolytic enzymes.
K e y words: cell coat - corneal endothelium - human cornea - acid mucopolysaccharide - ruthenium red - scanning electron microscopy - trabecular meshwork - trypsin - urokinase.
In an earlier publication (Schreder k Sperling 1977) the transmission electron microscopic observation of 60-150 nm thick layer of ruthenium red-osmium tetroxide precipitates, on the surface of normal human corneal endothelial cells, was reported. The demonstration was achieved by fixation of corneas in a mixture of ruthenium red and osmium tetroxide. Ruthenium red precipitates a number of negatively charged large polymers. In presence of osmium tetroxide a coupled reaction between ruthenium red, osmium tetroxide and acid polysaccharide occurs (Luft 1964, 1966, 197 la,b). This study was undertaken in order to gain additional information on the ruthenium red-osmium tetroxide precipitates on human endothelial cells by scanning electron microscopic observations. Received December 24, 1977.
161
Steen
Rq' jncobsen mid Stefjen S p e r l i n g Material and Methods
The material comprised 6 normal human corneas. Corneas were judged normal when no history of eye disease appeared from the case record and when inspection revealed a normal cornea and a normal anterior chamber. Corneas were obtained 8 to 36 h post mortem. Whole eyes were removed from the bodies after storage at 16-22OC for 8-10 h, later at 4OC. The corneas with an adjacent rim of sclera were removed atraumatically from the globes (Sperling 1977). The coating material was visualized by fixation of the tissue in equal parts of ruthenium red, 15 p. p. m. in distilled water, 5 O/o osmium tetroxyde, and 0.2 N cacodylate buffer (pH 7.3) for I & h at 25OC. Some tissue was fixed in 3 O/o glutaraldehyde or 3.6 O/o formaldehyde in 0.15 M Ssrensens phosphate buffer (pH 7.4) for 2 h at 25OC. After fixation the tissue was transferred to cacodylate buffer and dehydrated in graded ethanol, critical point dried from carbon dioxide, vacuum coated with gold and examined in a Cambridge Stereoscan GOO, scanning electron microscope. One cornea was incubated in a balanced salt solution for 30 min at 25OC before fixation. The salt solution contained NaCI, KCI, MgS04, Na,HPO,, CaCI,, glucose and phenol red. It was buffered to pH 7.45 by Tris (Ackroyd 1964). Other corneas were incubated in the salt solution with urokinase (2500 ,dml) or trypsin (25 mg/ml) for 30 min at 25OC.
Resu Its W h e n t h e tissue was fixed in glutaraldehyde or in formaldehyde the surface of endothelial cells o n cornea and o n the strands of t h e trabeculum w a s seen (Figs. 1 a n d 3). After fixation i n ruthenium red-osmium tetroxide the surface
Fig. 1. F i g . 2. Fig. 1 . Surface of corneal endothelial cells fixed in formaldehyde. Magnification 110 x. F i g . 2. The surface of corneal endothelial cells after fixation in ruthenium red-osmium tetroxyde. Magnification 80 x.
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Coat on Corneal Endotltelirrni
Fig. 3. Trabecular meshwork. Fixed in glutaraldehyde. Crystals between the strands in the meshwork. Magnification 1700 x.
patern of endothelial cells was obscured. A coherent layer coated the entire endothelial surface (Figs. 2 and 4). The smallest structures recognized in the material were spherical bodies of sizes 100-200 nm. The bodies were pparently linked together in aggregates
Fig. 4. The surface of the trabecular meshwork after fixation in ruthenium red-osmium tetroxyde. Magnification 1900 x.
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Steen R0j Jacobsen and S t e f l e n Sperling
Fig. 6 . Fig. 5 . Fig. 5 . Higher magnification of the coating material from the preparation shown in Fig. 4. The smallest structures visible are spherical bodies with diameters 100-200 mm. Magnification 10 000 x. Fig. 6 . A strand of the coating material protruding 80-100 p m from the main surface. From the preparation shown in Fig. 4. Magnification 500 x.
Fig. 7 . Fig. 8. Fig. 7 . Coating material partially removed from the corneal cells by trypsin. Magnification 525x. Fig. 8. Remanents of coating material on strands of the trabecular meshwork in a cornea treated by trypsin. Fixed in ruthenium red-osmium tetroxide. Magnification 850 x.
164
Coat on Corneal Endothelium
Fig. 9. Fig. 10. Fig. 9. The coating material after treatment by urokinase. Magnification 80 x. Fig. 10. Higher magnification of an area from the preparation in Fig. 9. The smallest structures visible are spherical bodies. Magnification 1800 x.
without preferred orientation (Figs. 4 and 5 ) . Strands and clumps of the material protruded 80-100 nm from the main surface of the layer (Fig. 6). In order to study the structural basis for the observed granular material coreas were treated with urokinase or trypsin before fixation. After enzymatic treatment the coating maerial was absent in some areas (Figs. 7 and 8). Remaining material appeared as whirled interwoven threads (Fig. 9). The micro structure of the threads was the same as the structure of the coherent surface layer (Fig. 10). T h e structure of the coating material was unaffected by incubation in the balanced salt solution.
Discussion T h e surface structure of corneal endothelial cells and of the trabeculum as it appears in a scanning electron microscope after glutaraldehyde fixation was described in detail by Svedbergh & Bill (1972) and by Bill & Svedbergh (1972). Ruthenium red precipitates a number of negatively charged large polymers and some lipids but fails to precipitate neutral polysaccharides and aliphatic compounds (Luft 1964, 1966, 197 la,b). The staining by ruthenium red-osmium 165
Stcen Koj Jacobsen nnd S t e f f e n Sperling
tetroxide points towards the presence of polysaccharides or lipids in the coating material. A polysaccharide component in the coating material was suggested by KlouEek (1967) after staining of the coat by colloidal iron. Polysaccharide in the coating material was also inferred by Wolff (1968) on basis of partial digestion of the material by hyaluronidase and metachromatic staining of material retained on plaster of Paris replicas. Urokinase and trypsin are proteolytic enzymes acting on peptide links without enzymatic activity towards polysaccharides or lipids. T h e structural change induced by proteolytic enzymes indicates that the coating material contains peptides. Surface coats containing lipids, proteins and polysaccharides have been observed on a number of other cells. This subject have been rewieved by Parsons & Subjeck (1972) and treated in detail by Cook & Stoddart (1973). This study shows that ruthenium red-osmium tetroxide precipiates in a substance on endothelial cells on cornea and in the trabeculum. Although no indication of the in uivo configuration of the substance is found the profound change of the gross structure of the substance after treatment by urokinase or trypsin implies a structural basis for the apparent configuration of the coating material. Further studies of the structure, the composition and the physiological role of the coating material are in progress.
Acknowledgment This work was supported by a grant from The Danish Committee for Prevetition of Blindness.
References Ackroyd J. F. (1961) Immunological methods, p. 212. A symposium organized by the Council for International Organization of Medical Sciences ets. Blackwell Scientific Publication, Oxford. Bill A. & Svedbergh B. (1972) Scanning electron microscopic studies of the trabecular meshwork and the canal of Schlemm - an attempt to localize the main resistance to outflow of aqueous humor in man. Acta ophthal. (Kbh.) 50, 295-320. Cook G. M. W. & Stoddart R. W. (1973) Surface carbohydrates of the eukaryotlc cell. Academic Press, London & New York. Klourek F. (1967) The corneal endothelium. Acta Univ. Carol. Med. 13, 321-375. Luft J. H. (1964) Electron microscopy of cell extraneous coats as revealed by ruthenium red staining. J . 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. 166
Luft J. H. (1971a) Ruthenium red and violet. I . Chemistry, purilication, methods of use for electron microscopy and mechanism of action. Anat. Rrc. 171, 347-368. Luft J. H. (1971b) Ruthenium red and violet. 11. Fine structural localization in animal tissue. Anat. Rec. 171, 369-416. Parsons D. F. & Subjeck J. R. (1972) T h e morphology of the polysaccharide coat of mammalian cells. Biochim. hiophys. A c l n ( A m s t . ) 265, 85-1 13. Schreder H. D. & Sperling S. (1977) Polysaccharide coating of human corneal endothelium. Actn o ~ h t h n l (Kblz.) . 55, 819-825. Sperling S. (1977) A simple apparatus for controlled rate corneal freezing. Actn oplzthal. (Kblz.) :Ti, 1-8. Wolff J. (1968) Inner surface of regions in the anterior chamber. Docrim. ophthnl. 2,i, 113-247.
Author’s address: Steffen Sperling, Department of Ophthalmology, Arhus Kommunehospital, University of Aarhus, DK-8000 Arhus C, Denmark.
167
Ophtlialniology (Head: Niels Elilers), Arhus Kommrrneliospital. and 2Department of Palaeoecology (Head: Nils Spjeldnes), In.ctiticte of Ecology and Genetics, University of Arhus, Denmark IDepartment
of
SCANNING ELECTRON MICROSCOPIC OBSERVATIONS
OF A MUCOPOLYSACCHARIDE COATING ON HUMAN CORNEAL ENDOTHELIUM BY
STEEN ROJ JACOBSEN1 and STEFFEN SPERLING?
Scanning electron microscopic observations of human corneal endothelium revealed a binding of ruthenium red-osmium tetroxide to a material on the inner surface of the endothelial cells on the cornea and the trabecular meshwork. The coating material was partially digested by proteolytic enzymes.
K e y words: cell coat - corneal endothelium - human cornea - acid mucopolysaccharide - ruthenium red - scanning electron microscopy - trabecular meshwork - trypsin - urokinase.
In an earlier publication (Schreder k Sperling 1977) the transmission electron microscopic observation of 60-150 nm thick layer of ruthenium red-osmium tetroxide precipitates, on the surface of normal human corneal endothelial cells, was reported. The demonstration was achieved by fixation of corneas in a mixture of ruthenium red and osmium tetroxide. Ruthenium red precipitates a number of negatively charged large polymers. In presence of osmium tetroxide a coupled reaction between ruthenium red, osmium tetroxide and acid polysaccharide occurs (Luft 1964, 1966, 197 la,b). This study was undertaken in order to gain additional information on the ruthenium red-osmium tetroxide precipitates on human endothelial cells by scanning electron microscopic observations. Received December 24, 1977.
161
Steen
Rq' jncobsen mid Stefjen S p e r l i n g Material and Methods
The material comprised 6 normal human corneas. Corneas were judged normal when no history of eye disease appeared from the case record and when inspection revealed a normal cornea and a normal anterior chamber. Corneas were obtained 8 to 36 h post mortem. Whole eyes were removed from the bodies after storage at 16-22OC for 8-10 h, later at 4OC. The corneas with an adjacent rim of sclera were removed atraumatically from the globes (Sperling 1977). The coating material was visualized by fixation of the tissue in equal parts of ruthenium red, 15 p. p. m. in distilled water, 5 O/o osmium tetroxyde, and 0.2 N cacodylate buffer (pH 7.3) for I & h at 25OC. Some tissue was fixed in 3 O/o glutaraldehyde or 3.6 O/o formaldehyde in 0.15 M Ssrensens phosphate buffer (pH 7.4) for 2 h at 25OC. After fixation the tissue was transferred to cacodylate buffer and dehydrated in graded ethanol, critical point dried from carbon dioxide, vacuum coated with gold and examined in a Cambridge Stereoscan GOO, scanning electron microscope. One cornea was incubated in a balanced salt solution for 30 min at 25OC before fixation. The salt solution contained NaCI, KCI, MgS04, Na,HPO,, CaCI,, glucose and phenol red. It was buffered to pH 7.45 by Tris (Ackroyd 1964). Other corneas were incubated in the salt solution with urokinase (2500 ,dml) or trypsin (25 mg/ml) for 30 min at 25OC.
Resu Its W h e n t h e tissue was fixed in glutaraldehyde or in formaldehyde the surface of endothelial cells o n cornea and o n the strands of t h e trabeculum w a s seen (Figs. 1 a n d 3). After fixation i n ruthenium red-osmium tetroxide the surface
Fig. 1. F i g . 2. Fig. 1 . Surface of corneal endothelial cells fixed in formaldehyde. Magnification 110 x. F i g . 2. The surface of corneal endothelial cells after fixation in ruthenium red-osmium tetroxyde. Magnification 80 x.
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Coat on Corneal Endotltelirrni
Fig. 3. Trabecular meshwork. Fixed in glutaraldehyde. Crystals between the strands in the meshwork. Magnification 1700 x.
patern of endothelial cells was obscured. A coherent layer coated the entire endothelial surface (Figs. 2 and 4). The smallest structures recognized in the material were spherical bodies of sizes 100-200 nm. The bodies were pparently linked together in aggregates
Fig. 4. The surface of the trabecular meshwork after fixation in ruthenium red-osmium tetroxyde. Magnification 1900 x.
163
Steen R0j Jacobsen and S t e f l e n Sperling
Fig. 6 . Fig. 5 . Fig. 5 . Higher magnification of the coating material from the preparation shown in Fig. 4. The smallest structures visible are spherical bodies with diameters 100-200 mm. Magnification 10 000 x. Fig. 6 . A strand of the coating material protruding 80-100 p m from the main surface. From the preparation shown in Fig. 4. Magnification 500 x.
Fig. 7 . Fig. 8. Fig. 7 . Coating material partially removed from the corneal cells by trypsin. Magnification 525x. Fig. 8. Remanents of coating material on strands of the trabecular meshwork in a cornea treated by trypsin. Fixed in ruthenium red-osmium tetroxide. Magnification 850 x.
164
Coat on Corneal Endothelium
Fig. 9. Fig. 10. Fig. 9. The coating material after treatment by urokinase. Magnification 80 x. Fig. 10. Higher magnification of an area from the preparation in Fig. 9. The smallest structures visible are spherical bodies. Magnification 1800 x.
without preferred orientation (Figs. 4 and 5 ) . Strands and clumps of the material protruded 80-100 nm from the main surface of the layer (Fig. 6). In order to study the structural basis for the observed granular material coreas were treated with urokinase or trypsin before fixation. After enzymatic treatment the coating maerial was absent in some areas (Figs. 7 and 8). Remaining material appeared as whirled interwoven threads (Fig. 9). The micro structure of the threads was the same as the structure of the coherent surface layer (Fig. 10). T h e structure of the coating material was unaffected by incubation in the balanced salt solution.
Discussion T h e surface structure of corneal endothelial cells and of the trabeculum as it appears in a scanning electron microscope after glutaraldehyde fixation was described in detail by Svedbergh & Bill (1972) and by Bill & Svedbergh (1972). Ruthenium red precipitates a number of negatively charged large polymers and some lipids but fails to precipitate neutral polysaccharides and aliphatic compounds (Luft 1964, 1966, 197 la,b). The staining by ruthenium red-osmium 165
Stcen Koj Jacobsen nnd S t e f f e n Sperling
tetroxide points towards the presence of polysaccharides or lipids in the coating material. A polysaccharide component in the coating material was suggested by KlouEek (1967) after staining of the coat by colloidal iron. Polysaccharide in the coating material was also inferred by Wolff (1968) on basis of partial digestion of the material by hyaluronidase and metachromatic staining of material retained on plaster of Paris replicas. Urokinase and trypsin are proteolytic enzymes acting on peptide links without enzymatic activity towards polysaccharides or lipids. T h e structural change induced by proteolytic enzymes indicates that the coating material contains peptides. Surface coats containing lipids, proteins and polysaccharides have been observed on a number of other cells. This subject have been rewieved by Parsons & Subjeck (1972) and treated in detail by Cook & Stoddart (1973). This study shows that ruthenium red-osmium tetroxide precipiates in a substance on endothelial cells on cornea and in the trabeculum. Although no indication of the in uivo configuration of the substance is found the profound change of the gross structure of the substance after treatment by urokinase or trypsin implies a structural basis for the apparent configuration of the coating material. Further studies of the structure, the composition and the physiological role of the coating material are in progress.
Acknowledgment This work was supported by a grant from The Danish Committee for Prevetition of Blindness.
References Ackroyd J. F. (1961) Immunological methods, p. 212. A symposium organized by the Council for International Organization of Medical Sciences ets. Blackwell Scientific Publication, Oxford. Bill A. & Svedbergh B. (1972) Scanning electron microscopic studies of the trabecular meshwork and the canal of Schlemm - an attempt to localize the main resistance to outflow of aqueous humor in man. Acta ophthal. (Kbh.) 50, 295-320. Cook G. M. W. & Stoddart R. W. (1973) Surface carbohydrates of the eukaryotlc cell. Academic Press, London & New York. Klourek F. (1967) The corneal endothelium. Acta Univ. Carol. Med. 13, 321-375. Luft J. H. (1964) Electron microscopy of cell extraneous coats as revealed by ruthenium red staining. J . 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. 166
Luft J. H. (1971a) Ruthenium red and violet. I . Chemistry, purilication, methods of use for electron microscopy and mechanism of action. Anat. Rrc. 171, 347-368. Luft J. H. (1971b) Ruthenium red and violet. 11. Fine structural localization in animal tissue. Anat. Rec. 171, 369-416. Parsons D. F. & Subjeck J. R. (1972) T h e morphology of the polysaccharide coat of mammalian cells. Biochim. hiophys. A c l n ( A m s t . ) 265, 85-1 13. Schreder H. D. & Sperling S. (1977) Polysaccharide coating of human corneal endothelium. Actn o ~ h t h n l (Kblz.) . 55, 819-825. Sperling S. (1977) A simple apparatus for controlled rate corneal freezing. Actn oplzthal. (Kblz.) :Ti, 1-8. Wolff J. (1968) Inner surface of regions in the anterior chamber. Docrim. ophthnl. 2,i, 113-247.
Author’s address: Steffen Sperling, Department of Ophthalmology, Arhus Kommunehospital, University of Aarhus, DK-8000 Arhus C, Denmark.
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