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Quantitative and comparative ultrastructure of the vertebrate cornea I. Urodele amphibia
TISSUE & CELL 1976 8 (4) 591-602 Published by Longman Group Ltd. Printed in Great Britain
LUKAS
H. MARGARITIS”,
THEMISTOCLES K. POLlTOF”r and JOHN X. KOLlOPOULOS$
QUANTITATIVE AND COMPARATIVE ULTRASTRUCTURE OF THE VERTEBRATE CORNEA I. URODELE AMPHIBIA ABSTRACT. The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet’s membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the cornea1 surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet’s membrane and the epithelium are believed to represent the primitive situation in the process of cornea1 evolution.
Introduction
transparency throughout the life cycle of some organisms have been observed and correlated to the cornea1 ultrastructure (Pederson et al., 1971). This work introduces a series of studies with the ultimate goal of investigating the variations in cornea1 ultrastructure throughout vertebrates. Since amphibia mark the transition from the aquatic to the land life, we decided to investigate and compare the cornea of the Urodele amphibian Triturus c. cristatus with other corneas (Jakus, 1956; Kaye, 1962; Goldman and Benedek, 1967; Walls 1967; Maurice, 1969a; Dohlman, 1971; Margaritis et al., 1975).
the vertebrates examined so far, the cornea shows a strikingly uniform morphology, i.e. it consists of an epithelium, an underlying basement membrane, a randomly organized collagenous layer (Bowman’s layer), a collagenous stroma more or less organized in lamellae, a second basement membrane (Descemet’s membrane) and an endothelium. The participation of these layers in the transparency of the cornea has been investigated (Goldman extensively and Benedek, 1967; Maurice, 1969a, b; Smith, 1969; Cox et al., 1971), and variations of this THROUGHOUT
From the Dept. of Biology, University of Athens, Greece, and the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A. Present address: * Dept. of Biology, University of Athens, Panepistimiopolis-Kouponia, Athens-621, Greece. t Dept. of Physics, Cornell University, Ithaca, N.Y., U.S.A. $ University Eye Clinic, National Ophthalmological Center, Mesogion-170, Holargos, Athens, Greece.
Materials and Methods
Eight corneas were studied out of an equal number of salamanders Triturus c. cristatus. After dissection the tissues were fixed as shown (Table 1). Dehydration in ethanol was followed by embedding in EponAraldite (Margaritis, 1974). Sections were
Received 5 April 1976. Revised 16 August 1976. 591
MARGARITIS,
592
POLITOF
AND
KOLIOPOULOS
Table 1. Fixation methods used during this investigation Fixative A 2 % glutaraldehyde 0.1 M SCB pH 7.4 90 min, 20°C
Wash A 6 % sucrose 0.1 M SCB pH 7.4 40 min, 4°C
2 % glutaraldehyde 800 ppm Ruthenium Red 0.1 M SCB pH 7.4* 90 min, 20°C
6% sucrose 600 ppm Ruthenium Red 0.1 M SCB 40 min, 4°C
2 % glutaraldehyde 1% LaNOat 0.1 M SCB pH 7.4 6 hr, 20°C
6 % sucrose 1% LaN03 0.1 M SCB pH 7.4 overnight 4°C
Fixative B
Wash B
Abbreviation
-
-
G
2 % OS04 aqueous 90 min, 4°C 3 % KMn04 0.1 M SCB pH 7.4 20 min, 4°C
As wash A
GO
As wash A
GPM
2 % OS04 400 ppm Ruthenium Red aqueous 90 min. 4°C
As wash A
GRRO
-
-
GLa
* Luft (197la). t Revel and Karnovsky (1967). cut on a MT-l Porter-Blum ultramicrotome with glass knives and were stained either with alkaline methylene blue for light microscopy and quantitative analysis or with uranyl acetate (Watson, 1958) followed by lead citrate (Venable and Coggeshall, 1965) for examination under the Philips electron microscopes EM200 and EM301 operating at 80 kV. The latter microscope was equipped with a goniometer stage which facilitated the observations of the occluding junctions. Micrographs were recorded on DuPont S-Litho Ortho sheet film and on Kodak electron image glass plates. Magnification calibration was performed with a carbon grating replica (Polysciences, Inc.). During light microscopy with a Leitz Ortholux microscope, quantitative analysis of the cornea1 layers was carried out by means of a micrometer slide and a micrometric occular (Leitz, Wezlar). At least ten measurements were taken at different regions of the layer under consideration from each cross-sectioned cornea. Mean values from the eight corneas and standard deviations
were subsequently calculated, the last factor representing both the physical variations in thicknesses and the errors of measurements.
Results I. Quantitative data At the very beginning of this investigation it became clear that this cornea differs in many essential points from mammalian corneas, so that a quantitative analysis and comparison appeared to be necessary. The layers of the salamander cornea show similarities and differences when compared with those of other vertebrate corneas (Table 2). The epithelium appears relatively thick and the basement membrane extraordinarily thick (cu.
3700 A) when compared to the mammalian corneas but are comparable to the corresponding layers of the elasmobranch cornea. Conversely Descemet’s membrane and the stroma are extremely thin in the amphibian and elasmobranch corneas. In the salamander the thickness of the lamellae is about 1.1 f 0.2 p; a slight gradient in fiber thickness has been detected: the fibers in
* Calculated t Margaritis $ Calculated 4 Calculated
from from
et al.
from
10-20 105 0.3tH.44 No data 200-250
Bowman Stroma Descemet Endothelium Total
4@-55 zzo.14
-15 42 EO.15 _ Goldman and Benedek (1967). (1975). Maurice (1969a). Dohlman (1971).
lOtx137 0.28-0.42
Epithelium Basement membrane
Layer
Elasmobranch (shark)* Thickness % of total (P) 30.7k3.7 0.5kO.l 67.2* 10.0 0.7kO.l 0*9?0-4 -
24‘251.9 0.37*0.10 53+7 0.56+0.07 o-74+0.3 79+9
Urodele amphibian (Triturus cristatus) Thickness % of total (P)
280+8 7.4kO.4 3-s+o-4 329+11
38.5k2.7 0~10+0~03
8553 2.1270.14 1.1 _+O*l -
11.7kO.9 0~03~0~009
Mammalian (rabbit)? Thickness % of total (P)
Cornea
_N52$ 0~01-0~03$ ?0~06§ z12 _N450 5-101 -5% 530 f 40
El0 ro-004 ~0.011 !z2.3 ~85.6 El.5 -1 -
Mammalian (human) Thickness % of total (CL)
-
:
z
z 2
: E
G
0 crl
Table 2. Quantitative comparison of saIamander’s corneal layers with other vertebrate corneas. It is clear that aN layers show variations in thicknesses and 2 percentages throughout vertebrates ln
2
2
2
r
c ?
594
MARGARITIS,
the middle lamellae are approximately 10% thicker than the rest. The mean value of the total collagen fibers was calculated as 267f 16 A after glutaraldehyde-Ruthenium Red-osmium tetroxide (GRRO) fixation and 301 f 29 A after glutaraldehyde-potassium permanganate(GPM) fixation whereas the striations of the collagen fibers show a peridocity of about 700 A after the last fixation method. II. Ultrastructural data In cross-section, the salamander cornea is about 80 p thick and consists of the following layers (Fig. 1) : (a) Epithelium. This cornea has three layers of epithelial cells; flattened basal cells, flattened and slightly polygonal intermediate cells and finally superficial almost cuboidal cells (Figs. 1, 2). Occluding junctions were observed at the outer region of adjacent epithelial cells (Fig. 3) which were otherwise
POLITOF
AND
KOLIOPOULOS
attached to each other through desmosomes. The surface of the epithelium is differentiated into microvilli with attached microfilaments (Fig. 4). The two outermost cell layers show a polarity in the distribution of dense vesicles (Fig. 2) which exhibit a granular content when RR is used during fixation (Fig. 4). The filaments attached to microvilli as well as the granular content of the vesicles are less obvious after GO fixation (Fig. S), almost invisible after G fixation (Fig. 6, although some material within the vesicle has been preserved), and totally invisible following GPM fixation (Fig. 7). All epithelial cells contain many mitochondria, tonofilaments and rough endoplasmic reticulum. No colloidal lanthanum (La) tracer could be detected inside the intercellular space after GLa fixation. (b) Basement membrane (basal lamina). The epithelium is separated from the stroma by a
Fig. 1. Light micrograph of cross-sectioned salamander cornea. Epithelium is shown to consist of three cell layers, i.e. basal flattened cells, intermediate slightly polygonal cells and superficial almost cuboidal cells. The basement membrane is visible (arrow) even at this low power light micrograph. x 600. Fig. 2. Shape differentiation of cells throughout epithelium is very limited, unlike other corneas. Also in this cornea a large number of dense vesicles is preferentially distributed at one hemisphere of the two outermost cell layers. The basement membrane shows moderate relative electron density (asterisk). GRRO fixation. x 6500. Fig. 3. The anterior intercellular region of the superficial cells has been differentiated into occluding junctions which normally are rarely observed. However by tilting the section along an axis parallel to the border between the cells these pentalaminar structures are clearly visible (arrow). Desmosomes form the lower end of the junctional complex. A, 30” tilt; B,.9” tilt; C, D, 2” tilt. G fixation. A, B, C, x 55,000; D, x 130,000. Fig. 4. Extracellular fine fillaments attached to the microvilli of the superficial cells can be detected when Ruthenium Red is included during fixation. Another result of the mentioned method is the preservation of granular dense vesicles (arrow). GRRO fixation. x 38,000. Fig. 5. The extracellular fine filaments demonstrated after GRRO fixation are absent following GO fixation. Also lower electron density show the intracellular vesicles. GO fixation. x 38,000. Fig. 6. A dramatic network of tonofilaments is revealed following G fixation. The extracellular filaments are absent whereas the dense vesicles look empty, although some dense material has been preserved (arrow). G fixation. x 38,000. Fig. 7. Total extraction of the vesicle material, absence of the extracellular fine filaments and the intracellular tonofilaments occurs when GPM is the fixative. GPM fixation. x 38,000.
ULTRASTRUCTURE
OF
AMPHIBIAN
CORNEA
relatively thick basement membrane (Figs. 2, 8) which shows the same relative electron density regardless of the applied fixation method (Figs. 8-11). Small granules were observed in the intracellular clefts of the basal epithelial cells with gradually increasing density towards the basement membrane (Fig. 8). (c) Substantia propria (stroma). The stroma consists of about 40 highly organized collagenous lamellae perpendicularly oriented to one another (Fig. 12). The lamellae are composed of hexagonally arranged collagen fibers which are interconnected by fine filaments most pronounced when RR is included during fixation (Fig. 13). No randomly oriented collagen fibers (Bowman’s layer in some corneas) were detected at the outer region of the stroma facing the basement membrane. Finally, a very thin Descemet’s membrane of granular structure (Fig. 14) separates the stroma from the endothelium being its basement membrane. Discussion I. General Unlike mammalian or even Anuran amphibian cornea (Kaye, 1962) we didn’t observe extended differentiation of the epithelial cells as far as shape is concerned. The use of RR during fixation enabled us to identify as mucopolysaccharide-rich (Luft, 1971b) the filaments on the epithelial surface as well as the material inside the dense vesicles. Penetration of RR through the intact plasma membrane and into the vesicles appears paradoxical. However, a similar unexplained penetration of RR has been reported previously (Luft, 1971b). Although the extracellular filaments are considered to represent material secreted for the lubrication of cornea1 surface (Holy and Lemp, 1971) from the Harderian gland (Tansley, 1965), the fate of the intracellular dense vesicles is obscure. Since the intermediate epithelial cells show such vesicles distributed with the same polarity, and considering that these cells are destined to replace the superficial ones after a certain period of time, we come to the conclusion that the material of the vesicles is secreted in some unknown way and may contribute to the lubrication process. Thus it is clear that
597
the epithelium provides more than a smooth protective refracting surface for the eye (Smelter and Ozanics, 1965). The vesicle content seems to be of proteinaceous nature as well (glycoprotein ?): it is partly preserved after G fixation, which is known to cross-link proteins (Hopwood, 1972), and totally extracted after PM fixation which is considered to cause fragmentation of proteins (Hake, 1965; Lenard and Singer, 1968). The observed polarity in the distribution of the vesicles could be retained by means of a potential in the epithelial cells known to exist in rabbit (Maurice, 1967) as well as in other corneas (Ehlers, 1970). The intercellular permeability of the epithelial cells was found to be very low, as demonstrated by the inability of lanthanum and Ruthenium Red to penetrate. A similar situation has been observed in the rabbit cornea (Leuenberger, 1973). A very low intercellular permeability has been shown to be effectively achieved by the existence of occluding junctions (Farquhar and Palade, 1963) which were observed during this study at the intercellular outer region of the superficial epithelial cells. Although we did not make use of any purely histochemical reactions in order to identify the chemical character of the basement membrane, we come to the following conclusions after consideration of what is currently known about the action of the applied fixatives: this membrane (a) is not entirely proteinaceous, since it does not lack electron density after GPM fixation which is known to destroy proteins (Hake, 1965 ; Lenard and Singer, 1968; Margaritis and Moudrianakis, 1974), and (b) is not rich in unsaturated lipids or disulfide bonds, because it does not show greater electron density following GO fixation (Stoeckenius, 1959; Hake, 1965). Thus it seems that the composition of this membrane fits the general description of the basement membranes (Kefalides, 1973); part of the material may be carbohydrate (fair reaction with PM, Glaeser and Mel, 1964; good reaction with RR, Luft, 1971a), and part protein (good reaction with G and GO, Hopwood, 1972). Suggestive for basement membrane synthesis is also the appearance of the extracellular glycogen-like granules. More
598
MARGARITIS,
evidence, however, has been obtained by amplification of that synthesis in response to ultraviolet light irradiation (Margaritis et al., 1976). No extensive sutural fibers throughout stroma could be detected in this cornea as was reported to exist in the dogfish (Goldman and Benedek, 1967). The relative uniformity in the diameter of the collagen fibers throughout stroma has been observed also in the chick (Hay and Revel, 1969); in the human cornea the variation is major (Jakus, 1961) whereas in the dogfish it is not significant (Goldman and Benedek, 1967). No periodic substructure could be identified in Descemet’s membrane as has been reported for human cornea (Jakus, 1956). II. Evolution This amphibian cornea has a relatively thick basement membrane and a thin Descemet’s membrane. Thus, the thickness of Descemet’s membrane is roughly double that of the basement membrane, whereas in mammalian corneas the ratio is more than SO-fold; moreover, Descemet’s membrane is roughly equal to the endothelium in thickness, whereas in mammalian corneas it is at least twice as thick. The fact that a Descemet’s membrane is either very thin (frog, Kaye, 1962; shark, Goldman and Benedek, 1967) or totally absent (lizard, Walls, 1967; sea lamprey, Pederson et al., 1971; teleost fish, Tripathi, 1974) in some primitive vertebrates, suggests
POLITOF
AND
KOLIOPOULOS
that it was developed late during evolution. It is secreted by an endothelium which during earlier evolutionary stage possibly had the function of controlling cornea1 hydration (Friedman, 1973). A thick basement membrane has also been reported in elasmobranchs (Goldman and Benedek, 1967) and in sea lampreys (Horn et al., 1969), whereas in the frog the thickness is moderate (Kaye, 1962). Thus it seems possible that a thick basement membrane is an exclusive feature either of aquatic and amphibious animals or of primitive vertebrates, although a systematic study is not available as yet. As far as the thickness of the epithelium is concerned it is believed that a thick epithelium is important in resisting the transcorneal flux of water in aquatic animals; the thickness is considerable in fish (trout, carp, pike) and chondrichthies as well (Tripathi, 1974). This feature has also been retained in the cornea of the frog (Kaye, 1962). The vertebrate eye originated in aquatic organisms (Walls, 1967) and amphibia mark the transition from aquatic to terrestrial life in the evolution of the vertebrate phylum. The big change in the eye during evolution came when animals left the water for land; in this new milieu one important problem was to protect the cornea from damage, which now had become the main refracting structure (Tansley, 1965). This might be achieved by a thicker stroma as in mammals.
Fig. 8. A thick basement membrane (cu. 3700 A) marks the border between epithelium and stroma while no Bowman layer is evident unlike other corneas; collagen fibers start to be organized into lamellae at the very beginning of the stroma. Dense granules are very often observed in the intercellular spaces of the basal cells occasionally associated with the basement membrane (arrow). GRRO fixation. x 30,000. Figs. 9-11. No great differences are visible regarding the relative electron density of the basement membrane after various fixation methods (compare also with Fig. 8). Fig. 9; GO fixation; Fig. 10, G fixation; Fig. 11. GPM fixation. x 30,000. Figs. 12-13. Cross section of stromal lamellae. The collagen fibers tend to form hexagonal lattice and are interconnected with fine filaments. Groups of other intermediate thickness fibers are also observed (circle). GRRO fixation. Fig. 12, x 50,000; Fig. 13, x 115,000. Fig. 14. No apparent brane. GRRO fixation.
substructure x 300,000.
is evident in a cross-sectioned
Descemet’s
mem-
-
‘t‘
ULTRASTRUCTURE
OF
AMPHIBIAN
CORNEA
The stroma is relatively thin in this amphibian (-40 lamellae) and in purely aquatic vertebrates (shark, 25 lamellae, Goldman and Benedek, 1967; sea lamprey, 2: 13 lamellae, Horn et al., 1969). As far as Bowman’s layer is concerned, it is unclear whether or not there is any correlation between its existence and cornea1 evolution. Some teleost fish (Tripathi, 1974) as well as chondrichthies (Goldman and Benedek, 1967) and sea lampreys (Horn et al., 1969) do have Bowman’s layer, whereas on the other hand some other vertebrate corneas do not (rat, Jakus, 1954; mouse, Whitear, 1960; frog, Kaye, 1962; marsupials and avian, Walls, 1967; rabbit, Margaritis et al., 1975). The absence of this layer from various vertebrate corneas scattered throughout evolution may be
601
explained by the fact that whenever Bowman’s layer is damaged it regenerates no more as such (Maurice, 1969a). Further systematic studies are required throughout in order to clarify the differences in ultrastructure among corneas throughout vertebrates. Acknowledgements
This work was supported by a grant from the Empirikion foundation of Athens Greece. We thank Dr F. C. Kafatos for his valuable suggestions. Completion of the work was performed in the laboratory of Dr D. Branton (The Biological Laboratories, Harvard University), to whom we are deeply indebted.
References Cox, J. L., FARRELL, R. A., HART, R. W.
and LANGHAM, M. E. 1971. The transparency of the mammalian cornea. J. Physiol., 210,601-616. DOHLMAN, C. H. 1971. The function of the cornea1 epithelium in health and disease. Invest. Ophthal., 10, 383-407. EHLERS, N. 1970. Some comparative studies on the mammalian cornea1 epithelium. Acra Ophthal., 48, 821-828. FARQUHAR, M. G. and PALADE, G. E. 1963. Junctional complexes in various epithelia. J. Cell Biol., 17, 375412. FRIEDMAN, M. H. 1973. Critique of current theories of cornea1 hydration control. J. theor. Biol., 42,287-306. GLAESER, R. M. and MEL, H. C. 1964. The electrophoretic behaviour of Os04-fixed, KMnOl-fixed rat erythrocytes. Biochim. Biophys. Acta, 79,606-621. GOLDMAN, J. N. and BENEDEK, G. B. 1967. The relationship between morphology and transparency in the unswelling cornea1 stroma of the shark. Invest.Ophthal., 6, 574-600. HAKE, T. 1965. Studies on the reactions of 0~04 and KMn04 with amino acids, proteins and peptides. Lab. Invest.,14 (6), 1208-1212. HAY, E. D. and REVEL, J. P. 1969. Fine Structure of the Developing Avian Cornea. Karger, Base1 and New York. HOLY, F. J. and LEMP, M. A. 1971. Wettability and wetting of cornea1 epithelium. Expl. Eye RES., 11, 239-250. HOPWOOD, D. 1972. Theoretical and practical aspects of glutaraldehyde fixation. Hid. J., 4, 267-303. HORN, VAN, D. L., EDELHAUSER,H. F. and SCHULTZ, R. 0. 1969. Ultrastructure of the primary spectacle and cornea of the sea lamprey. J. Ultrustruct. Res., 26, 454-464. JAKUS, M. A. 1954. Studies on the cornea. I. The fine structure of the rat cornea. Am. J. Ophthal., 38 (No. 1, part II), 40-52. JAKUS, M. A. 1956. Studies on the cornea. II. The fine structure of Descemet’s membrane. J. biophys. biochem. Cytol., 2, Suppl. 243-255. JAKUS, M. A. 1961. The fine structure of the human cornea. In The Structure ofthe Eye (ed. G. K. Shelser), pp. 343-366. Academic Press, New York and London. KAYE, G. I. 1962. Studies on the cornea. 111. The fine structure of the frog cornea and the uptake and transport of colloidal particles by the cornea in viva. J. Cell Biol., 15, 241-258. KEFALIDES,N. A. 1973. Structure and biosynthesis of basement membranes. Int. Rev. Corm. Tissue Res.. 6, 63-104. LENARD, J. and SINGER, S. J. 1968. Alteration of the conformation of proteins in red blood cell membranes and in solution by fixatives used in electron microscopy. J. Cell Biol., 37, 117-121.
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LEUENBERGER,D. M. 1973. Lanthanum hydroxide tracer studies on rat cornea1 endothelium. Expl. Eye Res., 15,85-91. LUFT, J. H. 1971a. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anaf. Rec., 171, 347-368. LUFT, J. H. 1971b. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anar. Rec., 171, 369-415. MARGARITIS,L. H. 1974. Programmed synthesis of specific proteins in cellular differentiation. A contribution to the study of chorion formation in the follicles of Drosophila melanogaster. Ph.D. Thesis, University of Athens, Greece. MARGARITIS, L. H. and MOUDRIANAKIS, E. N. 1974. Studies on glutaraldehyde-potassium permanganate (GPM) fixation for the preservation and ultrastructural identification of nucleic acids. Abstract. In First Balkan Congress on Electron Microscopy, Sarajevo, 22-25 May, pp. 99-100. MARGARITIS,L. H., POLITOF, Th. K. and KOLIOPOULOS,J. X. 1975. Quantitative changes and ultrastructural alterations of the cornea after exposure to ultraviolet light. 1. Effects on rabbits. Minerva Medica Grecu, 3 (3), 295-305.
MARGARITIS,L. H., POLITOF, Th. K. and KOL~OPOULOS,J. X. 1976. Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light. II. Effects on Amphibia: elucidation of desmosomal structure and basement membrane synthesis. Tissue & Cell, 8, 603-614. MAURICE, D. M. 1967. Epithehal potential of the cornea. Expl. Eye Res., 6, 138-140. MAURICE, D. M. 1969a. The cornea and sclera. In The Eye, Vol. I. Vegetative Psyciology and Biochemistry (ed. H. Davson), pp. 489-600. Academic Press, New York and London. MAURICE, D. M. 1969b. The transparency of the cornea1 stroma. Vision Res., 10, 107-108. PEDERSON,H. J., HORN, VAN, D. L. and EDELHAUSER,H. F. 1971. Ultrastructural changes associated with loss of transparency in the primary spectacle and cornea of the spawning sea lamprey. Expl. Eye Res., 12.1477150. REVEL, J. P. and KARNOVSKY, M. 1967. Hexagonal array of subunits in intracellular junctions of the mouse heart and liver. J. Cell Biol., 33, C7-Cl2. SMELSER,G. K. and OZANICS, V. 1965. New concepts in anatomy and histology of the cornea. In The Cornen, World Congress (ed. J. H. King and J. W. McTigue), pp. I-20, 163-356, Butterworths, Washington. SMITH, J. N. 1969. The transparency of the cornea1 stroma. Vision Res., 9, 393-397. STOECKENIUS,W. 1959. An electron microscope study of myelin figures. J. biophys. hiochem. Cy/o/., 5, 491-500.
TANSLEY,K. 1965. Vision in Vertebrates. Chapman and Hall, Ltd, London. TRIPATHI, R. C. 1974. Comparative physiology and anatomy of the outflow pathway. In The Eye, Vol. 5. Comparative Physiology (ed. H. Davson and L. T. Graham), pp. 1633356. Academic Press, New York and London. VENABLE,J. and COGGESHALL. R. 1965. A simplified lead citrate stain fat use in electron microscopy. J. Cell Biol., 25, 407413.
WALLS, G. L. 1967. The Vertebrate Eye and its Adaptive Radiation. Hafner Publishing Company, New York and London. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem.
Cytol., 4, 475478.
WHITEAR, M. 1960. An electron microscope vation. J. Anat., 94, 387-409.
study of the cornea
in mice with special reference
to the inner-
LUKAS
H. MARGARITIS”,
THEMISTOCLES K. POLlTOF”r and JOHN X. KOLlOPOULOS$
QUANTITATIVE AND COMPARATIVE ULTRASTRUCTURE OF THE VERTEBRATE CORNEA I. URODELE AMPHIBIA ABSTRACT. The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet’s membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the cornea1 surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet’s membrane and the epithelium are believed to represent the primitive situation in the process of cornea1 evolution.
Introduction
transparency throughout the life cycle of some organisms have been observed and correlated to the cornea1 ultrastructure (Pederson et al., 1971). This work introduces a series of studies with the ultimate goal of investigating the variations in cornea1 ultrastructure throughout vertebrates. Since amphibia mark the transition from the aquatic to the land life, we decided to investigate and compare the cornea of the Urodele amphibian Triturus c. cristatus with other corneas (Jakus, 1956; Kaye, 1962; Goldman and Benedek, 1967; Walls 1967; Maurice, 1969a; Dohlman, 1971; Margaritis et al., 1975).
the vertebrates examined so far, the cornea shows a strikingly uniform morphology, i.e. it consists of an epithelium, an underlying basement membrane, a randomly organized collagenous layer (Bowman’s layer), a collagenous stroma more or less organized in lamellae, a second basement membrane (Descemet’s membrane) and an endothelium. The participation of these layers in the transparency of the cornea has been investigated (Goldman extensively and Benedek, 1967; Maurice, 1969a, b; Smith, 1969; Cox et al., 1971), and variations of this THROUGHOUT
From the Dept. of Biology, University of Athens, Greece, and the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A. Present address: * Dept. of Biology, University of Athens, Panepistimiopolis-Kouponia, Athens-621, Greece. t Dept. of Physics, Cornell University, Ithaca, N.Y., U.S.A. $ University Eye Clinic, National Ophthalmological Center, Mesogion-170, Holargos, Athens, Greece.
Materials and Methods
Eight corneas were studied out of an equal number of salamanders Triturus c. cristatus. After dissection the tissues were fixed as shown (Table 1). Dehydration in ethanol was followed by embedding in EponAraldite (Margaritis, 1974). Sections were
Received 5 April 1976. Revised 16 August 1976. 591
MARGARITIS,
592
POLITOF
AND
KOLIOPOULOS
Table 1. Fixation methods used during this investigation Fixative A 2 % glutaraldehyde 0.1 M SCB pH 7.4 90 min, 20°C
Wash A 6 % sucrose 0.1 M SCB pH 7.4 40 min, 4°C
2 % glutaraldehyde 800 ppm Ruthenium Red 0.1 M SCB pH 7.4* 90 min, 20°C
6% sucrose 600 ppm Ruthenium Red 0.1 M SCB 40 min, 4°C
2 % glutaraldehyde 1% LaNOat 0.1 M SCB pH 7.4 6 hr, 20°C
6 % sucrose 1% LaN03 0.1 M SCB pH 7.4 overnight 4°C
Fixative B
Wash B
Abbreviation
-
-
G
2 % OS04 aqueous 90 min, 4°C 3 % KMn04 0.1 M SCB pH 7.4 20 min, 4°C
As wash A
GO
As wash A
GPM
2 % OS04 400 ppm Ruthenium Red aqueous 90 min. 4°C
As wash A
GRRO
-
-
GLa
* Luft (197la). t Revel and Karnovsky (1967). cut on a MT-l Porter-Blum ultramicrotome with glass knives and were stained either with alkaline methylene blue for light microscopy and quantitative analysis or with uranyl acetate (Watson, 1958) followed by lead citrate (Venable and Coggeshall, 1965) for examination under the Philips electron microscopes EM200 and EM301 operating at 80 kV. The latter microscope was equipped with a goniometer stage which facilitated the observations of the occluding junctions. Micrographs were recorded on DuPont S-Litho Ortho sheet film and on Kodak electron image glass plates. Magnification calibration was performed with a carbon grating replica (Polysciences, Inc.). During light microscopy with a Leitz Ortholux microscope, quantitative analysis of the cornea1 layers was carried out by means of a micrometer slide and a micrometric occular (Leitz, Wezlar). At least ten measurements were taken at different regions of the layer under consideration from each cross-sectioned cornea. Mean values from the eight corneas and standard deviations
were subsequently calculated, the last factor representing both the physical variations in thicknesses and the errors of measurements.
Results I. Quantitative data At the very beginning of this investigation it became clear that this cornea differs in many essential points from mammalian corneas, so that a quantitative analysis and comparison appeared to be necessary. The layers of the salamander cornea show similarities and differences when compared with those of other vertebrate corneas (Table 2). The epithelium appears relatively thick and the basement membrane extraordinarily thick (cu.
3700 A) when compared to the mammalian corneas but are comparable to the corresponding layers of the elasmobranch cornea. Conversely Descemet’s membrane and the stroma are extremely thin in the amphibian and elasmobranch corneas. In the salamander the thickness of the lamellae is about 1.1 f 0.2 p; a slight gradient in fiber thickness has been detected: the fibers in
* Calculated t Margaritis $ Calculated 4 Calculated
from from
et al.
from
10-20 105 0.3tH.44 No data 200-250
Bowman Stroma Descemet Endothelium Total
4@-55 zzo.14
-15 42 EO.15 _ Goldman and Benedek (1967). (1975). Maurice (1969a). Dohlman (1971).
lOtx137 0.28-0.42
Epithelium Basement membrane
Layer
Elasmobranch (shark)* Thickness % of total (P) 30.7k3.7 0.5kO.l 67.2* 10.0 0.7kO.l 0*9?0-4 -
24‘251.9 0.37*0.10 53+7 0.56+0.07 o-74+0.3 79+9
Urodele amphibian (Triturus cristatus) Thickness % of total (P)
280+8 7.4kO.4 3-s+o-4 329+11
38.5k2.7 0~10+0~03
8553 2.1270.14 1.1 _+O*l -
11.7kO.9 0~03~0~009
Mammalian (rabbit)? Thickness % of total (P)
Cornea
_N52$ 0~01-0~03$ ?0~06§ z12 _N450 5-101 -5% 530 f 40
El0 ro-004 ~0.011 !z2.3 ~85.6 El.5 -1 -
Mammalian (human) Thickness % of total (CL)
-
:
z
z 2
: E
G
0 crl
Table 2. Quantitative comparison of saIamander’s corneal layers with other vertebrate corneas. It is clear that aN layers show variations in thicknesses and 2 percentages throughout vertebrates ln
2
2
2
r
c ?
594
MARGARITIS,
the middle lamellae are approximately 10% thicker than the rest. The mean value of the total collagen fibers was calculated as 267f 16 A after glutaraldehyde-Ruthenium Red-osmium tetroxide (GRRO) fixation and 301 f 29 A after glutaraldehyde-potassium permanganate(GPM) fixation whereas the striations of the collagen fibers show a peridocity of about 700 A after the last fixation method. II. Ultrastructural data In cross-section, the salamander cornea is about 80 p thick and consists of the following layers (Fig. 1) : (a) Epithelium. This cornea has three layers of epithelial cells; flattened basal cells, flattened and slightly polygonal intermediate cells and finally superficial almost cuboidal cells (Figs. 1, 2). Occluding junctions were observed at the outer region of adjacent epithelial cells (Fig. 3) which were otherwise
POLITOF
AND
KOLIOPOULOS
attached to each other through desmosomes. The surface of the epithelium is differentiated into microvilli with attached microfilaments (Fig. 4). The two outermost cell layers show a polarity in the distribution of dense vesicles (Fig. 2) which exhibit a granular content when RR is used during fixation (Fig. 4). The filaments attached to microvilli as well as the granular content of the vesicles are less obvious after GO fixation (Fig. S), almost invisible after G fixation (Fig. 6, although some material within the vesicle has been preserved), and totally invisible following GPM fixation (Fig. 7). All epithelial cells contain many mitochondria, tonofilaments and rough endoplasmic reticulum. No colloidal lanthanum (La) tracer could be detected inside the intercellular space after GLa fixation. (b) Basement membrane (basal lamina). The epithelium is separated from the stroma by a
Fig. 1. Light micrograph of cross-sectioned salamander cornea. Epithelium is shown to consist of three cell layers, i.e. basal flattened cells, intermediate slightly polygonal cells and superficial almost cuboidal cells. The basement membrane is visible (arrow) even at this low power light micrograph. x 600. Fig. 2. Shape differentiation of cells throughout epithelium is very limited, unlike other corneas. Also in this cornea a large number of dense vesicles is preferentially distributed at one hemisphere of the two outermost cell layers. The basement membrane shows moderate relative electron density (asterisk). GRRO fixation. x 6500. Fig. 3. The anterior intercellular region of the superficial cells has been differentiated into occluding junctions which normally are rarely observed. However by tilting the section along an axis parallel to the border between the cells these pentalaminar structures are clearly visible (arrow). Desmosomes form the lower end of the junctional complex. A, 30” tilt; B,.9” tilt; C, D, 2” tilt. G fixation. A, B, C, x 55,000; D, x 130,000. Fig. 4. Extracellular fine fillaments attached to the microvilli of the superficial cells can be detected when Ruthenium Red is included during fixation. Another result of the mentioned method is the preservation of granular dense vesicles (arrow). GRRO fixation. x 38,000. Fig. 5. The extracellular fine filaments demonstrated after GRRO fixation are absent following GO fixation. Also lower electron density show the intracellular vesicles. GO fixation. x 38,000. Fig. 6. A dramatic network of tonofilaments is revealed following G fixation. The extracellular filaments are absent whereas the dense vesicles look empty, although some dense material has been preserved (arrow). G fixation. x 38,000. Fig. 7. Total extraction of the vesicle material, absence of the extracellular fine filaments and the intracellular tonofilaments occurs when GPM is the fixative. GPM fixation. x 38,000.
ULTRASTRUCTURE
OF
AMPHIBIAN
CORNEA
relatively thick basement membrane (Figs. 2, 8) which shows the same relative electron density regardless of the applied fixation method (Figs. 8-11). Small granules were observed in the intracellular clefts of the basal epithelial cells with gradually increasing density towards the basement membrane (Fig. 8). (c) Substantia propria (stroma). The stroma consists of about 40 highly organized collagenous lamellae perpendicularly oriented to one another (Fig. 12). The lamellae are composed of hexagonally arranged collagen fibers which are interconnected by fine filaments most pronounced when RR is included during fixation (Fig. 13). No randomly oriented collagen fibers (Bowman’s layer in some corneas) were detected at the outer region of the stroma facing the basement membrane. Finally, a very thin Descemet’s membrane of granular structure (Fig. 14) separates the stroma from the endothelium being its basement membrane. Discussion I. General Unlike mammalian or even Anuran amphibian cornea (Kaye, 1962) we didn’t observe extended differentiation of the epithelial cells as far as shape is concerned. The use of RR during fixation enabled us to identify as mucopolysaccharide-rich (Luft, 1971b) the filaments on the epithelial surface as well as the material inside the dense vesicles. Penetration of RR through the intact plasma membrane and into the vesicles appears paradoxical. However, a similar unexplained penetration of RR has been reported previously (Luft, 1971b). Although the extracellular filaments are considered to represent material secreted for the lubrication of cornea1 surface (Holy and Lemp, 1971) from the Harderian gland (Tansley, 1965), the fate of the intracellular dense vesicles is obscure. Since the intermediate epithelial cells show such vesicles distributed with the same polarity, and considering that these cells are destined to replace the superficial ones after a certain period of time, we come to the conclusion that the material of the vesicles is secreted in some unknown way and may contribute to the lubrication process. Thus it is clear that
597
the epithelium provides more than a smooth protective refracting surface for the eye (Smelter and Ozanics, 1965). The vesicle content seems to be of proteinaceous nature as well (glycoprotein ?): it is partly preserved after G fixation, which is known to cross-link proteins (Hopwood, 1972), and totally extracted after PM fixation which is considered to cause fragmentation of proteins (Hake, 1965; Lenard and Singer, 1968). The observed polarity in the distribution of the vesicles could be retained by means of a potential in the epithelial cells known to exist in rabbit (Maurice, 1967) as well as in other corneas (Ehlers, 1970). The intercellular permeability of the epithelial cells was found to be very low, as demonstrated by the inability of lanthanum and Ruthenium Red to penetrate. A similar situation has been observed in the rabbit cornea (Leuenberger, 1973). A very low intercellular permeability has been shown to be effectively achieved by the existence of occluding junctions (Farquhar and Palade, 1963) which were observed during this study at the intercellular outer region of the superficial epithelial cells. Although we did not make use of any purely histochemical reactions in order to identify the chemical character of the basement membrane, we come to the following conclusions after consideration of what is currently known about the action of the applied fixatives: this membrane (a) is not entirely proteinaceous, since it does not lack electron density after GPM fixation which is known to destroy proteins (Hake, 1965 ; Lenard and Singer, 1968; Margaritis and Moudrianakis, 1974), and (b) is not rich in unsaturated lipids or disulfide bonds, because it does not show greater electron density following GO fixation (Stoeckenius, 1959; Hake, 1965). Thus it seems that the composition of this membrane fits the general description of the basement membranes (Kefalides, 1973); part of the material may be carbohydrate (fair reaction with PM, Glaeser and Mel, 1964; good reaction with RR, Luft, 1971a), and part protein (good reaction with G and GO, Hopwood, 1972). Suggestive for basement membrane synthesis is also the appearance of the extracellular glycogen-like granules. More
598
MARGARITIS,
evidence, however, has been obtained by amplification of that synthesis in response to ultraviolet light irradiation (Margaritis et al., 1976). No extensive sutural fibers throughout stroma could be detected in this cornea as was reported to exist in the dogfish (Goldman and Benedek, 1967). The relative uniformity in the diameter of the collagen fibers throughout stroma has been observed also in the chick (Hay and Revel, 1969); in the human cornea the variation is major (Jakus, 1961) whereas in the dogfish it is not significant (Goldman and Benedek, 1967). No periodic substructure could be identified in Descemet’s membrane as has been reported for human cornea (Jakus, 1956). II. Evolution This amphibian cornea has a relatively thick basement membrane and a thin Descemet’s membrane. Thus, the thickness of Descemet’s membrane is roughly double that of the basement membrane, whereas in mammalian corneas the ratio is more than SO-fold; moreover, Descemet’s membrane is roughly equal to the endothelium in thickness, whereas in mammalian corneas it is at least twice as thick. The fact that a Descemet’s membrane is either very thin (frog, Kaye, 1962; shark, Goldman and Benedek, 1967) or totally absent (lizard, Walls, 1967; sea lamprey, Pederson et al., 1971; teleost fish, Tripathi, 1974) in some primitive vertebrates, suggests
POLITOF
AND
KOLIOPOULOS
that it was developed late during evolution. It is secreted by an endothelium which during earlier evolutionary stage possibly had the function of controlling cornea1 hydration (Friedman, 1973). A thick basement membrane has also been reported in elasmobranchs (Goldman and Benedek, 1967) and in sea lampreys (Horn et al., 1969), whereas in the frog the thickness is moderate (Kaye, 1962). Thus it seems possible that a thick basement membrane is an exclusive feature either of aquatic and amphibious animals or of primitive vertebrates, although a systematic study is not available as yet. As far as the thickness of the epithelium is concerned it is believed that a thick epithelium is important in resisting the transcorneal flux of water in aquatic animals; the thickness is considerable in fish (trout, carp, pike) and chondrichthies as well (Tripathi, 1974). This feature has also been retained in the cornea of the frog (Kaye, 1962). The vertebrate eye originated in aquatic organisms (Walls, 1967) and amphibia mark the transition from aquatic to terrestrial life in the evolution of the vertebrate phylum. The big change in the eye during evolution came when animals left the water for land; in this new milieu one important problem was to protect the cornea from damage, which now had become the main refracting structure (Tansley, 1965). This might be achieved by a thicker stroma as in mammals.
Fig. 8. A thick basement membrane (cu. 3700 A) marks the border between epithelium and stroma while no Bowman layer is evident unlike other corneas; collagen fibers start to be organized into lamellae at the very beginning of the stroma. Dense granules are very often observed in the intercellular spaces of the basal cells occasionally associated with the basement membrane (arrow). GRRO fixation. x 30,000. Figs. 9-11. No great differences are visible regarding the relative electron density of the basement membrane after various fixation methods (compare also with Fig. 8). Fig. 9; GO fixation; Fig. 10, G fixation; Fig. 11. GPM fixation. x 30,000. Figs. 12-13. Cross section of stromal lamellae. The collagen fibers tend to form hexagonal lattice and are interconnected with fine filaments. Groups of other intermediate thickness fibers are also observed (circle). GRRO fixation. Fig. 12, x 50,000; Fig. 13, x 115,000. Fig. 14. No apparent brane. GRRO fixation.
substructure x 300,000.
is evident in a cross-sectioned
Descemet’s
mem-
-
‘t‘
ULTRASTRUCTURE
OF
AMPHIBIAN
CORNEA
The stroma is relatively thin in this amphibian (-40 lamellae) and in purely aquatic vertebrates (shark, 25 lamellae, Goldman and Benedek, 1967; sea lamprey, 2: 13 lamellae, Horn et al., 1969). As far as Bowman’s layer is concerned, it is unclear whether or not there is any correlation between its existence and cornea1 evolution. Some teleost fish (Tripathi, 1974) as well as chondrichthies (Goldman and Benedek, 1967) and sea lampreys (Horn et al., 1969) do have Bowman’s layer, whereas on the other hand some other vertebrate corneas do not (rat, Jakus, 1954; mouse, Whitear, 1960; frog, Kaye, 1962; marsupials and avian, Walls, 1967; rabbit, Margaritis et al., 1975). The absence of this layer from various vertebrate corneas scattered throughout evolution may be
601
explained by the fact that whenever Bowman’s layer is damaged it regenerates no more as such (Maurice, 1969a). Further systematic studies are required throughout in order to clarify the differences in ultrastructure among corneas throughout vertebrates. Acknowledgements
This work was supported by a grant from the Empirikion foundation of Athens Greece. We thank Dr F. C. Kafatos for his valuable suggestions. Completion of the work was performed in the laboratory of Dr D. Branton (The Biological Laboratories, Harvard University), to whom we are deeply indebted.
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LEUENBERGER,D. M. 1973. Lanthanum hydroxide tracer studies on rat cornea1 endothelium. Expl. Eye Res., 15,85-91. LUFT, J. H. 1971a. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anaf. Rec., 171, 347-368. LUFT, J. H. 1971b. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anar. Rec., 171, 369-415. MARGARITIS,L. H. 1974. Programmed synthesis of specific proteins in cellular differentiation. A contribution to the study of chorion formation in the follicles of Drosophila melanogaster. Ph.D. Thesis, University of Athens, Greece. MARGARITIS, L. H. and MOUDRIANAKIS, E. N. 1974. Studies on glutaraldehyde-potassium permanganate (GPM) fixation for the preservation and ultrastructural identification of nucleic acids. Abstract. In First Balkan Congress on Electron Microscopy, Sarajevo, 22-25 May, pp. 99-100. MARGARITIS,L. H., POLITOF, Th. K. and KOLIOPOULOS,J. X. 1975. Quantitative changes and ultrastructural alterations of the cornea after exposure to ultraviolet light. 1. Effects on rabbits. Minerva Medica Grecu, 3 (3), 295-305.
MARGARITIS,L. H., POLITOF, Th. K. and KOL~OPOULOS,J. X. 1976. Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light. II. Effects on Amphibia: elucidation of desmosomal structure and basement membrane synthesis. Tissue & Cell, 8, 603-614. MAURICE, D. M. 1967. Epithehal potential of the cornea. Expl. Eye Res., 6, 138-140. MAURICE, D. M. 1969a. The cornea and sclera. In The Eye, Vol. I. Vegetative Psyciology and Biochemistry (ed. H. Davson), pp. 489-600. Academic Press, New York and London. MAURICE, D. M. 1969b. The transparency of the cornea1 stroma. Vision Res., 10, 107-108. PEDERSON,H. J., HORN, VAN, D. L. and EDELHAUSER,H. F. 1971. Ultrastructural changes associated with loss of transparency in the primary spectacle and cornea of the spawning sea lamprey. Expl. Eye Res., 12.1477150. REVEL, J. P. and KARNOVSKY, M. 1967. Hexagonal array of subunits in intracellular junctions of the mouse heart and liver. J. Cell Biol., 33, C7-Cl2. SMELSER,G. K. and OZANICS, V. 1965. New concepts in anatomy and histology of the cornea. In The Cornen, World Congress (ed. J. H. King and J. W. McTigue), pp. I-20, 163-356, Butterworths, Washington. SMITH, J. N. 1969. The transparency of the cornea1 stroma. Vision Res., 9, 393-397. STOECKENIUS,W. 1959. An electron microscope study of myelin figures. J. biophys. hiochem. Cy/o/., 5, 491-500.
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study of the cornea
in mice with special reference
to the inner-