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Fine Structure of the Human Cornea: Epithelium and Stroma*
FINE STRUCTURE OF T H E H U M A N CORNEA: EPITHELIUM A N D STROMA* C. C. T E N G , M.D. New York This communication will undertake a description of the fine structure of the corneal epithelial and stromal cells with particular emphasis on the pattern of the cellular structures. This is important both in identifying any particular type of cell in normal tissue and in describing pathologic changes as will be done in future reports. Before embarking on a study of corneal pathology using the electron microscope, a preliminary study of the fine structure of the normal animal and human cornea is indispensable. One has to experience for himself the delicacy of the techniques and the completely unfamiliar picture presented under the much greater magnification. This realization cannot be obtained merely from a perusal of the literature. These studies have been greatly facilitated by the use of Watson's lead and uranyl stains, which definitely improve the contrast and visibility of the cellular structures and which are only recently beginning to receive their deserved recognition in the field of electron microscopy of the eye. The structure of corneal collagen has drawn a great deal of attention from investigators, while, until recently, the cytology of the cornea has attracted much less attention. Notable work has been done by Sebruyns ( 1 9 5 0 ) , Van den Hooff ( 1 9 5 2 ) , Schwarz ( 1 9 5 3 ) , François, Rabaey and Vandermeersche (1954 and 1960), Rouiller, Danon and Ryter (1954, 1956 and 1961), Sheldon ( 1 9 5 6 ) , Ishida ( 1 9 5 8 ) , Kayes and Holmberg ( 1 9 6 0 ) , Iguchi ( 1 9 6 0 ) , Kobayash and Sato (1960) and Nakaizumi (1960 and 1961).
to our knowledge of the fine structure of the cornea. M A T E R I A L S
M E T H O D S
Three human eyes without any history of corneal disease were used in this study. All were removed because of a malignancy in the orbit or maxillary sinus. W e wish to express our appreciation to Drs. Edgar Frazell and Frank Gerrold of the MemorialSloan-Kettering Cancer Center -ιοί* ' permitting us to examine these eyes: Because of their co-operation we were able to get the tissue into fixative within three minutes after severance from the adjacent tissue, a freshness of tissue which is essential to electron microscope procedures, since even the slightest post-mortem change can make a great difference. The cornea was fixed in one-percent osmium tetroxide in veronal acetate buffer with a p H of 7.4 to 7.6 with sucrose. The tissue was cut into 1.0 by 1.0 mm. pieces, fixed for two hours, then dehydrated through a series of ethyl alcohol concentrations, after which it was embedded in nbutyl methacrylate. The embedded tissue was sectioned with a Porter-Blum microtome. Watson's lead stain and uranyl stains were used routinely. Uranyl acetate was found to be especially good for showing collagen fibers. Additional material for examination was obtained from normal portions of many pathologic corneas removed at keratoplasty in the Manhattan Eye, Ear and Throat Hospital. O B S E R V A T I O N S
Jakus, especially, has contributed greatly
O F C O R N E A (figs. 1-17) The fine structure of the corneal epithelium of the human is much like that of the rabbit. There are three main types of cells
E P I T H E L I U M
"•"From t h e laboratory o f T h e E y e - B a n k f o r S i g h t Restoration, Inc., M a n h a t t a n E y e , E a r and T h r o a t H o s p i t a l T h i s w o r k w a s supported b y grant B-2623 f r o m the P u b l i c H e a l t h Service.
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F i g s . 1, 2, 3 and 4 ( T e n g ) . Oblique of flat sections o f squamous and w i n g cell layers of the epithelium, s h o w i n g different degrees of disintegration of the squamous cells. ( 1 ) S h o w s quite advanced disintegration of the squamous cells. N o t e the high degree of tonofibril formation in the w i n g cells ( X 8,000). ( 2 ) A l s o s h o w s advanced disintegration of squamous cells on the surface (χό,ΟΟΟ). ( 3 ) N o t e less tonofibril formation in the w i n g cells, disintegration of squamous cells (χό,ΟΟΟ) ; ( S , superficial side of epit h e l i u m ) . ( 4 ) F l a t section, s h o w i n g the general pattern of ordinary ( A ) and secretory ( B ) epithelial cells in the basal layer of corneal epithelium (χό,ΟΟΟ)
FINE STRUCTURE OF H U M A N
which occur in layers—the superficial, squamous cells, the middle layer of wingshaped cells and the basal cells, which are columnar in shape. The same process of gradually pushing forward, changing shape, disintegration and shedding is seen in the human cornea just as it was described in the rabbit cornea. Because of this process of disintegration and shedding, the superficial epithelial cells present different appearances, according to the stage of the process caught in a particular specimen. The surface of the epithelium may be composed of wing-shaped cells, showing that the more superficial cells have only recently been shed. Or it may be composed of fine cell fragments, showing that the cells have recently disintegrated and are about to be shed (figs. 1, 2, and 3 ) . Epithelial cells seem to have a greater number of intracellular tonofibrils as they are pushed anteriorly from the basal layer (fig. 5 ) , and the amount of tonofibrils varies with different specimens. Occasionally we have observed this greater number of tonofibrils occurring earlier in the basal layer of cells. This may be a pathologic condition, but the control mechanism of tonofibril formation is not known and is not within the scope of this report. The organelles of human corneal epithelium are much the same as we described earlier in rabbit corneal epithelium, so only a brief description will be given here. The nucleus of the epithelial cell in the basal layer is round or oval in shape (figs. 4 and 17), with the nucleoplasm evenly distributed and enclosed in a double membrane. It often contains one or two nucleoli but not so often as was found in rabbit corneal epithelium. The nucleus changes in form and disintegrates before the cell is shed at the surface of the cornea. The mitochondria of the corneal epithelial cells take the form of irregular, slender filaments (figs. 5, 10 and 17). They are most thickly distributed at the posterior portion of the basal cells and around the nu-
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971
cleus. The cristae of the mitochondria are irregular, laminated, tubular or microvilli in form. The structures constituting the endoplasmic reticulum are comparatively few in the ordinary epithelial cell even at the basal layer (figs. 5, 10 and 15). They are composed of vesicles and tubules and have rough surfaces due to the attachment of Palade particles (microsomes). The Golgi complex (figs. 6 and 10) is usually very well developed in the basal cells of the corneal epithelium and is located at the apex of the nucleus. It is composed of an array or group of six elongated, smoothsurfaced profiles surrounded by circular or oval profiles. The Golgi complex is closely associated with the endoplasmic reticulum and a direct continuation can occasionally be traced (fig. 6 ) . The desmosomes of the human cornea are exactly the same as in the rabbit cornea, but they are much more numerous in the human (figs. 5, 7, 8, 9 and 12). It has been demonstrated that there are often a few fibers crossing the cell walls at the site of the desmosomes. Morphologically these fibers are similar in character to tonofibrils. The more tonofibrils there are in the cytoplasm, the more of these fibers can be found crossing the cell walls and often a connection between the two can be traced. Usually desmosomes disintegrate gradually as the cells are displaced from the middle layers to the superficial layers before being cast off (fig. 9 ) . The tonofibrils inside the cells disintegrate soon after this stage. The secretory, or dark, cells of the epithelium (figs. 10-14) are very interesting to observe. These cells are usually few in a normal human cornea, although, sometimes, especially in pathologic conditions, the number increases for reasons we do not yet fully understand. The secretory cell differs from the ordinary epithelial cells in that it is composed of a greater number of endoplasmic reticu-
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F i g . S ( T e n g ) . A l a r g e portion o f a single corneal epithelial cell f r o m the w i n g cell layer, s h o w i n g moderate increase in tonofibrils. ( D , d e s m o s o m e ; G, Golgi c o m p l e x ; M , m i t o c h o n d r i a ; N , n u c l e u s ; T , tonofibrils; E R , endoplasmic reticulum; X 3 3 , 0 0 0 . )
F i g s . 6, 7 and 8 ( T e n g ) . ( 6 ) Golgi c o m p l e x in epithelial cell of basal layer situated near the a p e x o f the cell close to the nucleus. S i x groups o f elongated smooth-surfaced profiles intermingled w i t h oval profiles a s vacuoles a r e s h o w n . I n places the close association w i t h the endoplasmic reticulum is evident. (G, G o l g i c o m p l e x ; E R , endoplasmic r e t i c u l u m ; X 2 4 . 0 0 0 . ) ( 7 and 8 ) D e s m o s o m e . T h e r e are definitely a Eew fibers of tonofibrils s h o w n p a s s i n g f r o m o n e cell t o another. B e s i d e s the d e s m o s o m e s , there are single übers, n o t i n bundle f o r m , p a s s i n g f r o m o n e cell t o another. ( D , d e s m o s o m e ; fig. 7, χ 3 9 , 0 0 0 ; fig. 8, X33.000.)
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F i g . 9 ( T e n g ) . A transitional stage between w i n g cell and squamous cell. Disintegration is evidenced by the presence of fragmented desmosomes in the intercellular spaces. T h i s causes separation o f the cell o n either side. T h e tonofibrils inside the w i n g cell are a s y e t unchanged. N o t e the pattern of the tonofibrils, their close association w i t h the organelles. ( D , d e s m o s o m e s ; dd, disintegrated d e s m o s o m e ; N , n u c l e u s ; T , tonofibrils; χ 3 3 , 0 0 0 . )
FINE STRUCTURE OF HUMAN
lum and tonofibril elements, so that it looks denser and darker. This increase in the amount of endoplasmic reticulum, Golgi complex elements, and so forth may be an indication that this cell is active in a secretory function. Its nucleus is also denser than that of an ordinary epithelial cell, and the cell shape is very different. Often the secretory cells send out irregular processes, extending between the cell walls of the other epithelial cells, and there are transient secretory capillaries continuous with these cell processes, as demonstrated in a previous communication. There are normal desmosomes connecting the ordinary epithelial cells with the secretory epithelial cells. Mitosis of the epithelial cells is often seen in the basal epithelial cell layer (figs. 15 and 16). The basement membrane of the corneal epithelium (figs. 18-23) in the human cornea is much like that of the rabbit. It consists of two major layers, a lipid layer and a layer of reticular fiber meshwork. The lipid layer is situated between the cell wall of the basal epithelial cells and the reticular fiber meshwork. Sometimes the lipid layer appears as two fine, dense lines. One is actually the epithelial cell wall and the posterior one is the lipid layer of the basement membrane. More often these two lines appear as one wider, dense line. The lipid layer is more osmophilic than the reticular fiber layer. The reticular fiber layer can be subdivided into three layers (figs. 22 and 2 3 ) . First there is a very thin, superficial layer formed of a compact network of reticular fibers supporting the lipid layer, which occasionally is very granular looking. The middle layer, usually thicker than the first layer, is composed of a loose meshwork of reticular fibers varying in texture, probably according to the amount of fluid collected in this layer at the time. Often there is vacuole formation. The posterior layer of reticular fibers is somewhat more compact than the middle layer, but less compact than
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the superficial layer. Normally the middle layer is the thickest of the three layers. In cases where there is an abnormal increase of tonofibrils in the basal epithelial cells these tonofibrils look very much like the reticular fibers and some even cross over the cell walls to mingle with the two layers of reticular fiber meshwork which make up the basement membrane. These tonofibrils which cross over and mix with the reticular fiber meshwork layers are often found in bundles which resemble desmosomes (fig. 2 2 ) . Bowman's layer of the stroma (fig. 21) has been well demonstrated by Jakus and our findings confirm hers. A s she described it, Bowman's layer consists of collagen fibers much like those in the deeper stroma, but arranged more at random. This layer is not, however, sharply demarcated from the superficial stroma, in which the collagen occurs in layers of parallel fibers. In the stroma the layers are arranged so that in any field of a section cut from a suitably oriented block, the fibrils in one lamella form essentially right angles with those in adjacent layers. W e also support Jakus's suggestion that Bowman's membrane should be called Bowman's layer of the stroma. The collagen fibrils of both Bowman's membrane and the stroma proper are composed of young collagen fibers with a width of 200-300 Â and a periodicity of 640 A. Stroma. Concerning the fibrous structure of the stroma (fig. 31) we will not say much, as it would only be a duplication of Jakus's and others' reports. W e would like, however, to dwell briefly on the morphology of the keratocyte and the wandering cell or monocyte. Fixed stromal cells or keratocytes (figs. 24-30) are flatter than most cells. In cross section they appear spindle-shaped. In flat sections the cytoplasm is seen to be full of fine structure elements. The shape of the cells may be very irregular due to the irregular cytoplasmic processes. On the other hand, occasionally they are quite oval or (Text
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F i g . 10 ( T e n g ) . P o r t i o n o f a dark o r secretory cell f r o m the basal layer. I n the right upper corner is a small portion of ordinary epithelial cell ( A ) f o r contrast. I n the secretory cell note the intracellular structures w h i c h are greater in number and m u c h m o r e compact than in the ordinary epithelial cell. ( E R endoplasmic r e t i c u l u m ; G, Golgi c o m p l e x ; M , m i t o c h o n d r i a ; T , tonofibrils; X 3 3 , 0 0 0 . )
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F i g . 11 ( T e n g ) . I n the left upper portion is part of a n ordinary epithelial cell, w h i l e the right l o w e r part of the picture s h o w s a portion of a secretory cell in the w i n g cell layer. N o t e the compact arrangem e n t of tonofibrils and endoplasmic reticulum in the dark cell. ( E R , endoplasmic r e t i c u l u m ; T , tonofibrils; N , nucleus; X33.000.)
F i g s . 12, 13 and 14 ( T e n g ) . ( 1 2 ) T h i s portion of a secretory cell, similar to F i g u r e s 10 and 11, s h o w s that the desmosomes and nuclei are similar in shape but denser in constituents than those structures in an ordinary epithelial cell. ( D , d e s m o s o m e ; N , n u c l e u s ; T , tonofibril; X 3 3 , 0 0 0 . ) ( 1 3 ) Disintegration of a secretory cell in the squamous cell layer is practically the same a s in the ordinary epithelial cell. N o t e disintegration of the cell wall, the organelles and the nucleus. ( S , superficial s i d e ; N , n u c l e u s ; X 16,000.) ( 1 4 ) T h i s cell has m o r e tonofibrils than that in F i g u r e 13, but both cells h a v e a great many free osmophilic particles in the debris. ( S , superficial s i d e ; N , n u c l e u s ; T , tonofibril; X 16,000.)
F i g s . 15, 16 and 17 ( T e n g ) . ( 1 5 and 1 6 ) P o r t i o n o f mitotic figure o f an epithelial cell in prophase s h o w s contraction and condensation of chromatin ( c h r o m a t i d e s ) . N o t e skeinlike chromât ides and double parallel lines o f chromatides ( A and B ) . ( C M , cell m e m b r a n e ; E R , endoplasmic reticulum; N , n u c l e u s ; N M , nucleus m e m b r a n e ; fig. 15, X 16,000; fig. 16, χ 6 6 , 0 0 0 . ) ( 1 7 ) T h i s epithelial cell w i t h apparently three nuclei m a y be a n accidental finding o r it m a y be a lobulated nucleus. T h i s picture is a g o o d illustration o f the normal structures o f a nucleus in a cell f r o m the basal layer. A l s o , the granular appearance o f normal cytoplasm, normal mitochondria and endoplasmic reticulum. ( E R , endoplasmic reticulum; M , mitochondria; N , n u c l e u s ; X 16,000.)
F i g . 18 ( T e n g ) . General v i e w of the basement membrane o f corneal epithelium, s h o w i n g a double dense layer and a faint reticular layer behind. I n the dense double layer ( i n s e t s A and B ) the anterior line is the cell membrane of the epithelium and the posterior line is the lipid layer of the basement m e m brane. T h e s e t w o usually appear a s one dense line. ( B M , basement membrane ; a, cell membrane ; b, lipid l a y e r ; c. reticular l a y e r ; B o m , B o w m a n ' s m e m b r a n e ; E , epithelium; M c , m o n o c y t e ; χ 2 4 , 0 0 0 ; insets, X48.000.)
F i g s . 19, 2 0 and 21 ( T e n g ) . ( 1 9 and 2 0 ) T w o m o r e v i e w s o f the basement membrane. I n these t w o pictures the cell membrane and the lipid layer o f the basement membrane appear a s one dense line. Behind this line is the thicker reticular layer, and irregular spaces o r vacuoles between are probably due t o the collection of fluid. T h e s e vacuoles a r e transitory. T h e monocyte is not a c o m m o n finding. ( B o m , B o w m a n ' s m e m b r a n e ; E , epithelium; L , lipid l a y e r ; M o , m o n o c y t e ; N F , nerve fibers; R, reticular; fig. 19, X 1 5 , 0 0 0 ; fig. 20, X 16,000.) ( 2 1 ) A portion o f B o w m a n ' s membrane. N o t e the random arrangement o f "young" collagen fibers, t h e s a m e type o f collagen fiber a s i n the stroma. ( 6 4 0 ± A ; 2 5 0 ± A w i d t h ; XSO.000.)
F i g s . 2 2 and 23 ( T e n g ) . ( 2 2 ) I n this c a s e there is a great increase of tonofibrils in the basal epithelial cells. T h e r e are comparatively less osmophilic or lipid granules in the lipid layer o f the basement membrane. I t is a good illustration o f : ( 1 ) similarity o f tonofibrils and reticular fibers o f basement m e m b r a n e ; ( 2 ) the division of reticular fiber m e s h w o r k into three layers, the thin compact anterior layer, the loose middle layer w i t h transient collection of fluid, and the posterior layer w h i c h is less c o m pact than the middle layer, as illustrated in F i g u r e 23. ( B o m , B o w m a n ' s m e m b r a n e ; B M , basement m e m b r a n e ; C, collagen fiber; L , lipid l a y e r ; M , m i t o c h o n d r i a ; R, reticular l a y e r ; T , tonofibrils; χ 3 3 , 0 0 0 . ) ( 2 3 ) D i a g r a m o f basement membrane.
Fig. 2 4 ( T e n g ) . A portion of a normal keratocyte. Inset represents a general v i e w o f this keratocyte o r stromal cell. ( C , c o l l a g e n fiber; C M , cell m e m b r a n e ; E R , endoplasmic r e t i c u l u m ; M , mitochondria; N , n u c l e u s ; T , tonofibrils; X 3 3 . 0 0 0 ; inset, X 12,000.)
F i g s . 25 and 26 ( T e n g ) . ( 2 5 ) A portion of keratocyte near t h e Golgi area. ( C , c e n t r i o l e ; plasmic reticulum; G, Golgi c o m p l e x ; M , m i t o c h o n d r i a ; N , n u c l e u s ; X 3 0 , 0 0 0 . ) ( 2 6 ) T h i s near the cell wall o f a keratocyte s h o w s the possibility of collagen fibers i n direct contact o r w i t h a cell wall. ( C , collagen fibers; C M , ectoplasm o r cell m e m b r a n e ; E R , endoplasmic X33,000.)
E R , endoflat section connection reticulum;
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F i g . 27 ( T e n g ) . Interlocking of t w o keratocyte cell-processes. ( N , n u c l e u s ; M , m i t o c h o n d r i a ; T , tonofibrils; C, collagen fibers; X 2 4 , 0 0 0 . )
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F i g s . 28 and 2 9 ( T e n g ) . ( 2 8 ) S y n c y t i u m of keratocyte processes. ( N , n u c l e u s ; χ 1 2 , 0 0 0 ) . ( 2 9 ) jugation between a keratocyte and a monocyte. ( K , k e r a t o c y t e ; M , m o n o c y t e ; χ 12,000.)
Con-
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F i g . 3 0 ( T e n g ) . Conjugation b e t w e e n a keratocyte and a m o n o c y t e H i g h e r p o w e r v i e w of marked area in F i g u r e 29.
(X30.000).
C.
C.
T E N G
F i g . 31 ( T e n g ) . P a t t e r n o f strata arrangement of stromal collagen
fibers
(X9,000).
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STRUCTURE OF
star-shaped with irregular processes—it depends on the level of the section. The shape of the nucleus is also affected in the same way. As to the nucleus, the central portion of the nucleoplasm is more or less uniform in density, but at the periphery there is a margin with some irregularity in the density. Usually the nucleus has a double nuclear membrane. In flat sections it presents a mosaic appearance with fine multiple perforations which in some sections show communication with the endoplasmic reticular system. The endoplasmic reticulum is rather well developed and evenly distributed. The double membrane of the endoplasmic reticulum system may be composed of tubular processes, so that in cross section they form a pattern more or less like round or oval profiles. These membranes are not very clearly presented, but the attached Palade particles are very osmophilic, large and discrete. In cross section of the tubules these particles may assume a rosette form and usually there are a few free Palade particles in the cytoplasm (fig. 24). The Golgi complex
of the keratocyte is
well developed. It is composed of fine vesicles and fine double membranes with smooth surfaces, and the close association with the endoplasmic reticulum is quite evident. The centriole is localized more or less at the center (fig. 25). The mitochondria are scattered throughout the cytoplasm in the form of very irregular long rod-shaped structures. The cristae also present irregular profiles. Normally the profile of the mitochondria is one of the important features in distinguishing a stromal cell from a monocyte. The pattern of the nucleus is another differentiating characteristic. The cytoplasm is usually granular, but often there are fibrillar structures, very similar to tonofibrils in appearance and size, and sometimes they are even attached to the cell membrane (fig. 24). One interesting point is that stromal col-
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lagen fibers normally seem to remain attached to the cell membrane. Figure 26 presents a more detailed picture; the area corresponding to the cell membrane may be precollagen fibers or ectoplasm attached to the cell wall. In some places these fibers appear to be directly connected with, or continuations of the endoplasmic reticulum. The other end of these precollagen fibers connects with collagen fibers outside the cell wall. This picture suggests that the keratocyte is responsible for the origin and production of collagen fibers and maintaining them in attachment. However, further investigation is necessary along these lines before a more definite conclusion can be reached. There are three characteristic pictures of keratocytes when they are in close proximity to each other: they may just be in contact without any reaction, they may meet each other with interlocked extended processes, without any demonstrable connection, or they may join in an apparent syncytium. Whether this syncytium is due to a meeting and fusing or whether, as is more generally believed, there was an incomplete separation after cell division, we cannot tell (figs. 27 and 28). On one occasion we noticed another phenomenon—a monocyte in contact with a keratocyte (figs. 29 and 30) had apparently indented its cell body. Even the nucleus of the keratocye within the cell wall seemed to be indented. No direct communication could be made out. It is possible, of course, that the indentation of the nucleus had been there before contact with the keratocyte. It will be interesting to see if we find more examples of this. NERVE
FIBERS
S T R O M A
I N
T H E
E P I T H E L I U M
A N D
(figs. 32-37)
The nerve supply of the cornea has been carefully and extensively studied in the past and recently the use of the new technique of silver stain was reported in this connection by Scharenberg, Wolter and Lassman. However, the fine structure of these nerves
F i g s . 32 and 33-a, b and c ( T e n g ) . F i g u r e s 3 2 through 37 s h o w different types of nerve
fibers.
( 3 2 ) T h e single fiber type of nerve fiber perforating the basement membrane w i t h a thick end-bulb which consists of larger mitochondria, vesicles and filaments. ( N F , nerve fiber; B M , basement m e m brane; E B , end-bulb; E , epithelium; X 10,000.) (33-a, b and c ) Represent three different sections f r o m serial sections of a thicker single type nerve fiber with a larger end-bulb and an accumulation o f multiple mitochondria and vesicles. T h e filaments o f the nerve fiber are very clear ( B M , basement m e m b r a n e ; V , vacuoles—possibly due to perforation of the nerve fiber, E B , end-bulb; M , mitochondria; N F , nerve fiber; X 10,000.)
F i g . 34-a and b ( T e n g ) . A second type of nerve fiber, consisting of multiple fibers, each single fiber b e i n g similar t o that in F i g u r e 33-a. T h e r e is a side s w e l l i n g (side-bulb) w i t h accumulated mitochondria and vesicles, as in the end-bulb in F i g u r e s 33-a and b. ( E , epithelium, S B , side-bulb; M , mitochondria; [a], X24.000; [b], X12.000.)
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F i g s . 35 and 36 ( T e n g ) . A third type o f nerve fiber. ( 3 5 ) G i v i n g a general v i e w (χό,ΟΟΟ). S h o w i n g a portion of F i g u r e 35 w i t h w h a t seems to be an end-bulb ( χ 2 4 , 0 0 0 ) .
(36)
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F i g . 3 7 ( T e n g ) . A portion of the s a m e type o f nerve fiber a s in F i g u r e s 35 and 36, w i t h Schwann's cell enveloped. T h e profile of this kind o f fiber is m u c h w i d e r and m o r e irregular in caliber than the second type. I t m a y run in a v e r y t w i s t e d course. T h e contents o f these nerve fibers are mitochondria and irregular vesicles w h i c h a r e v e r y similar t o t h o s e in the other n e r v e fibers, e x c e p t that the m i t o chondria are m o r e irregular. ( M , mitochondria; N F , nerve fiber; S, S c h w a n n ' s c e l l ; S T , stroma o r collagen fibers; V , v e s i c l e s ; X 2 4 , 0 0 0 . )
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has not y e t b e e n described. W i t h the help of o s m i c fixative the n e r v e fibers and their fine structures can be brought out clearly u n d e r the electron microscope, but at p r e s e n t w e are limiting ourselves to s o m e preliminary observations. T h e corneal n e r v e s appear u n d e r light m i c r o s c o p y as branches of the ciliary n e r v e s , e n t e r i n g the cornea f r o m the m i d d l e layers of the limbus. T h e y lose their m y e l i n sheath s o o n after e n t e r i n g the s t r o m a and divide into branches as they proceed centrally and anteriorly t o w a r d Bowman's membrane through w h i c h t h e y penetrate. T h e n e r v e fibers f o r m a p l e x u s beneath the epithelium and send u p branches b e t w e e n the epithelial cells, e n d i n g in round or p e a r - s h a p e d bulbs. S w e l l i n g s a l o n g the side of the thicker n e r v e fibers h a v e also been reported. I n our preliminary o b s e r v a t i o n s w e s a w three t y p e s o f n e r v e fibers. T h e first is c o m p o s e d o f a single fiber w i t h a pear-shaped terminal bulb. A n o t h e r t y p e consists of a bundle o f n e r v e fibers w i t h s w e l l i n g s a l o n g the sides that have a structure c o r r e s p o n d ing to that of a terminal bulb. B o t h o f these t w o types of n e r v e fibers are f o u n d b e t w e e n the epithelial cells and in the stroma. A third t y p e o f n e r v e fiber and e n d i n g , f o u n d o n l y in the stroma, consists o f m u c h thicker irregular fibers o f t e n s u r r o u n d e d b y S c h w a n n ' s cells. T h e first t y p e of single n e r v e fiber (figs. 3 2 and 3 3 ) is c o m p o s e d of a double m e m brane, the outer m e m b r a n e actually b e i n g the cell wall of the epithelium. I n s i d e these nerve fibers there are the fine filaments of neurofibrils, mitochondria, and e n d o p l a s m i c reticulum w i t h fine vesicles. T h e r e are o n l y s m o o t h s u r f a c e s o n the double m e m b r a n e of e n d o p l a s m i c reticulum. T h e s e c o n d t y p e of n e r v e fiber (fig. 3 4 ) consists o f a bundle o f n e r v e fibers j u s t like the fibers that appear singly. T h e bundles m a y contain around 1 0 single fibers. T h e s w e l l i n g s n o t e d a l o n g the side o f the first t y p e a l s o occur in this type, a n d they are
c o m p o s e d of type.
e l e m e n t s similar to the
first
T h e picture o f these t w o t y p e s of n e r v e fibers is quite clear and t h e y can b e described w i t h s o m e assurance. A third t y p e of n e r v e fiber, f o u n d o n l y in the stroma, presents a less clear picture. A c c o r d i n g to W o l t e r ' s description of h i s results u s i n g the silver stain technique, these fibers are much thicker and irregular in contour, f o r m i n g e n d i n g s o n l y in the stroma and a l w a y s acc o m p a n i e d b y regular S c h w a n n ' s cells. T h e picture w e f o u n d is o n e of much thicker fibers f o r m i n g a double m e m b r a n e e n c l o s i n g mitochondria, s m o o t h - s u r f a c e d end o p l a s m i c reticulum and fine vesicles as well as other n e r v e fibers. T h e r e w e r e also fibrils o r g r a n u l e s r e p r e s e n t i n g a c r o s s section of axofibrils. T h e m i t o c h o n d r i a h o w e v e r , are not so regular in shape a s in other t y p e s of n e r v e fibers. T h i s t y p e o f fiber is partially e n v e l o p e d b y S c h w a n n ' s cells (fig. 3 7 ) . I n the p r e v i o u s figures (figs. 3 5 and 3 6 ) , the irregular appearance o f the thick n e r v e fibers m a y be e x p l a i n e d b y partial or irregular c o v e r i n g b y S c h w a n n ' s cells and the irregular or t w i s t e d course o f the n e r v e fiber. T h e enlarged, s w o l l e n portion m a y be the end-bulb of this t y p e of n e r v e fiber, as suggested by Wolter. T h e anatomic o r i g i n s and different p h y s i ologic f u n c t i o n s o f these three t y p e s o f nerve fibers will m a k e a n interesting study. The monocyte. W a n d e r i n g cells can s o m e t i m e s be seen b e t w e e n the epithelial cells or in the stroma (figs. 3 8 - 4 1 ) . T h e s e are u s u ally m o n o c y t e s . It is k n o w n that p o l y m o r phonuclear l e u k o c y t e s m a y be o b s e r v e d in inflammatory conditions and other blood cells occur o n l y w h e n there are blood v e s sels i n v a d i n g the cornea. It is e a s y to d i s t i n g u i s h b e t w e e n a m o n o cyte and a keratocyte u n d e r electron m i c r o s copy. T h e n u c l e u s of the m o n o c y t e is oval or h o r s e s h o e - s h a p e d . I t s n u c l e o p l a s m varies in density w i t h the denser n u c l e o p l a s m f o r m i n g a definite but irregular b a n d subjacent
FINE STRUCTURE OF HUMAN
CORNEA
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F i g . 38 ( T e n g ) . A m o n o c y t e in the stroma. Compare w i t h the keratocyte. N o t e the differences of their uiclei and mitochondria. ( C , collagen fiber; G, Golgi c o m p l e x ; K, k e r a t o c y t e ; M , mitochondria; M o , nonocyte; N , nucleus; χ24,000.)
996
C. C. T E N G
F i g . 39 ( T e n g ) . A m o n o c y t e between the epithelial cells. ( Ν , n u c l e u s ; N e , n u c l e o l u s ; M , large m i t o chondria; G, area of Golgi c o m p l e x ; E , epithelium; χ 3 3 , 0 0 0 . )
FINE STRUCTURE OF HUMAN
CORNEA
997
F i g . 4 0 ( T e n g ) . T h e m a j o r portion of a m o n o c y t e between the epithelium and basement membrane w i t h a small portion of B o w m a n ' s membrane. T h i s specimen is f r o m a case of macular degeneration of the cornea. N o t e the early vacuole formation in the endoplasmic reticulum. ( B M , basement m e m b r a n e ; B o m , B o w m a n ' s m e m b r a n e ; E , epithelium; E R , endoplasmic reticulum; M , mitochondria; N , n u c l e u s ; V , v a c u o l e s with s e c r e t i o n ; χ 3 3 , 0 0 0 . )
998
C. C. T E N G
F i g . 41 ( T e n g ) . A m o n o c y t e in the stroma, a portion near a Golgi complex. ( C , collagen fiber; Ce, centriole; D , dense b o d y ; E R , endoplasmic reticulum; G, Golgi c o m p l e x ; M , mitochondria; Ν , n u c l e u s ; X33.000.)
FINE STRUCTURE OF H U M A N
to the nuclear membrane. In the central part of the nucleus this dense band appears to mix haphazardly with nucleoplasm of a lighter density. The mitochondria of these monocytes have a typical white blood cell pattern. They are larger and more regular than the mitochondria of corneal epithelium. The cristae of the mitochondria are also much more regular than that of the stromal cell. The endoplasmic reticulum of monocytes has a double membrane with attached, dense Palade particles. They are not very abundant ordinarily, but they increase with an increase in cellular activity. Golgi complex is very well developed in the monocytes judging from the abundance of smooth double membrane system and vesicles in the Golgi area. There is a wellformed pair of centrioles or diplosomes near the central area of the Golgi complex (fig. 4 1 ) in typical forms as in the other cell types. So in general, the structure of the monocyte is quite different from that of the stromal cell judging by its shape and the profiles of the nucleus and mitochondria. DISCUSSION
This study confirms many of our previous findings, including the picture of the disintegration of squamous cells before they are shed. The migration and eventual desquamation of epithelial cells has been well known for a long time, but was only recently scientifically demonstrated by Hanna and O'Brien using tritium labelled thymidine in rat, mouse, and dog corneal epithelium. The complete course from the basal layer to the superficial layer and the casting off takes from three and one half to seven days, according to these authors. W e also noticed in our study of the human corneal epithelium, as we had in the rabbit, that there is an increase in the number of tonofibrils in the wing cells. The fibers in the desmosomes are very similar to these tonofibrils, and where there is an
CORNEA
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increase in the number of tonofibrils, there are likely to be more visible fibers in the desmosomes, and at the same time, more fibers from the desmosomes can be seen crossing over both cell membranes and joining with the tonofibrils. This demonstrates their close relationship. They are very likely the same type of fibers, as we conjectured after our study of the rabbit cornea. The reticular fibers have been considered a type of collagen fiber with argyrophilic properties. Our observations indicate that morphologically the tonofibrils of the corneal epithelium are very similar to the reticular fibers in the basement membrane of the corneal epithelium. To us it seems that it is more likely that reticular fibers are produced from the epithelial cells than from fibroblasts in the stroma. Thus concerning the basement membrane of the corneal epithelium, this study has enabled us to confirm our previous findings and add some new concepts regarding the structure and origin of the basement membrane of the corneal epithelium. Between the cell wall of the basal epithelial cells and the layer of reticular fibers, there is a lipid layer, a thin but continuous layer of lipid substance, mainly plasmalogen. In the reticular fiber layer there is reticular fiber meshwork filled with mucoor glycoproteins. The superficial layer of the reticular fiber meshwork is the most compact layer and its supports the lipid layer. The middle layer of reticular fibers is the loosest mesh of the three layers. Sometimes there may be a collection of fluid ; otherwise it will look more compact but less compact than the third layer. Since the cell membrane of the corneal epithelium and the reticular fiber meshwork are connected only by a few desmosomes, in addition to the lipid substance between the layers, the cohesive force here may be mostly chemical in nature. It was suggested by Herrman and Hickman that lipoprotein is the main cause of adhesive force. In the connection between the reticular fibers and the
1000
C. C. T E N G
collagen fibers of Bowman's membrane there are many interlocked fibers at the time of contact. A s to the attachment between the reticular fiber layer and Bowman's layer, that is more likely due to interlocked fibers than to a chemical cementing factor. Katzin's experiment on electric impedance and fat solvents may have foreshadowed our current findings that fat solvents, such as butyl alcohol, ether, and so forth can cause separation of the epithelium from the stroma and at the same time lower the electric resistance. The impedance is due evidently to the presence of the lipid layer described. Judging from the evidence derived from studies of the electric impedance, the cell wall may not play an important role in impeding current flow because the intercellular spaces are comparatively large in the corneal epithelium and provide wide passage for electrolytes that carry the current. Only the lipid layer of the basement membrane is a real and continuous barrier to an electric current or chemical agent. Our histochemical studies in 1955 and the more recent work of Offret and Haye (1959) and Forgacs (1960) all seem to confirm this observation. Swan and White's work also emphasizes the importance of this lipid layer of the basement membrane, especially in relation to the selective permeability of the cornea to drugs. It may be that the major requirement for permeation of the cornea by a drug is fat solubility. This would be further evidence of the importance of the continuous lipid layer which forms the anterior layer of the basement membrane. Histologically we have observed the collection of fluid in front of and behind the basement membrane in pathologic specimens which suggests that the basement membrane is also very important in the hydration of the cornea. In histochemical studies of human pathologic material we have seen such changes of the basement membrane as variations in thickness, in density of staining property
and in uniformity. The membrane may be smooth or granular; it may have small breaks or large ones, or it may even be lost for a long interval. From all of this evidence and from the work on basement membrane in other tissues, we have come to believe that the basement membrane plays a very important role in the physiology and pathology of the cornea. However, due to the delicacy of this membrane and the fact that it is hard to demonstrate in most animal corneas, even using P A S stain, we must depend largely on the limited supply of human pathologic material for our studies. Now, by employing both histochemical and electron microscope techniques, the possibilities are very much wider and the prospects for future research are improved. With these techniques even animal corneas are useful because the appearance and changes in the basement membrane can be demonstrated nicely with the advantage of the high resolution offered by the electron microscope. THE
SECRETORY
CELL
In the current study we were able to observe more of the details of the cytoplasmic structure of the secretory cell than we described in a previous paper on the rabbit cornea. The fact that these cells contain more tonofibrils, endoplasmic reticulum and microsome particles may explain their distinguishing darker appearance. It may also explain the fact that they are P A S positive and have a more basophilic character. W e still have not been able to get a good photograph of the secretory function in the normal human cornea, but it was described in the study of the rabbit cornea and will be further described in our report on keratoconus. In a previous study we noticed that these secretory cells form transient secretory capillaries containing at least three types of granules: large osmophilic particles, finer particles and large fragments of cytoplasm somewhat resembling inclusion bodies. Morphologically the large particles are
FINE STRUCTURE OF HUMAN
similar to Palade particles in size and density, which may be the major lipid element of basement membrane. The finer particles may be constituents of muco- or glycoprotein in the meshwork of the reticular fiber layer. With all this information on the structure of the basement membrane, it is easier to understand its origin. As we observed, the reticular fibers of the basement membrane are probably basically the same as the tonofibrils of the epithelial cells and as the fibrils in the desmosomes. The lipid part of the basement membrane is very likely secreted from the dark cells of the epithelium. Thus all these findings corroborate the observations of Redslob (1948), Busacca (1949), and Vidal (1951) who all suggested that the basement membrane is of epithelial origin. They based their opinions on the difference in staining properties of the basement membrane and Bowman's membrane and, most important, they observed that the basement membrane regenerated with the corneal epithelium. S Y N C Y T I U M
O F
K E R A T O C Y T E S
Syncytium, intercellular bridges or protoplasmic anastomosis between keratocytes was suggested by Scharenberg, reporting in his silver carbonate preparation studies. This phenomenon has been noted in spermatocytes and can explain the synchronous differentiation in the process of development. This would also indicate tnat it plays a role in the conduction of action potential from one cell to another, probably by the transmission of metabolic material from cell to cell. This finding also helps to explain
CORNEA
1001
the progression of pathologic changes by spreading pathogenic agents rapidly from cell to cell. This may be of particular importance in the development of lattice degeneration of the cornea to be reported on later. The morphogenesis of this syncytium is also an interesting point for conjecture. Some researchers believe that the attachment is due to incomplete separation after cell division, but this has not yet been definitely demonstrated in the corneal stromal cells. S U M M A R Y
1. Fine structure of the human corneal epithelium and stroma is discussed with emphasis on the different types of cells of the epithelium. The dark or secretory epithelial cell is described in greater detail than in our previous communication. 2. The fine structure of the basement membrane is elaborated again, and the close relationship of the basement membrane to the epithelium rather than the stroma is emphasized. 3. The fine structure of the stromal cell or keratocyte is described and the intercellular relationship illustrated. 4. Three types of nerve fibers found in the epithelium and stroma are briefly reported. 5. The fine structure of the monocyte in the epithelium and stroma is described. 210 East 64th Street (21 ) . ACKNOWLEDGMENT
I a m greatly indebted to N e i l H a r d y , Irene H u g h e s , M a x K l i n g e r , and W i l l i a m R i c e f o r their co-operation and assistance.
R E F E R E N C E S
Busacca, Α . : T h e basal membrane o f the corneal epithelium. Bull. soc. Franc, ophtal., 6 2 : 1 3 3 , 1949. F a w c e t t , D . W . , Ito, S., and Slautterbach : T h e occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation. J. Biophysic. & B i o c h e m . Cytol., 5:453, 1959. Forgacs, J.: Clinical and experimental study o f the basal membrane of the corneal epithelium in keratitis bullousa. Brit. J. Ophth., 4 4 : 3 8 5 , 1960. François, J., Rabaey, M., and V a n d e r m e e r s c h e : L'ultrastructure des tissue oculares a u microscope électronique: I I . E t u d e de la cornée et de la sclérotique. Ophthalmologica, 1 2 7 : 7 4 - 8 5 , 1954. François, J., and Rabaey, M . : T h e anatomy of the cornea. I n Transparency o f the Cornea. ( E d i t e d by S i r S t e w a r t D u k e E l d e r and E . S . P e r k i n s . ) Springfield, 111., T h o m a s , 1960, p. 7.
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Friedenwald, J. S , and Buschke, W . : Mitotic and w o u n d healing activities o f corneal epithelium. Arch. Ophth., 3 2 : 4 1 0 , 1944. H a n n a , C , and O'Brien, J. E . : Cell production and migration in the epithelial layer o f cornea. Arch. O p h t h , 6 4 : 5 3 6 , 1960. H e r r m a n , H , and H i c k m a n , R. H . : T h e adhesion o f epithelium t o stroma in the cornea. Bull. Johns H o p k i n s H o s p , 8 2 : 2 0 8 , 1948. Iguchi, S . : Electron microscope study of corneal tissue. A c t a S o c . O p h t h , Jap. ,64:1294, 1960. Ishida, T . : A study of corneal tissue by electron microscope. A c t a S o c . O p h t h , J a p , 62:1324-1331, 2220-2227, 1958. Jakus, Μ . Α . : Studies o n the cornea: I. T h e fine structure of the rat cornea. A m . J. O p h t h , 3 8 : 4 0 , 1954. : Studies o n the c o r n e a : A c t a X V I I concilium O p h t h , 1954, pp. 461-464. — : Studies on the cornea. J. Biophys. & Biochem. C y t o l , 2 : 2 4 3 (suppl. 1 ) , 1956. : T h e fine structure of the human cornea. I n T h e Structure o f the E y e . ( E d i t e d by S m e l s e r . ) N e w York, A c a d e m i c P r e s s , 1961, p. 343. Katzin, H . M . : A study of the electrical resistance properties o f corneal epithelium. A m . J. O p h t h , 3 4 : 1 1 5 9 , 1951. K a y e s , J , and H o l m b e r g , Α . : T h e fine structure of Bowman's layer and basement membrane of the corneal epithelium. A m . J. O p h t h , 50:1013-1021, 1960. Kobayash, S . : Electron microscopy of corneal epithelium. A c t a S o c . O p h t h , J a p , 6 4 : 1 2 8 6 , 1960. L a s s m a n , G. : D i e Innervation der Hornhaut. A r c h . f. O p h t h , 162:565-609, 1961. L a T e s s a , A . J , T e n g , C. C , and Katzin, Η . M . : T h e histochemistry of the basement membrane of the cornea. A m . J. O p h t h , 3 8 : 1 7 5 , 1954. Lauber, H . : Handbuch der mikroskopischen A n a t o m i e des M e n s c h e n ( M ö l l e n d o r f f ) . 3 ( 2 ) : 4 3 - 5 9 , 1936. Nakaizumi, Y . : Electromicroscopic investigation o f the cornea. I. Corneal stroma. A c t a S o c . O p h t h , J a p , 6 4 : 1 0 6 6 , 1960. — ·: Electromicroscopic investigation of the cornea: I I . B o w m a n ' s membrane. A c t a S o c . O p h t h , J a p , 6 5 : 7 9 , 1961. Off ret, G , and H a y e , C. : Basement membrane o f the corneal epithelium: Histopathologic study. Arch, o p h t a l , 19:126, 1959. Polack, F . M . : Morphology of the cornea. A m . J. O p h t h , 5 1 : 1 0 5 1 , 1961. Redslob (cited b y Busacca, A . ) : T h e basal membrane of the corneal epithelium. Bull. soc. Franc, o p h t a l , 6 2 : 1 3 3 , 1949. Rouiller, C , D a n o n , D , and Ryter, Α . : Application de la microscopic électrique a l'étude de la cornea. A c t a a n a t , 2 0 : 3 9 - 5 2 , 1954. Scharenberg, Κ. : T h e cells and nerves o f the human cornea. A m . J. O p h t h , 4 0 :368, 1955. Schwarz, W . : Electonen Mikroskopishe Untersuchungen ueber die Differenzierung der Cornea und Sklerafibrillen des Menschen. Ztschr. Z e l l f o r s c h , 3 8 : 7 8 - 8 6 , 1953. Sebruyns, M. : Ultrastructure de la cornea et du cristallin etudee au microscope électronique. A n n . o c u l , 183:483, 1950. Sheldon, H . : A n electron microscope study of epithelium in normal mature and immature mouse cornea. J. Biophysic & Biochem. C y t o l , 2 : 2 5 3 , 1956. Sheldon, H , and Zellerquist, H . : A n electron microscopy study o f the cornea in vitamin A deficient mouse. Bull. J o h n s H o p k i n s H o s p , 9 8 : 3 7 2 , 1956. S w a n , K. C , a n d W h i t e , N . G.: Corneal permeability. F a c t o r s affecting penetration of drugs into the cornea. A m . J. O p h t h , 2 5 :1043, 1942. T e n g , C. C . : T h e fine structure of corneal epithelium and basement membrane of the rabbit. A m . J. O p h t h , 5 1 : 2 7 8 , 1961. T e n g , G C , and Katzin, H . M . : T h e basement membrane o f the corneal epithelium. A m . J. O p h t h , 3 6 : 8 9 5 , 1953. Toussaint, D . : Etudes des nerfs corneens par D'verses techniques histogiques. Bull. S o c . beige o p h t a l , 1 2 0 : 5 8 9 , 1959. V a n den Hooff, Α . : Electron microscopy o f cornea and sclera connective tissue. P r o c . K o n . M e d . A k . & W e t . S e r . C , 5 5 : 6 2 8 , 1952. Vidal, F . : Histological division o f the corneal epithelium. Ophth. I b e r o . - A m , 1 3 : 2 0 1 , 1951. W a t s o n , M . L . : Staining o f tissue section f o r electron microscopy w i t h heavy metal: I. J. Biophysic & Biochem. C y t o l , 4 : 4 7 5 , 1 9 5 8 ; I I . J. Biophysic & Biochem. C y t o l , 4:727, 1958. W o l t e r , J. R . : U e b e r den A u f b a u der Nervenbündel in der H o r n h a u t des kaninchen A u g e n . Klin. Monatsbl. f. A u g e n h , 2 2 9 : 2 0 5 6 , 1956. : S i l v e r carbonate technique f o r a demonstration of ocular histology. I n T h e Structure of the E y e ( E d i t e d by S m e l s e r . ) N e w York, Academic P r e s s , 1961, p. 133.
to our knowledge of the fine structure of the cornea. M A T E R I A L S
M E T H O D S
Three human eyes without any history of corneal disease were used in this study. All were removed because of a malignancy in the orbit or maxillary sinus. W e wish to express our appreciation to Drs. Edgar Frazell and Frank Gerrold of the MemorialSloan-Kettering Cancer Center -ιοί* ' permitting us to examine these eyes: Because of their co-operation we were able to get the tissue into fixative within three minutes after severance from the adjacent tissue, a freshness of tissue which is essential to electron microscope procedures, since even the slightest post-mortem change can make a great difference. The cornea was fixed in one-percent osmium tetroxide in veronal acetate buffer with a p H of 7.4 to 7.6 with sucrose. The tissue was cut into 1.0 by 1.0 mm. pieces, fixed for two hours, then dehydrated through a series of ethyl alcohol concentrations, after which it was embedded in nbutyl methacrylate. The embedded tissue was sectioned with a Porter-Blum microtome. Watson's lead stain and uranyl stains were used routinely. Uranyl acetate was found to be especially good for showing collagen fibers. Additional material for examination was obtained from normal portions of many pathologic corneas removed at keratoplasty in the Manhattan Eye, Ear and Throat Hospital. O B S E R V A T I O N S
Jakus, especially, has contributed greatly
O F C O R N E A (figs. 1-17) The fine structure of the corneal epithelium of the human is much like that of the rabbit. There are three main types of cells
E P I T H E L I U M
"•"From t h e laboratory o f T h e E y e - B a n k f o r S i g h t Restoration, Inc., M a n h a t t a n E y e , E a r and T h r o a t H o s p i t a l T h i s w o r k w a s supported b y grant B-2623 f r o m the P u b l i c H e a l t h Service.
A N D
970
C. C. T E N G
F i g s . 1, 2, 3 and 4 ( T e n g ) . Oblique of flat sections o f squamous and w i n g cell layers of the epithelium, s h o w i n g different degrees of disintegration of the squamous cells. ( 1 ) S h o w s quite advanced disintegration of the squamous cells. N o t e the high degree of tonofibril formation in the w i n g cells ( X 8,000). ( 2 ) A l s o s h o w s advanced disintegration of squamous cells on the surface (χό,ΟΟΟ). ( 3 ) N o t e less tonofibril formation in the w i n g cells, disintegration of squamous cells (χό,ΟΟΟ) ; ( S , superficial side of epit h e l i u m ) . ( 4 ) F l a t section, s h o w i n g the general pattern of ordinary ( A ) and secretory ( B ) epithelial cells in the basal layer of corneal epithelium (χό,ΟΟΟ)
FINE STRUCTURE OF H U M A N
which occur in layers—the superficial, squamous cells, the middle layer of wingshaped cells and the basal cells, which are columnar in shape. The same process of gradually pushing forward, changing shape, disintegration and shedding is seen in the human cornea just as it was described in the rabbit cornea. Because of this process of disintegration and shedding, the superficial epithelial cells present different appearances, according to the stage of the process caught in a particular specimen. The surface of the epithelium may be composed of wing-shaped cells, showing that the more superficial cells have only recently been shed. Or it may be composed of fine cell fragments, showing that the cells have recently disintegrated and are about to be shed (figs. 1, 2, and 3 ) . Epithelial cells seem to have a greater number of intracellular tonofibrils as they are pushed anteriorly from the basal layer (fig. 5 ) , and the amount of tonofibrils varies with different specimens. Occasionally we have observed this greater number of tonofibrils occurring earlier in the basal layer of cells. This may be a pathologic condition, but the control mechanism of tonofibril formation is not known and is not within the scope of this report. The organelles of human corneal epithelium are much the same as we described earlier in rabbit corneal epithelium, so only a brief description will be given here. The nucleus of the epithelial cell in the basal layer is round or oval in shape (figs. 4 and 17), with the nucleoplasm evenly distributed and enclosed in a double membrane. It often contains one or two nucleoli but not so often as was found in rabbit corneal epithelium. The nucleus changes in form and disintegrates before the cell is shed at the surface of the cornea. The mitochondria of the corneal epithelial cells take the form of irregular, slender filaments (figs. 5, 10 and 17). They are most thickly distributed at the posterior portion of the basal cells and around the nu-
CORNEA
971
cleus. The cristae of the mitochondria are irregular, laminated, tubular or microvilli in form. The structures constituting the endoplasmic reticulum are comparatively few in the ordinary epithelial cell even at the basal layer (figs. 5, 10 and 15). They are composed of vesicles and tubules and have rough surfaces due to the attachment of Palade particles (microsomes). The Golgi complex (figs. 6 and 10) is usually very well developed in the basal cells of the corneal epithelium and is located at the apex of the nucleus. It is composed of an array or group of six elongated, smoothsurfaced profiles surrounded by circular or oval profiles. The Golgi complex is closely associated with the endoplasmic reticulum and a direct continuation can occasionally be traced (fig. 6 ) . The desmosomes of the human cornea are exactly the same as in the rabbit cornea, but they are much more numerous in the human (figs. 5, 7, 8, 9 and 12). It has been demonstrated that there are often a few fibers crossing the cell walls at the site of the desmosomes. Morphologically these fibers are similar in character to tonofibrils. The more tonofibrils there are in the cytoplasm, the more of these fibers can be found crossing the cell walls and often a connection between the two can be traced. Usually desmosomes disintegrate gradually as the cells are displaced from the middle layers to the superficial layers before being cast off (fig. 9 ) . The tonofibrils inside the cells disintegrate soon after this stage. The secretory, or dark, cells of the epithelium (figs. 10-14) are very interesting to observe. These cells are usually few in a normal human cornea, although, sometimes, especially in pathologic conditions, the number increases for reasons we do not yet fully understand. The secretory cell differs from the ordinary epithelial cells in that it is composed of a greater number of endoplasmic reticu-
972
C. C. T E N G
F i g . S ( T e n g ) . A l a r g e portion o f a single corneal epithelial cell f r o m the w i n g cell layer, s h o w i n g moderate increase in tonofibrils. ( D , d e s m o s o m e ; G, Golgi c o m p l e x ; M , m i t o c h o n d r i a ; N , n u c l e u s ; T , tonofibrils; E R , endoplasmic reticulum; X 3 3 , 0 0 0 . )
F i g s . 6, 7 and 8 ( T e n g ) . ( 6 ) Golgi c o m p l e x in epithelial cell of basal layer situated near the a p e x o f the cell close to the nucleus. S i x groups o f elongated smooth-surfaced profiles intermingled w i t h oval profiles a s vacuoles a r e s h o w n . I n places the close association w i t h the endoplasmic reticulum is evident. (G, G o l g i c o m p l e x ; E R , endoplasmic r e t i c u l u m ; X 2 4 . 0 0 0 . ) ( 7 and 8 ) D e s m o s o m e . T h e r e are definitely a Eew fibers of tonofibrils s h o w n p a s s i n g f r o m o n e cell t o another. B e s i d e s the d e s m o s o m e s , there are single übers, n o t i n bundle f o r m , p a s s i n g f r o m o n e cell t o another. ( D , d e s m o s o m e ; fig. 7, χ 3 9 , 0 0 0 ; fig. 8, X33.000.)
974
C. C. T E N G
F i g . 9 ( T e n g ) . A transitional stage between w i n g cell and squamous cell. Disintegration is evidenced by the presence of fragmented desmosomes in the intercellular spaces. T h i s causes separation o f the cell o n either side. T h e tonofibrils inside the w i n g cell are a s y e t unchanged. N o t e the pattern of the tonofibrils, their close association w i t h the organelles. ( D , d e s m o s o m e s ; dd, disintegrated d e s m o s o m e ; N , n u c l e u s ; T , tonofibrils; χ 3 3 , 0 0 0 . )
FINE STRUCTURE OF HUMAN
lum and tonofibril elements, so that it looks denser and darker. This increase in the amount of endoplasmic reticulum, Golgi complex elements, and so forth may be an indication that this cell is active in a secretory function. Its nucleus is also denser than that of an ordinary epithelial cell, and the cell shape is very different. Often the secretory cells send out irregular processes, extending between the cell walls of the other epithelial cells, and there are transient secretory capillaries continuous with these cell processes, as demonstrated in a previous communication. There are normal desmosomes connecting the ordinary epithelial cells with the secretory epithelial cells. Mitosis of the epithelial cells is often seen in the basal epithelial cell layer (figs. 15 and 16). The basement membrane of the corneal epithelium (figs. 18-23) in the human cornea is much like that of the rabbit. It consists of two major layers, a lipid layer and a layer of reticular fiber meshwork. The lipid layer is situated between the cell wall of the basal epithelial cells and the reticular fiber meshwork. Sometimes the lipid layer appears as two fine, dense lines. One is actually the epithelial cell wall and the posterior one is the lipid layer of the basement membrane. More often these two lines appear as one wider, dense line. The lipid layer is more osmophilic than the reticular fiber layer. The reticular fiber layer can be subdivided into three layers (figs. 22 and 2 3 ) . First there is a very thin, superficial layer formed of a compact network of reticular fibers supporting the lipid layer, which occasionally is very granular looking. The middle layer, usually thicker than the first layer, is composed of a loose meshwork of reticular fibers varying in texture, probably according to the amount of fluid collected in this layer at the time. Often there is vacuole formation. The posterior layer of reticular fibers is somewhat more compact than the middle layer, but less compact than
975
CORNEA
the superficial layer. Normally the middle layer is the thickest of the three layers. In cases where there is an abnormal increase of tonofibrils in the basal epithelial cells these tonofibrils look very much like the reticular fibers and some even cross over the cell walls to mingle with the two layers of reticular fiber meshwork which make up the basement membrane. These tonofibrils which cross over and mix with the reticular fiber meshwork layers are often found in bundles which resemble desmosomes (fig. 2 2 ) . Bowman's layer of the stroma (fig. 21) has been well demonstrated by Jakus and our findings confirm hers. A s she described it, Bowman's layer consists of collagen fibers much like those in the deeper stroma, but arranged more at random. This layer is not, however, sharply demarcated from the superficial stroma, in which the collagen occurs in layers of parallel fibers. In the stroma the layers are arranged so that in any field of a section cut from a suitably oriented block, the fibrils in one lamella form essentially right angles with those in adjacent layers. W e also support Jakus's suggestion that Bowman's membrane should be called Bowman's layer of the stroma. The collagen fibrils of both Bowman's membrane and the stroma proper are composed of young collagen fibers with a width of 200-300 Â and a periodicity of 640 A. Stroma. Concerning the fibrous structure of the stroma (fig. 31) we will not say much, as it would only be a duplication of Jakus's and others' reports. W e would like, however, to dwell briefly on the morphology of the keratocyte and the wandering cell or monocyte. Fixed stromal cells or keratocytes (figs. 24-30) are flatter than most cells. In cross section they appear spindle-shaped. In flat sections the cytoplasm is seen to be full of fine structure elements. The shape of the cells may be very irregular due to the irregular cytoplasmic processes. On the other hand, occasionally they are quite oval or (Text
continues
on page
989)
976
C. C. T E N G
F i g . 10 ( T e n g ) . P o r t i o n o f a dark o r secretory cell f r o m the basal layer. I n the right upper corner is a small portion of ordinary epithelial cell ( A ) f o r contrast. I n the secretory cell note the intracellular structures w h i c h are greater in number and m u c h m o r e compact than in the ordinary epithelial cell. ( E R endoplasmic r e t i c u l u m ; G, Golgi c o m p l e x ; M , m i t o c h o n d r i a ; T , tonofibrils; X 3 3 , 0 0 0 . )
FINE STRUCTURE OF HUMAN
CORNEA
977
F i g . 11 ( T e n g ) . I n the left upper portion is part of a n ordinary epithelial cell, w h i l e the right l o w e r part of the picture s h o w s a portion of a secretory cell in the w i n g cell layer. N o t e the compact arrangem e n t of tonofibrils and endoplasmic reticulum in the dark cell. ( E R , endoplasmic r e t i c u l u m ; T , tonofibrils; N , nucleus; X33.000.)
F i g s . 12, 13 and 14 ( T e n g ) . ( 1 2 ) T h i s portion of a secretory cell, similar to F i g u r e s 10 and 11, s h o w s that the desmosomes and nuclei are similar in shape but denser in constituents than those structures in an ordinary epithelial cell. ( D , d e s m o s o m e ; N , n u c l e u s ; T , tonofibril; X 3 3 , 0 0 0 . ) ( 1 3 ) Disintegration of a secretory cell in the squamous cell layer is practically the same a s in the ordinary epithelial cell. N o t e disintegration of the cell wall, the organelles and the nucleus. ( S , superficial s i d e ; N , n u c l e u s ; X 16,000.) ( 1 4 ) T h i s cell has m o r e tonofibrils than that in F i g u r e 13, but both cells h a v e a great many free osmophilic particles in the debris. ( S , superficial s i d e ; N , n u c l e u s ; T , tonofibril; X 16,000.)
F i g s . 15, 16 and 17 ( T e n g ) . ( 1 5 and 1 6 ) P o r t i o n o f mitotic figure o f an epithelial cell in prophase s h o w s contraction and condensation of chromatin ( c h r o m a t i d e s ) . N o t e skeinlike chromât ides and double parallel lines o f chromatides ( A and B ) . ( C M , cell m e m b r a n e ; E R , endoplasmic reticulum; N , n u c l e u s ; N M , nucleus m e m b r a n e ; fig. 15, X 16,000; fig. 16, χ 6 6 , 0 0 0 . ) ( 1 7 ) T h i s epithelial cell w i t h apparently three nuclei m a y be a n accidental finding o r it m a y be a lobulated nucleus. T h i s picture is a g o o d illustration o f the normal structures o f a nucleus in a cell f r o m the basal layer. A l s o , the granular appearance o f normal cytoplasm, normal mitochondria and endoplasmic reticulum. ( E R , endoplasmic reticulum; M , mitochondria; N , n u c l e u s ; X 16,000.)
F i g . 18 ( T e n g ) . General v i e w of the basement membrane o f corneal epithelium, s h o w i n g a double dense layer and a faint reticular layer behind. I n the dense double layer ( i n s e t s A and B ) the anterior line is the cell membrane of the epithelium and the posterior line is the lipid layer of the basement m e m brane. T h e s e t w o usually appear a s one dense line. ( B M , basement membrane ; a, cell membrane ; b, lipid l a y e r ; c. reticular l a y e r ; B o m , B o w m a n ' s m e m b r a n e ; E , epithelium; M c , m o n o c y t e ; χ 2 4 , 0 0 0 ; insets, X48.000.)
F i g s . 19, 2 0 and 21 ( T e n g ) . ( 1 9 and 2 0 ) T w o m o r e v i e w s o f the basement membrane. I n these t w o pictures the cell membrane and the lipid layer o f the basement membrane appear a s one dense line. Behind this line is the thicker reticular layer, and irregular spaces o r vacuoles between are probably due t o the collection of fluid. T h e s e vacuoles a r e transitory. T h e monocyte is not a c o m m o n finding. ( B o m , B o w m a n ' s m e m b r a n e ; E , epithelium; L , lipid l a y e r ; M o , m o n o c y t e ; N F , nerve fibers; R, reticular; fig. 19, X 1 5 , 0 0 0 ; fig. 20, X 16,000.) ( 2 1 ) A portion o f B o w m a n ' s membrane. N o t e the random arrangement o f "young" collagen fibers, t h e s a m e type o f collagen fiber a s i n the stroma. ( 6 4 0 ± A ; 2 5 0 ± A w i d t h ; XSO.000.)
F i g s . 2 2 and 23 ( T e n g ) . ( 2 2 ) I n this c a s e there is a great increase of tonofibrils in the basal epithelial cells. T h e r e are comparatively less osmophilic or lipid granules in the lipid layer o f the basement membrane. I t is a good illustration o f : ( 1 ) similarity o f tonofibrils and reticular fibers o f basement m e m b r a n e ; ( 2 ) the division of reticular fiber m e s h w o r k into three layers, the thin compact anterior layer, the loose middle layer w i t h transient collection of fluid, and the posterior layer w h i c h is less c o m pact than the middle layer, as illustrated in F i g u r e 23. ( B o m , B o w m a n ' s m e m b r a n e ; B M , basement m e m b r a n e ; C, collagen fiber; L , lipid l a y e r ; M , m i t o c h o n d r i a ; R, reticular l a y e r ; T , tonofibrils; χ 3 3 , 0 0 0 . ) ( 2 3 ) D i a g r a m o f basement membrane.
Fig. 2 4 ( T e n g ) . A portion of a normal keratocyte. Inset represents a general v i e w o f this keratocyte o r stromal cell. ( C , c o l l a g e n fiber; C M , cell m e m b r a n e ; E R , endoplasmic r e t i c u l u m ; M , mitochondria; N , n u c l e u s ; T , tonofibrils; X 3 3 . 0 0 0 ; inset, X 12,000.)
F i g s . 25 and 26 ( T e n g ) . ( 2 5 ) A portion of keratocyte near t h e Golgi area. ( C , c e n t r i o l e ; plasmic reticulum; G, Golgi c o m p l e x ; M , m i t o c h o n d r i a ; N , n u c l e u s ; X 3 0 , 0 0 0 . ) ( 2 6 ) T h i s near the cell wall o f a keratocyte s h o w s the possibility of collagen fibers i n direct contact o r w i t h a cell wall. ( C , collagen fibers; C M , ectoplasm o r cell m e m b r a n e ; E R , endoplasmic X33,000.)
E R , endoflat section connection reticulum;
FINE STRUCTURE OF HUMAN
CORNEA
985
F i g . 27 ( T e n g ) . Interlocking of t w o keratocyte cell-processes. ( N , n u c l e u s ; M , m i t o c h o n d r i a ; T , tonofibrils; C, collagen fibers; X 2 4 , 0 0 0 . )
986
C. C. T E N G
F i g s . 28 and 2 9 ( T e n g ) . ( 2 8 ) S y n c y t i u m of keratocyte processes. ( N , n u c l e u s ; χ 1 2 , 0 0 0 ) . ( 2 9 ) jugation between a keratocyte and a monocyte. ( K , k e r a t o c y t e ; M , m o n o c y t e ; χ 12,000.)
Con-
FINE STRUCTURE OF HUMAN
987
CORNEA
F i g . 3 0 ( T e n g ) . Conjugation b e t w e e n a keratocyte and a m o n o c y t e H i g h e r p o w e r v i e w of marked area in F i g u r e 29.
(X30.000).
C.
C.
T E N G
F i g . 31 ( T e n g ) . P a t t e r n o f strata arrangement of stromal collagen
fibers
(X9,000).
FINE
STRUCTURE OF
star-shaped with irregular processes—it depends on the level of the section. The shape of the nucleus is also affected in the same way. As to the nucleus, the central portion of the nucleoplasm is more or less uniform in density, but at the periphery there is a margin with some irregularity in the density. Usually the nucleus has a double nuclear membrane. In flat sections it presents a mosaic appearance with fine multiple perforations which in some sections show communication with the endoplasmic reticular system. The endoplasmic reticulum is rather well developed and evenly distributed. The double membrane of the endoplasmic reticulum system may be composed of tubular processes, so that in cross section they form a pattern more or less like round or oval profiles. These membranes are not very clearly presented, but the attached Palade particles are very osmophilic, large and discrete. In cross section of the tubules these particles may assume a rosette form and usually there are a few free Palade particles in the cytoplasm (fig. 24). The Golgi complex
of the keratocyte is
well developed. It is composed of fine vesicles and fine double membranes with smooth surfaces, and the close association with the endoplasmic reticulum is quite evident. The centriole is localized more or less at the center (fig. 25). The mitochondria are scattered throughout the cytoplasm in the form of very irregular long rod-shaped structures. The cristae also present irregular profiles. Normally the profile of the mitochondria is one of the important features in distinguishing a stromal cell from a monocyte. The pattern of the nucleus is another differentiating characteristic. The cytoplasm is usually granular, but often there are fibrillar structures, very similar to tonofibrils in appearance and size, and sometimes they are even attached to the cell membrane (fig. 24). One interesting point is that stromal col-
HUMAN
989
CORNEA
lagen fibers normally seem to remain attached to the cell membrane. Figure 26 presents a more detailed picture; the area corresponding to the cell membrane may be precollagen fibers or ectoplasm attached to the cell wall. In some places these fibers appear to be directly connected with, or continuations of the endoplasmic reticulum. The other end of these precollagen fibers connects with collagen fibers outside the cell wall. This picture suggests that the keratocyte is responsible for the origin and production of collagen fibers and maintaining them in attachment. However, further investigation is necessary along these lines before a more definite conclusion can be reached. There are three characteristic pictures of keratocytes when they are in close proximity to each other: they may just be in contact without any reaction, they may meet each other with interlocked extended processes, without any demonstrable connection, or they may join in an apparent syncytium. Whether this syncytium is due to a meeting and fusing or whether, as is more generally believed, there was an incomplete separation after cell division, we cannot tell (figs. 27 and 28). On one occasion we noticed another phenomenon—a monocyte in contact with a keratocyte (figs. 29 and 30) had apparently indented its cell body. Even the nucleus of the keratocye within the cell wall seemed to be indented. No direct communication could be made out. It is possible, of course, that the indentation of the nucleus had been there before contact with the keratocyte. It will be interesting to see if we find more examples of this. NERVE
FIBERS
S T R O M A
I N
T H E
E P I T H E L I U M
A N D
(figs. 32-37)
The nerve supply of the cornea has been carefully and extensively studied in the past and recently the use of the new technique of silver stain was reported in this connection by Scharenberg, Wolter and Lassman. However, the fine structure of these nerves
F i g s . 32 and 33-a, b and c ( T e n g ) . F i g u r e s 3 2 through 37 s h o w different types of nerve
fibers.
( 3 2 ) T h e single fiber type of nerve fiber perforating the basement membrane w i t h a thick end-bulb which consists of larger mitochondria, vesicles and filaments. ( N F , nerve fiber; B M , basement m e m brane; E B , end-bulb; E , epithelium; X 10,000.) (33-a, b and c ) Represent three different sections f r o m serial sections of a thicker single type nerve fiber with a larger end-bulb and an accumulation o f multiple mitochondria and vesicles. T h e filaments o f the nerve fiber are very clear ( B M , basement m e m b r a n e ; V , vacuoles—possibly due to perforation of the nerve fiber, E B , end-bulb; M , mitochondria; N F , nerve fiber; X 10,000.)
F i g . 34-a and b ( T e n g ) . A second type of nerve fiber, consisting of multiple fibers, each single fiber b e i n g similar t o that in F i g u r e 33-a. T h e r e is a side s w e l l i n g (side-bulb) w i t h accumulated mitochondria and vesicles, as in the end-bulb in F i g u r e s 33-a and b. ( E , epithelium, S B , side-bulb; M , mitochondria; [a], X24.000; [b], X12.000.)
992
C. C. T E N G
F i g s . 35 and 36 ( T e n g ) . A third type o f nerve fiber. ( 3 5 ) G i v i n g a general v i e w (χό,ΟΟΟ). S h o w i n g a portion of F i g u r e 35 w i t h w h a t seems to be an end-bulb ( χ 2 4 , 0 0 0 ) .
(36)
FINE STRUCTURE OF HUMAN
CORNEA
993
F i g . 3 7 ( T e n g ) . A portion of the s a m e type o f nerve fiber a s in F i g u r e s 35 and 36, w i t h Schwann's cell enveloped. T h e profile of this kind o f fiber is m u c h w i d e r and m o r e irregular in caliber than the second type. I t m a y run in a v e r y t w i s t e d course. T h e contents o f these nerve fibers are mitochondria and irregular vesicles w h i c h a r e v e r y similar t o t h o s e in the other n e r v e fibers, e x c e p t that the m i t o chondria are m o r e irregular. ( M , mitochondria; N F , nerve fiber; S, S c h w a n n ' s c e l l ; S T , stroma o r collagen fibers; V , v e s i c l e s ; X 2 4 , 0 0 0 . )
994
C. C. T E N G
has not y e t b e e n described. W i t h the help of o s m i c fixative the n e r v e fibers and their fine structures can be brought out clearly u n d e r the electron microscope, but at p r e s e n t w e are limiting ourselves to s o m e preliminary observations. T h e corneal n e r v e s appear u n d e r light m i c r o s c o p y as branches of the ciliary n e r v e s , e n t e r i n g the cornea f r o m the m i d d l e layers of the limbus. T h e y lose their m y e l i n sheath s o o n after e n t e r i n g the s t r o m a and divide into branches as they proceed centrally and anteriorly t o w a r d Bowman's membrane through w h i c h t h e y penetrate. T h e n e r v e fibers f o r m a p l e x u s beneath the epithelium and send u p branches b e t w e e n the epithelial cells, e n d i n g in round or p e a r - s h a p e d bulbs. S w e l l i n g s a l o n g the side of the thicker n e r v e fibers h a v e also been reported. I n our preliminary o b s e r v a t i o n s w e s a w three t y p e s o f n e r v e fibers. T h e first is c o m p o s e d o f a single fiber w i t h a pear-shaped terminal bulb. A n o t h e r t y p e consists of a bundle o f n e r v e fibers w i t h s w e l l i n g s a l o n g the sides that have a structure c o r r e s p o n d ing to that of a terminal bulb. B o t h o f these t w o types of n e r v e fibers are f o u n d b e t w e e n the epithelial cells and in the stroma. A third t y p e o f n e r v e fiber and e n d i n g , f o u n d o n l y in the stroma, consists o f m u c h thicker irregular fibers o f t e n s u r r o u n d e d b y S c h w a n n ' s cells. T h e first t y p e of single n e r v e fiber (figs. 3 2 and 3 3 ) is c o m p o s e d of a double m e m brane, the outer m e m b r a n e actually b e i n g the cell wall of the epithelium. I n s i d e these nerve fibers there are the fine filaments of neurofibrils, mitochondria, and e n d o p l a s m i c reticulum w i t h fine vesicles. T h e r e are o n l y s m o o t h s u r f a c e s o n the double m e m b r a n e of e n d o p l a s m i c reticulum. T h e s e c o n d t y p e of n e r v e fiber (fig. 3 4 ) consists o f a bundle o f n e r v e fibers j u s t like the fibers that appear singly. T h e bundles m a y contain around 1 0 single fibers. T h e s w e l l i n g s n o t e d a l o n g the side o f the first t y p e a l s o occur in this type, a n d they are
c o m p o s e d of type.
e l e m e n t s similar to the
first
T h e picture o f these t w o t y p e s of n e r v e fibers is quite clear and t h e y can b e described w i t h s o m e assurance. A third t y p e of n e r v e fiber, f o u n d o n l y in the stroma, presents a less clear picture. A c c o r d i n g to W o l t e r ' s description of h i s results u s i n g the silver stain technique, these fibers are much thicker and irregular in contour, f o r m i n g e n d i n g s o n l y in the stroma and a l w a y s acc o m p a n i e d b y regular S c h w a n n ' s cells. T h e picture w e f o u n d is o n e of much thicker fibers f o r m i n g a double m e m b r a n e e n c l o s i n g mitochondria, s m o o t h - s u r f a c e d end o p l a s m i c reticulum and fine vesicles as well as other n e r v e fibers. T h e r e w e r e also fibrils o r g r a n u l e s r e p r e s e n t i n g a c r o s s section of axofibrils. T h e m i t o c h o n d r i a h o w e v e r , are not so regular in shape a s in other t y p e s of n e r v e fibers. T h i s t y p e o f fiber is partially e n v e l o p e d b y S c h w a n n ' s cells (fig. 3 7 ) . I n the p r e v i o u s figures (figs. 3 5 and 3 6 ) , the irregular appearance o f the thick n e r v e fibers m a y be e x p l a i n e d b y partial or irregular c o v e r i n g b y S c h w a n n ' s cells and the irregular or t w i s t e d course o f the n e r v e fiber. T h e enlarged, s w o l l e n portion m a y be the end-bulb of this t y p e of n e r v e fiber, as suggested by Wolter. T h e anatomic o r i g i n s and different p h y s i ologic f u n c t i o n s o f these three t y p e s o f nerve fibers will m a k e a n interesting study. The monocyte. W a n d e r i n g cells can s o m e t i m e s be seen b e t w e e n the epithelial cells or in the stroma (figs. 3 8 - 4 1 ) . T h e s e are u s u ally m o n o c y t e s . It is k n o w n that p o l y m o r phonuclear l e u k o c y t e s m a y be o b s e r v e d in inflammatory conditions and other blood cells occur o n l y w h e n there are blood v e s sels i n v a d i n g the cornea. It is e a s y to d i s t i n g u i s h b e t w e e n a m o n o cyte and a keratocyte u n d e r electron m i c r o s copy. T h e n u c l e u s of the m o n o c y t e is oval or h o r s e s h o e - s h a p e d . I t s n u c l e o p l a s m varies in density w i t h the denser n u c l e o p l a s m f o r m i n g a definite but irregular b a n d subjacent
FINE STRUCTURE OF HUMAN
CORNEA
995
F i g . 38 ( T e n g ) . A m o n o c y t e in the stroma. Compare w i t h the keratocyte. N o t e the differences of their uiclei and mitochondria. ( C , collagen fiber; G, Golgi c o m p l e x ; K, k e r a t o c y t e ; M , mitochondria; M o , nonocyte; N , nucleus; χ24,000.)
996
C. C. T E N G
F i g . 39 ( T e n g ) . A m o n o c y t e between the epithelial cells. ( Ν , n u c l e u s ; N e , n u c l e o l u s ; M , large m i t o chondria; G, area of Golgi c o m p l e x ; E , epithelium; χ 3 3 , 0 0 0 . )
FINE STRUCTURE OF HUMAN
CORNEA
997
F i g . 4 0 ( T e n g ) . T h e m a j o r portion of a m o n o c y t e between the epithelium and basement membrane w i t h a small portion of B o w m a n ' s membrane. T h i s specimen is f r o m a case of macular degeneration of the cornea. N o t e the early vacuole formation in the endoplasmic reticulum. ( B M , basement m e m b r a n e ; B o m , B o w m a n ' s m e m b r a n e ; E , epithelium; E R , endoplasmic reticulum; M , mitochondria; N , n u c l e u s ; V , v a c u o l e s with s e c r e t i o n ; χ 3 3 , 0 0 0 . )
998
C. C. T E N G
F i g . 41 ( T e n g ) . A m o n o c y t e in the stroma, a portion near a Golgi complex. ( C , collagen fiber; Ce, centriole; D , dense b o d y ; E R , endoplasmic reticulum; G, Golgi c o m p l e x ; M , mitochondria; Ν , n u c l e u s ; X33.000.)
FINE STRUCTURE OF H U M A N
to the nuclear membrane. In the central part of the nucleus this dense band appears to mix haphazardly with nucleoplasm of a lighter density. The mitochondria of these monocytes have a typical white blood cell pattern. They are larger and more regular than the mitochondria of corneal epithelium. The cristae of the mitochondria are also much more regular than that of the stromal cell. The endoplasmic reticulum of monocytes has a double membrane with attached, dense Palade particles. They are not very abundant ordinarily, but they increase with an increase in cellular activity. Golgi complex is very well developed in the monocytes judging from the abundance of smooth double membrane system and vesicles in the Golgi area. There is a wellformed pair of centrioles or diplosomes near the central area of the Golgi complex (fig. 4 1 ) in typical forms as in the other cell types. So in general, the structure of the monocyte is quite different from that of the stromal cell judging by its shape and the profiles of the nucleus and mitochondria. DISCUSSION
This study confirms many of our previous findings, including the picture of the disintegration of squamous cells before they are shed. The migration and eventual desquamation of epithelial cells has been well known for a long time, but was only recently scientifically demonstrated by Hanna and O'Brien using tritium labelled thymidine in rat, mouse, and dog corneal epithelium. The complete course from the basal layer to the superficial layer and the casting off takes from three and one half to seven days, according to these authors. W e also noticed in our study of the human corneal epithelium, as we had in the rabbit, that there is an increase in the number of tonofibrils in the wing cells. The fibers in the desmosomes are very similar to these tonofibrils, and where there is an
CORNEA
999
increase in the number of tonofibrils, there are likely to be more visible fibers in the desmosomes, and at the same time, more fibers from the desmosomes can be seen crossing over both cell membranes and joining with the tonofibrils. This demonstrates their close relationship. They are very likely the same type of fibers, as we conjectured after our study of the rabbit cornea. The reticular fibers have been considered a type of collagen fiber with argyrophilic properties. Our observations indicate that morphologically the tonofibrils of the corneal epithelium are very similar to the reticular fibers in the basement membrane of the corneal epithelium. To us it seems that it is more likely that reticular fibers are produced from the epithelial cells than from fibroblasts in the stroma. Thus concerning the basement membrane of the corneal epithelium, this study has enabled us to confirm our previous findings and add some new concepts regarding the structure and origin of the basement membrane of the corneal epithelium. Between the cell wall of the basal epithelial cells and the layer of reticular fibers, there is a lipid layer, a thin but continuous layer of lipid substance, mainly plasmalogen. In the reticular fiber layer there is reticular fiber meshwork filled with mucoor glycoproteins. The superficial layer of the reticular fiber meshwork is the most compact layer and its supports the lipid layer. The middle layer of reticular fibers is the loosest mesh of the three layers. Sometimes there may be a collection of fluid ; otherwise it will look more compact but less compact than the third layer. Since the cell membrane of the corneal epithelium and the reticular fiber meshwork are connected only by a few desmosomes, in addition to the lipid substance between the layers, the cohesive force here may be mostly chemical in nature. It was suggested by Herrman and Hickman that lipoprotein is the main cause of adhesive force. In the connection between the reticular fibers and the
1000
C. C. T E N G
collagen fibers of Bowman's membrane there are many interlocked fibers at the time of contact. A s to the attachment between the reticular fiber layer and Bowman's layer, that is more likely due to interlocked fibers than to a chemical cementing factor. Katzin's experiment on electric impedance and fat solvents may have foreshadowed our current findings that fat solvents, such as butyl alcohol, ether, and so forth can cause separation of the epithelium from the stroma and at the same time lower the electric resistance. The impedance is due evidently to the presence of the lipid layer described. Judging from the evidence derived from studies of the electric impedance, the cell wall may not play an important role in impeding current flow because the intercellular spaces are comparatively large in the corneal epithelium and provide wide passage for electrolytes that carry the current. Only the lipid layer of the basement membrane is a real and continuous barrier to an electric current or chemical agent. Our histochemical studies in 1955 and the more recent work of Offret and Haye (1959) and Forgacs (1960) all seem to confirm this observation. Swan and White's work also emphasizes the importance of this lipid layer of the basement membrane, especially in relation to the selective permeability of the cornea to drugs. It may be that the major requirement for permeation of the cornea by a drug is fat solubility. This would be further evidence of the importance of the continuous lipid layer which forms the anterior layer of the basement membrane. Histologically we have observed the collection of fluid in front of and behind the basement membrane in pathologic specimens which suggests that the basement membrane is also very important in the hydration of the cornea. In histochemical studies of human pathologic material we have seen such changes of the basement membrane as variations in thickness, in density of staining property
and in uniformity. The membrane may be smooth or granular; it may have small breaks or large ones, or it may even be lost for a long interval. From all of this evidence and from the work on basement membrane in other tissues, we have come to believe that the basement membrane plays a very important role in the physiology and pathology of the cornea. However, due to the delicacy of this membrane and the fact that it is hard to demonstrate in most animal corneas, even using P A S stain, we must depend largely on the limited supply of human pathologic material for our studies. Now, by employing both histochemical and electron microscope techniques, the possibilities are very much wider and the prospects for future research are improved. With these techniques even animal corneas are useful because the appearance and changes in the basement membrane can be demonstrated nicely with the advantage of the high resolution offered by the electron microscope. THE
SECRETORY
CELL
In the current study we were able to observe more of the details of the cytoplasmic structure of the secretory cell than we described in a previous paper on the rabbit cornea. The fact that these cells contain more tonofibrils, endoplasmic reticulum and microsome particles may explain their distinguishing darker appearance. It may also explain the fact that they are P A S positive and have a more basophilic character. W e still have not been able to get a good photograph of the secretory function in the normal human cornea, but it was described in the study of the rabbit cornea and will be further described in our report on keratoconus. In a previous study we noticed that these secretory cells form transient secretory capillaries containing at least three types of granules: large osmophilic particles, finer particles and large fragments of cytoplasm somewhat resembling inclusion bodies. Morphologically the large particles are
FINE STRUCTURE OF HUMAN
similar to Palade particles in size and density, which may be the major lipid element of basement membrane. The finer particles may be constituents of muco- or glycoprotein in the meshwork of the reticular fiber layer. With all this information on the structure of the basement membrane, it is easier to understand its origin. As we observed, the reticular fibers of the basement membrane are probably basically the same as the tonofibrils of the epithelial cells and as the fibrils in the desmosomes. The lipid part of the basement membrane is very likely secreted from the dark cells of the epithelium. Thus all these findings corroborate the observations of Redslob (1948), Busacca (1949), and Vidal (1951) who all suggested that the basement membrane is of epithelial origin. They based their opinions on the difference in staining properties of the basement membrane and Bowman's membrane and, most important, they observed that the basement membrane regenerated with the corneal epithelium. S Y N C Y T I U M
O F
K E R A T O C Y T E S
Syncytium, intercellular bridges or protoplasmic anastomosis between keratocytes was suggested by Scharenberg, reporting in his silver carbonate preparation studies. This phenomenon has been noted in spermatocytes and can explain the synchronous differentiation in the process of development. This would also indicate tnat it plays a role in the conduction of action potential from one cell to another, probably by the transmission of metabolic material from cell to cell. This finding also helps to explain
CORNEA
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the progression of pathologic changes by spreading pathogenic agents rapidly from cell to cell. This may be of particular importance in the development of lattice degeneration of the cornea to be reported on later. The morphogenesis of this syncytium is also an interesting point for conjecture. Some researchers believe that the attachment is due to incomplete separation after cell division, but this has not yet been definitely demonstrated in the corneal stromal cells. S U M M A R Y
1. Fine structure of the human corneal epithelium and stroma is discussed with emphasis on the different types of cells of the epithelium. The dark or secretory epithelial cell is described in greater detail than in our previous communication. 2. The fine structure of the basement membrane is elaborated again, and the close relationship of the basement membrane to the epithelium rather than the stroma is emphasized. 3. The fine structure of the stromal cell or keratocyte is described and the intercellular relationship illustrated. 4. Three types of nerve fibers found in the epithelium and stroma are briefly reported. 5. The fine structure of the monocyte in the epithelium and stroma is described. 210 East 64th Street (21 ) . ACKNOWLEDGMENT
I a m greatly indebted to N e i l H a r d y , Irene H u g h e s , M a x K l i n g e r , and W i l l i a m R i c e f o r their co-operation and assistance.
R E F E R E N C E S
Busacca, Α . : T h e basal membrane o f the corneal epithelium. Bull. soc. Franc, ophtal., 6 2 : 1 3 3 , 1949. F a w c e t t , D . W . , Ito, S., and Slautterbach : T h e occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation. J. Biophysic. & B i o c h e m . Cytol., 5:453, 1959. Forgacs, J.: Clinical and experimental study o f the basal membrane of the corneal epithelium in keratitis bullousa. Brit. J. Ophth., 4 4 : 3 8 5 , 1960. François, J., Rabaey, M., and V a n d e r m e e r s c h e : L'ultrastructure des tissue oculares a u microscope électronique: I I . E t u d e de la cornée et de la sclérotique. Ophthalmologica, 1 2 7 : 7 4 - 8 5 , 1954. François, J., and Rabaey, M . : T h e anatomy of the cornea. I n Transparency o f the Cornea. ( E d i t e d by S i r S t e w a r t D u k e E l d e r and E . S . P e r k i n s . ) Springfield, 111., T h o m a s , 1960, p. 7.
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Friedenwald, J. S , and Buschke, W . : Mitotic and w o u n d healing activities o f corneal epithelium. Arch. Ophth., 3 2 : 4 1 0 , 1944. H a n n a , C , and O'Brien, J. E . : Cell production and migration in the epithelial layer o f cornea. Arch. O p h t h , 6 4 : 5 3 6 , 1960. H e r r m a n , H , and H i c k m a n , R. H . : T h e adhesion o f epithelium t o stroma in the cornea. Bull. Johns H o p k i n s H o s p , 8 2 : 2 0 8 , 1948. Iguchi, S . : Electron microscope study of corneal tissue. A c t a S o c . O p h t h , Jap. ,64:1294, 1960. Ishida, T . : A study of corneal tissue by electron microscope. A c t a S o c . O p h t h , J a p , 62:1324-1331, 2220-2227, 1958. Jakus, Μ . Α . : Studies o n the cornea: I. T h e fine structure of the rat cornea. A m . J. O p h t h , 3 8 : 4 0 , 1954. : Studies o n the c o r n e a : A c t a X V I I concilium O p h t h , 1954, pp. 461-464. — : Studies on the cornea. J. Biophys. & Biochem. C y t o l , 2 : 2 4 3 (suppl. 1 ) , 1956. : T h e fine structure of the human cornea. I n T h e Structure o f the E y e . ( E d i t e d by S m e l s e r . ) N e w York, A c a d e m i c P r e s s , 1961, p. 343. Katzin, H . M . : A study of the electrical resistance properties o f corneal epithelium. A m . J. O p h t h , 3 4 : 1 1 5 9 , 1951. K a y e s , J , and H o l m b e r g , Α . : T h e fine structure of Bowman's layer and basement membrane of the corneal epithelium. A m . J. O p h t h , 50:1013-1021, 1960. Kobayash, S . : Electron microscopy of corneal epithelium. A c t a S o c . O p h t h , J a p , 6 4 : 1 2 8 6 , 1960. L a s s m a n , G. : D i e Innervation der Hornhaut. A r c h . f. O p h t h , 162:565-609, 1961. L a T e s s a , A . J , T e n g , C. C , and Katzin, Η . M . : T h e histochemistry of the basement membrane of the cornea. A m . J. O p h t h , 3 8 : 1 7 5 , 1954. Lauber, H . : Handbuch der mikroskopischen A n a t o m i e des M e n s c h e n ( M ö l l e n d o r f f ) . 3 ( 2 ) : 4 3 - 5 9 , 1936. Nakaizumi, Y . : Electromicroscopic investigation o f the cornea. I. Corneal stroma. A c t a S o c . O p h t h , J a p , 6 4 : 1 0 6 6 , 1960. — ·: Electromicroscopic investigation of the cornea: I I . B o w m a n ' s membrane. A c t a S o c . O p h t h , J a p , 6 5 : 7 9 , 1961. Off ret, G , and H a y e , C. : Basement membrane o f the corneal epithelium: Histopathologic study. Arch, o p h t a l , 19:126, 1959. Polack, F . M . : Morphology of the cornea. A m . J. O p h t h , 5 1 : 1 0 5 1 , 1961. Redslob (cited b y Busacca, A . ) : T h e basal membrane of the corneal epithelium. Bull. soc. Franc, o p h t a l , 6 2 : 1 3 3 , 1949. Rouiller, C , D a n o n , D , and Ryter, Α . : Application de la microscopic électrique a l'étude de la cornea. A c t a a n a t , 2 0 : 3 9 - 5 2 , 1954. Scharenberg, Κ. : T h e cells and nerves o f the human cornea. A m . J. O p h t h , 4 0 :368, 1955. Schwarz, W . : Electonen Mikroskopishe Untersuchungen ueber die Differenzierung der Cornea und Sklerafibrillen des Menschen. Ztschr. Z e l l f o r s c h , 3 8 : 7 8 - 8 6 , 1953. Sebruyns, M. : Ultrastructure de la cornea et du cristallin etudee au microscope électronique. A n n . o c u l , 183:483, 1950. Sheldon, H . : A n electron microscope study of epithelium in normal mature and immature mouse cornea. J. Biophysic & Biochem. C y t o l , 2 : 2 5 3 , 1956. Sheldon, H , and Zellerquist, H . : A n electron microscopy study o f the cornea in vitamin A deficient mouse. Bull. J o h n s H o p k i n s H o s p , 9 8 : 3 7 2 , 1956. S w a n , K. C , a n d W h i t e , N . G.: Corneal permeability. F a c t o r s affecting penetration of drugs into the cornea. A m . J. O p h t h , 2 5 :1043, 1942. T e n g , C. C . : T h e fine structure of corneal epithelium and basement membrane of the rabbit. A m . J. O p h t h , 5 1 : 2 7 8 , 1961. T e n g , G C , and Katzin, H . M . : T h e basement membrane o f the corneal epithelium. A m . J. O p h t h , 3 6 : 8 9 5 , 1953. Toussaint, D . : Etudes des nerfs corneens par D'verses techniques histogiques. Bull. S o c . beige o p h t a l , 1 2 0 : 5 8 9 , 1959. V a n den Hooff, Α . : Electron microscopy o f cornea and sclera connective tissue. P r o c . K o n . M e d . A k . & W e t . S e r . C , 5 5 : 6 2 8 , 1952. Vidal, F . : Histological division o f the corneal epithelium. Ophth. I b e r o . - A m , 1 3 : 2 0 1 , 1951. W a t s o n , M . L . : Staining o f tissue section f o r electron microscopy w i t h heavy metal: I. J. Biophysic & Biochem. C y t o l , 4 : 4 7 5 , 1 9 5 8 ; I I . J. Biophysic & Biochem. C y t o l , 4:727, 1958. W o l t e r , J. R . : U e b e r den A u f b a u der Nervenbündel in der H o r n h a u t des kaninchen A u g e n . Klin. Monatsbl. f. A u g e n h , 2 2 9 : 2 0 5 6 , 1956. : S i l v e r carbonate technique f o r a demonstration of ocular histology. I n T h e Structure of the E y e ( E d i t e d by S m e l s e r . ) N e w York, Academic P r e s s , 1961, p. 133.