Documenta Ophthalmologica 47,1 : 69-87, 1979 CYTOLOGICAL STUDY OF SENILE CATARACT J. FRANCOIS & V. VICTORIA-TRONCOSO
(Ghent, Belgium) INTRODUCTION The lens is transparent thanks to the perfect metabolic equilibrium of its cells, the small amount of interstitial substance, the uniformity of the refractive indices, the homogeneous distribution of its proteins and the regularity of the capsular surface at the level of the anterior and posterior zones. According to Franqois and Goes (1968), the biometrical study of the lens by means of ultrasonography shows that it has an antero-posterior diameter of 3.71 mm up to the age of twenty-five years, of 4.16 mm between twentyfive and fifty years and of 4.53 mm after the age of fifty years. Hogan and Alvarado (1971) found an average weight of 65 mg in children, and of 250 mg in adults. That difference demonstrates a high activity, both from the point of view of cellular division and from that of biosynthesis, since the lens can double its weight during the first year of life, the growthrate becoming slower subsequently (Franqois, 1959). We investigated the cytological aspects of the lens epithelium, first histologically under the electron microscope by means of flat mountings and sections, and afterwards in tissue cultures, which we examined from both the microscopic and the histochemical points of view. We compared histologically, on the one hand, normal human lenses and, on the other hand, lenses with senile cataracts at the same stage of evolution. We intended in that manner to localize exactly the various cellular structures of the lens epithelium and to determine the more active zones which would produce richer tissue cultures.
MATERIAL AND METHODS We studied ten lenses from cadavers, removed from eyes, the corneal endothelium of which had a viability of at least 70%, what justifies the assumption that the lens was in good condition, because the corneal endothelium is particularly sensitive. Furthermore, we studied forty cataractous lenses, removed during surgical extractions.
69
Six normal lenses and six pathologic lenses were examined as described here below: 1. Fixation in to for one hour in glutaraldehyde or 10% formol, the two products being buffered with 0.2 M phosphate (pH 7.2). 2. Dissection of the lens at various zones, as showed in the topographical diagram of Fig. 1.: (1) anterior cortical, (2) posterior cortical, (3) anterior equator and (4) posterior equator. The dissection was carried out under a binocular microscope of magnification x 40, using a bistoury, Katzin's curved scissors and a spatula. In order to prevent the lens' adhering to the tip of the instruments, they must be kept continuously moistened.
Fig. 1. Topographical schema. 70
3. The cortex pieces were very carefully spread, by means of the spatula, on quartz slides and again fixed for from twelve to twenty-four hours in the same fixative. 4. These fiat mountings, dehydrated or not, were observed directly, without staining, or after staining with haematoxylin-eosin, either under the optical microscope or the phasecontrast microscope. Four normal lenses and twelve with cortical cataracts were prepared for examination by the electron microscope. After having been fixed with glutaraldehyde and dissected, they were again fixed, first in glutaraldehyde and afterwards in osmic acid, before being dehydrated and included according to Spurr's method. The cadavers lenses, as well as the cataractous lenses were examined at the biomicroscope, so as to locate the changes according to the topographical diagram (Fig. 1), and to relate them to those observed under the optical and electron microscopes. For the histotypical culture of the lens epithelium, we developed the method described herebelow. As control material, we used normal rabbit (10) and human (5) lenses. The eyes were first sterilized in the following solution: Sodium penicillin 'G' 1 000 000 IU Streptomycin 1g Ritter's solution 100 ml Each eye was immersed successively in two baths for thirty minutes. The eyes were dissected under sterile conditions in a chamber sterilized by UV light for twenty-four hours and under a laminar-draught hood equipped with filters capable of retaining bacteria. First, we removed the cornea. Next, the iris was detached from its base by means of two pincets. The zonular fibres were cut. The vitreo-lens ligament, which is highly developed in the rabbit, was dissected by means of a spatula. The lenses were treated in the same fashion as the cataractous human lenses. The lenses with senile cataracts were removed during classical operations. It is indispensable that the extraction be effected by either Arruga's or Green's forceps, because the cryode irreversibly damages all the cells within the cone of congelation. The lens was then dissected under a x 40 binocular microscope, according to the diagram of Fig. 1. In turn, (1) the capsule and the anterior epithelium, (2) the peripheral sector and (3) the posterior capsule were removed. These thin specimens were then spread on thin sheets of glass, in order to obtain monolayers. These lamellae were placed in Leighton tubes, to which carbon dioxide gas was applied. Next, a thin layer of plasma clot was spread on the lamellae. The plasma clot had the following composition:
71
Chicken plasma 1 part Chicken embryo extract (Difco) 1 part The chicken plasma was obtained by adding, without shaking, 4.5 ml of reconstituting fluid (Difco) to a flask of lyophilised chicken plasma and was then incubated at 37~ for thirty minutes. The chicken embryo extract was prepared by adding 2 ml of TC reconstituting fluid (Difco) to 8 ml of a sterile Earle's solution. That mixture was incubated at 37~ for thirty minutes and centrifuged at 2 5 0 0 - 3 0 0 0 RPM for ten minutes. Finally, the glass lamellae were removed from the Leighton tubes at the instant of implantation. They were placed on filter paper fixed to the bottom of a Petri dish of diameter 20 cm. When the specimen, which consisted of a very fragile thin membrane, was being spread, it was necessary to make sure that the capsule was underneath and the epithelium on top. With the help of a spatula, which had to be kept continuously wetted with saline to avoid its adhering to the specimen, the specimen was spread out, taking great care to avoid curling of its edges. The lamellae carrying the specimens were then reintroduced into the Leighton tubes, each monolayer containing two specimens. One drop of chicken embryo extract was added to each specimen. The whole was then placed in the incubator at 37~ for two and a half hours. Finally, the following culture medium was added: TC 199 (Difco) Hanks (BSS) Chicken embryo extract Homologous serum To 100 ml of this medium is added: L-proline L-hydroxyproline Glycin Glucose
40 40 7.5 12.5 4.8 1.2 6.0 0.5
% % % % mg mg mg g
The growth of the cultures, their mobility and the behaviour of the cells were studied by micro-cinematography (Wild equipment with inverted microscope and incorporated Bolex 16-mm film-camera). The microscope was placed in a special incubator at a temperature of 37~ The optimum interval between two successive pictures was 10, 15, 20 and 30 seconds. A photo-electric cell incorporated in the microscope tube indicated the exposure times (0.8 or 1 second). We used Kodachrome 40 type-A professional film having a sensitivity of 18 DIN.
72
RESULTS
L Histology of the normal adult lens A. Lens Epithelium When flat mountings are examined, it is seen that the normal anterior epithelium has the form of a very regular mosaic, the distances between the nuclei being identical (hexagonal arrangement). The intercellular spaces are very narrow and almost invisible. The cytoplasm is finely granular, but homogeneous. The chromatin of the nuclei is clotted. At the level of the equatorial epithelium, the cells form loops and move toward the back. They show a less homogeneous cytoplasm, containing granules of various sizes. They appear to be more active than at the level of the anterior surface. Under the dark-field microscope, it is possible to see very clearly the homogeneous distribution of the granular cytoplasmic material in the cells of the anterior epithelium. The distribution of the fibre-cells is seen in Fig. 2. At the level of the embryonic nucleus, there are two sutures in the form of a Y, one at each pole of the nucleus. They are orientated perpendicularly one to the other. These
Fig. 2. Flat mounting of normal lens epithelium at the level of the equator. The cells have a shape in the form of a loop or a fork and run from the anterior part to the posterior one (obj. x 50).
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sutures represent the contact points of the fibre-cells originating from the opposite pole. These cells are distributed as follows: 1. The fibre-cells that originate in the long branch of the anterior suture join up with the short branch of the posterior suture. 2. The fibre-cells that originate at the level of the apex of the long branch of the anterior suture join up with the angle between the short branches of the posterior suture. 3. The fibre-cells that originate at the level of the apex of a short branch of the anterior suture join up with the angle formed by the short branch and the long branch of the posterior suture. 4. In the intermediate and adult nuclei, the sutures become more complex, and they have a larger number of branches, but the systematisation of the fibre-cells remains essentially the same. A histological fact to be borne in mind is that the sutures do not represent the junction line of fibres located in a single plane, as the biomicroscopic examination might lead to assume; there are, in fact, several planes of cells, joining at the same level. The epithelial cells are localized at the level of the anterior cortex and the equator, the other parts of the lens being occupied by fibres, which are merely cytoplasmic extensions of the anterior cortical cells. There are no cells, but only fibres, in the posterior cortex. The adult lens contains 2100 to 2300 fibre-cells, of length 12 000/am, width 7/am and thickness 4.6/am. The electron microscope shows that the equatorial epithelial cells, whose flattened nuclei are arranged obliquely and whose cytoplasmic fibre is shorter, contain a larger number of cytoplasmic organelles than the anterior epithelial cells, what demonstrates a greater cellular activity. Their cytoplasmic matrix is finely granular. The ribosomes, which are generally attached to the endoplasmic membranes, are either isolated or grouped. The mitochondria have well-developed crests. They are small and of either rounded or elongated shape. The Golgi's apparatus is well developed. Its cisterns are flattened and surrounded by numerous vesicles. Lysosomes are rare. Multivesicular bodies occur in the older lenses, their small vesicles being arranged in chains. There are no desmosomes between the ceils, which are attached one to another by interlocking processes. Cytoplasmic organelles are rare in the ceils of the anterior cortex, either because the cytoplasm is relatively abundant, or because their metabolism is weak. Some pinocytose vesicles can be observed. In the cortical layers, the nucleus is surrounded by a membrane, which is not seen in the deeper layers, where only chromatin fragments of medium electron density are found.
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B. Capsule The capsule displays a glycoproteinic structure with disseminated collagenous elements. At the electron microscope, its structure appears to be relatively homogeneous, and in some places collagen, especially of the longspacing-fibre type, and clear spaces, are to be seen.
C. Zonular system Zinn's zonule belongs neither embryologically nor histologically to the lens. Embryologically, it forms part of the tertiary vitreous, because the mucopolysaccharides and the collagen bundles develop at the same time, after the atrophy of the Druault fasciculus. Histologically, it is impossible, in the adult, to separate the zonular fibres from the mucopolysaccharides in which they are included. The examination of flat mountings shows that there are two annular bundles of fibres, one of them anterior and the other posterior, extending from the anterior or posterior pre-equatorial zones of the lens toward the anterior part of the ciliary body. At the phase-contrast microscope, the thickness of the fibres is variable. At the polarisation microscope, it is seen that their birefringence is the same as that of adult collagen. At the electron microscope, it is observed that each zonular fibre consists of several delicate collagen fibrils of fairly uniform diameter, whose periodicity is that of adult collagen. When the zonular bundles reach the capsule, they arrange themselves in lamellae, which constitute the superficial peripheral layer of the capsule.
II. Histopathology of the cataractous lens Examination o f flat mounting The epithelium of the cataractous lens displays the following changes: 1. The intercellular spaces are much enlarged, and contain foamy deposits (fig. 3). 2. Between the cells there are giant vacuoles, which may attain 20 to 40 ~m and force the adjacent cells toward the periphery (fig. 4). 3. There are also cells whose cytoplasm contains large irregularly distributed clots of high optical density. 4. Even the ceils that appear to be more or less normal are arranged in irregular mosaics. 5. In some places, the cells are destroyed and replaced by a foamy deposit. These various abnormal cells may occupy more or less extensive areas,
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Fig. 3, Flat mounting of the anterior lens epithelium in a case of cortical cataract. Foamy intercellular deposits are seen (obj. x 50).
Fig. 4. Flat mounting of the cataractous lens epithelium. Large intercellular vacuole (obj. x 50).
76
which may lie alongside normal areas. The more or less large proportion of pathological areas depends upon the extent of the cataract.
Electron microscopy Examination at the electron microscope shows the following changes (figs.5, 6 and 7): 1. The ceUular destruction in the cataractous lens stimulates the cellular migration, with the result that nucleated cells are found at the level of the posterior cortex. 2. The intercellular spaces are first dilated. They have a fusiform shape and afterwards a more rounded form. These vesicular dilations measure from 5 to 50 #m. Their walls are constituted by the cellular membranes of the neighbouring cells. These vesicles are empty or may contain membranous debris. 3. Many of the cells contain small particles of the lysosomal type, measuring between 0.2 and 0.5 /~m. Their electron density is average, and their appearance is smooth. 4. The endoplasmic reticulum is well developed. Its membranes display several folds and contain some ribosomes. 5. The Golgi's apparatus is as developed as in a normal cell. 6. In many of the cells, the nucleus is destroyed, the cellular membrane ruptured and the structure disorganized. 7. More or less large areas of destroyed fibres are observed. These necrotic areas are produced by the neighbouring lysosomes. 8. There are swollen mitochondria in the course of destruction alongside normal mitochondria. The most remarkable characteristic of the cataractous lens is the fact that there are foci of developing cells in the posterior cortex and that cytoplasmic organelles are found in all the areas, as indicated in the topographical diagram (Fig. 1).
IlL Cytology of the epithelium of normal and cataractous lenses in tissue culture There are two types of lens cultures: 1. The organotypic culture, whereby the lens is cultivated in render possible physiological studies on its metabolism and studies on its transparence. 2. The histotypic culture, whereby the proliferation of the outside the organ, which renders possible cytological studies
to, so as to biophysical cells occurs on the bio-
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Fig. 5. Electron-microscopy. Cortical cataract at the posterior pole. Important development of lysosome-like particles and cytolysis (x 20.000).
synthesis and catabolism of the cell material, as well as on the cellular respiration and metabolism. The importance of lens cultures was stressed by Nordmann (1954, 1962),
78
Fig. 6. Electron-microscopy. Cortical cataract at the anterior intermediary region. Macro- and microvacuolisation. Membranous debris in some vacuoles (x 30.000).
Pirie and Van Heyningen (1956), Lucas (1965), Francois and VictoriaTroncoso (1976). Our studies on lens cultures, which increase the activity of the cells and
79
Fig. 7. Electron-microscopy. Anterior epithelium of cortical cataract. Cellular destruction and presence of numerous vacuolar spaces (x 30.000).
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accelerate the pathological process of the cells of the cataractous lens, have a triple objective: 1. The microscopic and histochemical study of the cataractous lens, compared with that of the normal lens. 2. The study of the growth of the cultures, relating to the anterior, equatorial or posterior topography of the lens tissue. 3. The study of the vacuolar and lysosomal systems in cataracts.
Growth o f the lens in tissue culture 1. Normal lens epithelium. It is easy to obtain a growth of the anterior and peripheral epithelium. As might be foreseen, the posterior capsule does not grow. Growth occurs in the specimen after the first twenty-four hours. The cells are seen to take on a rounded shape and their cytoplasm to develop. After between forty-eight and seventy-two hours, they multiply outside the specimen in a centrifugal pattern and follow a very precise direction. They form an irregular network around the specimen, the cell prolongations tending to form very elongated fibres, which frequently lie alongside and adhere to the neighbouring fibres. 2. Pathological lens epithelium. We placed ten cataractous lenses in tissue culture. The growth was slow and irregular. During the first forty-eight hours, rounded cells which burst were seen in the specimen, whereas others migrated in a very anarchic fashion toward the edge of the specimen. It was toward the fourth day that a few isolated cells outside the specimen were to be seen, but sometimes that phenomenon did not occur until about the eighth day. Unlike what was ob~ served for normal epithelial cells, the anterior epithelium grew only rarely, whereas the posterior capsule grew very frequently. The peripheral epithelium grew irregularly; one half might grow and the other half not.
Characteristics or fresh epithelial cells examined at the phase-contrast microscope 1. Normal epithelium. Essentially two types of epithelial cells are found, having numerous intermediate forms:
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a) Elongated or fibrillar cells. These measure from 60 to 100 pm. Generally, they group themselves in twos or threes, seeking to form systems of fibres. They contain large numbers of very fine granulations, as well as mitochondria. b) Cells with abundant cytoplasm. These are generally polygonal and occasionally star-shaped. They measure from 30 to 70 #m. They also contain large numbers of very fine granulations, as well as regularly star-shaped mitochondria. 2. Pathological epithelium. Anisomorphism and irregular distribution are characteristics of these cells, which remain isolated, with the exception of the elongated cells, Which tend to behave as the elongated cells of the normal epithelium. Four types of cells can be distinguished: elongated cells, rounded cells, rectangular or starshaped cells and irregular cells.
a) Elongated cells. These have characteristics similar to those of the elongated cells of the normal epithelium, and they also have a tendency to form fibres. Some of them nevertheless display small vacuoles of 0 . 5 - 1 . 5 pm.
b) Rounded cells. These are highly pathological cells, which contain little cytoplasm. The cytoplasm is above all constituted of granules, which move rapidly. The nucleus is globular. These ceils inevitably separate from the lamella of the culture and move. c) Rectangular or star-shaped cells. The cytoplasm is rather abundant. The branches of the star-shaped forms are short. Large vacuoles of from 1 to 3 p m are observed, in many cases arranged in chains and optically empty. There are also small granulations and irregularly distributed mitochondria, being pressed back by the vacuoles.
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d) Irregular cells. A large variety of cells may be observed, all having different sizes: fusiform, hammer-shaped, club-shaped, geographical form, etc. The nucleus is generally displaced toward one extremity of the cell.
Microcinematography of pathological epithelial cells At the beginning of the growth within the specimen, the cells are grouped in irregular masses. Their cytoplasm contains granules in rapid motion. Later on, some of the cells become isolated in order to elongate and to attempt to form fibres, but because their adherence to the surface of the glass is weak, many cells become detached, whereas others take up a polygonal or irregular shape. Although some of the cells can become elongated, they nevertheless in many cases remain cut off from one part of their cytoplasm. These are the cells which become rounded, detach and die. Whereas, in the cultures of normal epithelium, the migration is centrifugal, we observe, in the cultures of pathological epithelium, some cells which move successively in different directions, their movements becoming thus anarchic. Vacuoles develop inside the cells. Small vacuoles can merge with one another. These vacuoles are very mobile and change their shapes when the cell moves. The intracytoplasmic granules move rapidly, although their displacement is rather small. Pinocytosis phenomena are observed in many cases. DISCUSSION The histological study of normal and pathological lenses has made it possible to establish the following facts. By comparison with the normal lens, the characteristics of the cataractous lens are (figs 8, 9 and 10): 1. Anomalies in the distribution of the cells, which display an irregular migration and are found in the posterior cortex, where normally they are absent. 2. There are cytoplasmic organelles in all the cortical areas indicated in the topographical diagram, whereas in the normal lens, they are only found at the level of the equator. 3. At the electron microscope, enlargement of the intercellular spaces is observed, and large intercellular vacuoles with storage of a foamy material are found in the flat mountings. It is most probably a matter of deterioration of the polyanionic surface of the intercellular glycoproteins. The inter-
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Fig. 8. Electron microscopy. Tissue culture of the posterior epithelium in a case of cortical cataract. Development of lysosomes in the cell fibres (x 24.000). cellular transport must be very slow at that level. These intercellular vacuoles correspond without doubt to the clear cortical slits and to the vacuoles that can be observed at the biomicroscope. 4. Intracellular vacuoles and deposits exist, which indicates an irreversible deterioration of the vacuolar system. That vacuolisation is in relation with
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Fig. 9. Electron microscopy. Tissue culture of cortical cataract. Development of multivesicular bodies (x 24.000). the important development of the lysosomal system and is responsible for the observed cytolytic phenomena. These severely damaged cells are nevertheless alive, as demonstrated by microcinematography (figs 8 and 9). 5. There is a serious deterioration of the cellular membranes of the lens epithelium. 6. The great development of the endoplasmic reticulum indicates an intense biosynthetic activity. 7. Cytolysis with destruction of the mitochondria and nuclei, as well as cell proliferation, are observed. As regards Zinn's zonule, it has to be stressed that it is a mesodermic structure, associated embryologically and histologically with the vitreous. The zonule consists of adult collagen, arranged in bundles, which are immersed in the mucopolysaccharides of the vitreous. As regards the lens in tissue culture, we were able to demonstrate histochemically, in the normal epithelium. 1. The presence of glycoproteins in the lysosomal matrix. 2. The presence of a large number of lysosomes containing acid phosphatases. 3. The presence of glucose-6-phosphate dehydrogenase and lacto-dehydrogenase in the mitochondria.
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Fig. 10. Scanning microscopy. Tissue culture of a normal lens. t"lat aspect of the cells, which show very long cytoplasmic extensions (x 3000).
We compared the normal epithelium and the epithelium of senile cataracts with the help of seriated photographs and microcinematography. We may say that: 1. There is a deterioration of the cell tropism in the cultures of pathological epithelium, the movements of the cells becoming anarchic. 2. There is anisomorphism and anisometry not found in the cultures of normal epithelium. 3. There is a deterioration of the vacuolar system with a tendency to the formation of large vacuoles. This phenomenon is commonly observed in storage diseases and is associated with a deterioration of the lysosomal system. 4. A tendency is observed to form fibres in the most normal cells, but not in the vacuolised cells. The formation of fibres may be considered as a sign of cellular differentiation, whereas the formation of vacuoles is a sign of degeneration and of dedifferentiation. These phenomena ought to be found
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in the epithelium in situ, because they occur during the initial phase of the culture, that is to say, at the time when the cells are still in contact with their natural support, the capsule. 5. The large vacuoles indicate the presence of stored products and that storage is progressive. There is also, in senile cataract, an abnormal migration of cells toward places where they are not found normally, that is to say, at the level of the posterior capsule. Finally, there is a close relationship between the degree of clinical evolution of the cataract and the degree of cellular deterioration. The principal interest of this research is to demonstrate that the culture of normal or pathologic lens epithelium gives constant results, making a cytological study possible. The deteriorations of the lens epithelium in senile cataract continue to occur in tissue culture, with the consequence that we have thus an ideal experimental method of determining whether the cataract is a purely epithelial disease and of stating which cellular organelles are responsible for it. SUMMARY Cortical cataracts are essentially characterized by a cellular deterioration of the lens epithelium with hypertrophy of the vacuolar system, cytolysis and cell proliferation, as well as by an intercellular storage of foamy material and the presence of intracellular deposits. REFERENCI~S Francois J. Les cataractes cong6nitales. Ed. Masson, Paris, 1959. Francois J. & Goes F. L'ultrasonographie dans le diagnostic des affections oculaires. Possibilit6s et limitation de la technique. Bull. Soc. Belge Ophtal. 150:600-614 (1968). Francois, J. & Victoria-Troncoso V. Epithelium of the adult lens in tissue culture. In: Progress of lens biochemistry research, pp. 39-46. Doc. Ophthal. Proc. Series, Junk, The Hague, pp. 39-46, 1976. Hogan M.J., Alvarado A.B. & Weddell J.E. Histology of the human eye, Ed. W.B. Saunders, Philadelphia, 1971. Lucas D.R. Special cytology of the eye. In: Cells and tissues in culture. Methods, Biology and Physiology, Ed. E.N. Willmer, vol. II, Academic Press, London-N.Y., 1965. Nordmann J. Biologie du cristallin. Masson, Paris, 1954. Nordmann J. Acquisitions r~centes dans le domaine de la biologie du cristallin. Adv. OphthaL, Karger, Basel 59: I 47, (1962). Pirie A. and Van Heyningen R. Biochemistry of the eye. Blackwell, Oxford, 1956. Author's address: Ophthalmological Clinic University of Ghent Ghent, Belgium
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(Ghent, Belgium) INTRODUCTION The lens is transparent thanks to the perfect metabolic equilibrium of its cells, the small amount of interstitial substance, the uniformity of the refractive indices, the homogeneous distribution of its proteins and the regularity of the capsular surface at the level of the anterior and posterior zones. According to Franqois and Goes (1968), the biometrical study of the lens by means of ultrasonography shows that it has an antero-posterior diameter of 3.71 mm up to the age of twenty-five years, of 4.16 mm between twentyfive and fifty years and of 4.53 mm after the age of fifty years. Hogan and Alvarado (1971) found an average weight of 65 mg in children, and of 250 mg in adults. That difference demonstrates a high activity, both from the point of view of cellular division and from that of biosynthesis, since the lens can double its weight during the first year of life, the growthrate becoming slower subsequently (Franqois, 1959). We investigated the cytological aspects of the lens epithelium, first histologically under the electron microscope by means of flat mountings and sections, and afterwards in tissue cultures, which we examined from both the microscopic and the histochemical points of view. We compared histologically, on the one hand, normal human lenses and, on the other hand, lenses with senile cataracts at the same stage of evolution. We intended in that manner to localize exactly the various cellular structures of the lens epithelium and to determine the more active zones which would produce richer tissue cultures.
MATERIAL AND METHODS We studied ten lenses from cadavers, removed from eyes, the corneal endothelium of which had a viability of at least 70%, what justifies the assumption that the lens was in good condition, because the corneal endothelium is particularly sensitive. Furthermore, we studied forty cataractous lenses, removed during surgical extractions.
69
Six normal lenses and six pathologic lenses were examined as described here below: 1. Fixation in to for one hour in glutaraldehyde or 10% formol, the two products being buffered with 0.2 M phosphate (pH 7.2). 2. Dissection of the lens at various zones, as showed in the topographical diagram of Fig. 1.: (1) anterior cortical, (2) posterior cortical, (3) anterior equator and (4) posterior equator. The dissection was carried out under a binocular microscope of magnification x 40, using a bistoury, Katzin's curved scissors and a spatula. In order to prevent the lens' adhering to the tip of the instruments, they must be kept continuously moistened.
Fig. 1. Topographical schema. 70
3. The cortex pieces were very carefully spread, by means of the spatula, on quartz slides and again fixed for from twelve to twenty-four hours in the same fixative. 4. These fiat mountings, dehydrated or not, were observed directly, without staining, or after staining with haematoxylin-eosin, either under the optical microscope or the phasecontrast microscope. Four normal lenses and twelve with cortical cataracts were prepared for examination by the electron microscope. After having been fixed with glutaraldehyde and dissected, they were again fixed, first in glutaraldehyde and afterwards in osmic acid, before being dehydrated and included according to Spurr's method. The cadavers lenses, as well as the cataractous lenses were examined at the biomicroscope, so as to locate the changes according to the topographical diagram (Fig. 1), and to relate them to those observed under the optical and electron microscopes. For the histotypical culture of the lens epithelium, we developed the method described herebelow. As control material, we used normal rabbit (10) and human (5) lenses. The eyes were first sterilized in the following solution: Sodium penicillin 'G' 1 000 000 IU Streptomycin 1g Ritter's solution 100 ml Each eye was immersed successively in two baths for thirty minutes. The eyes were dissected under sterile conditions in a chamber sterilized by UV light for twenty-four hours and under a laminar-draught hood equipped with filters capable of retaining bacteria. First, we removed the cornea. Next, the iris was detached from its base by means of two pincets. The zonular fibres were cut. The vitreo-lens ligament, which is highly developed in the rabbit, was dissected by means of a spatula. The lenses were treated in the same fashion as the cataractous human lenses. The lenses with senile cataracts were removed during classical operations. It is indispensable that the extraction be effected by either Arruga's or Green's forceps, because the cryode irreversibly damages all the cells within the cone of congelation. The lens was then dissected under a x 40 binocular microscope, according to the diagram of Fig. 1. In turn, (1) the capsule and the anterior epithelium, (2) the peripheral sector and (3) the posterior capsule were removed. These thin specimens were then spread on thin sheets of glass, in order to obtain monolayers. These lamellae were placed in Leighton tubes, to which carbon dioxide gas was applied. Next, a thin layer of plasma clot was spread on the lamellae. The plasma clot had the following composition:
71
Chicken plasma 1 part Chicken embryo extract (Difco) 1 part The chicken plasma was obtained by adding, without shaking, 4.5 ml of reconstituting fluid (Difco) to a flask of lyophilised chicken plasma and was then incubated at 37~ for thirty minutes. The chicken embryo extract was prepared by adding 2 ml of TC reconstituting fluid (Difco) to 8 ml of a sterile Earle's solution. That mixture was incubated at 37~ for thirty minutes and centrifuged at 2 5 0 0 - 3 0 0 0 RPM for ten minutes. Finally, the glass lamellae were removed from the Leighton tubes at the instant of implantation. They were placed on filter paper fixed to the bottom of a Petri dish of diameter 20 cm. When the specimen, which consisted of a very fragile thin membrane, was being spread, it was necessary to make sure that the capsule was underneath and the epithelium on top. With the help of a spatula, which had to be kept continuously wetted with saline to avoid its adhering to the specimen, the specimen was spread out, taking great care to avoid curling of its edges. The lamellae carrying the specimens were then reintroduced into the Leighton tubes, each monolayer containing two specimens. One drop of chicken embryo extract was added to each specimen. The whole was then placed in the incubator at 37~ for two and a half hours. Finally, the following culture medium was added: TC 199 (Difco) Hanks (BSS) Chicken embryo extract Homologous serum To 100 ml of this medium is added: L-proline L-hydroxyproline Glycin Glucose
40 40 7.5 12.5 4.8 1.2 6.0 0.5
% % % % mg mg mg g
The growth of the cultures, their mobility and the behaviour of the cells were studied by micro-cinematography (Wild equipment with inverted microscope and incorporated Bolex 16-mm film-camera). The microscope was placed in a special incubator at a temperature of 37~ The optimum interval between two successive pictures was 10, 15, 20 and 30 seconds. A photo-electric cell incorporated in the microscope tube indicated the exposure times (0.8 or 1 second). We used Kodachrome 40 type-A professional film having a sensitivity of 18 DIN.
72
RESULTS
L Histology of the normal adult lens A. Lens Epithelium When flat mountings are examined, it is seen that the normal anterior epithelium has the form of a very regular mosaic, the distances between the nuclei being identical (hexagonal arrangement). The intercellular spaces are very narrow and almost invisible. The cytoplasm is finely granular, but homogeneous. The chromatin of the nuclei is clotted. At the level of the equatorial epithelium, the cells form loops and move toward the back. They show a less homogeneous cytoplasm, containing granules of various sizes. They appear to be more active than at the level of the anterior surface. Under the dark-field microscope, it is possible to see very clearly the homogeneous distribution of the granular cytoplasmic material in the cells of the anterior epithelium. The distribution of the fibre-cells is seen in Fig. 2. At the level of the embryonic nucleus, there are two sutures in the form of a Y, one at each pole of the nucleus. They are orientated perpendicularly one to the other. These
Fig. 2. Flat mounting of normal lens epithelium at the level of the equator. The cells have a shape in the form of a loop or a fork and run from the anterior part to the posterior one (obj. x 50).
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sutures represent the contact points of the fibre-cells originating from the opposite pole. These cells are distributed as follows: 1. The fibre-cells that originate in the long branch of the anterior suture join up with the short branch of the posterior suture. 2. The fibre-cells that originate at the level of the apex of the long branch of the anterior suture join up with the angle between the short branches of the posterior suture. 3. The fibre-cells that originate at the level of the apex of a short branch of the anterior suture join up with the angle formed by the short branch and the long branch of the posterior suture. 4. In the intermediate and adult nuclei, the sutures become more complex, and they have a larger number of branches, but the systematisation of the fibre-cells remains essentially the same. A histological fact to be borne in mind is that the sutures do not represent the junction line of fibres located in a single plane, as the biomicroscopic examination might lead to assume; there are, in fact, several planes of cells, joining at the same level. The epithelial cells are localized at the level of the anterior cortex and the equator, the other parts of the lens being occupied by fibres, which are merely cytoplasmic extensions of the anterior cortical cells. There are no cells, but only fibres, in the posterior cortex. The adult lens contains 2100 to 2300 fibre-cells, of length 12 000/am, width 7/am and thickness 4.6/am. The electron microscope shows that the equatorial epithelial cells, whose flattened nuclei are arranged obliquely and whose cytoplasmic fibre is shorter, contain a larger number of cytoplasmic organelles than the anterior epithelial cells, what demonstrates a greater cellular activity. Their cytoplasmic matrix is finely granular. The ribosomes, which are generally attached to the endoplasmic membranes, are either isolated or grouped. The mitochondria have well-developed crests. They are small and of either rounded or elongated shape. The Golgi's apparatus is well developed. Its cisterns are flattened and surrounded by numerous vesicles. Lysosomes are rare. Multivesicular bodies occur in the older lenses, their small vesicles being arranged in chains. There are no desmosomes between the ceils, which are attached one to another by interlocking processes. Cytoplasmic organelles are rare in the ceils of the anterior cortex, either because the cytoplasm is relatively abundant, or because their metabolism is weak. Some pinocytose vesicles can be observed. In the cortical layers, the nucleus is surrounded by a membrane, which is not seen in the deeper layers, where only chromatin fragments of medium electron density are found.
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B. Capsule The capsule displays a glycoproteinic structure with disseminated collagenous elements. At the electron microscope, its structure appears to be relatively homogeneous, and in some places collagen, especially of the longspacing-fibre type, and clear spaces, are to be seen.
C. Zonular system Zinn's zonule belongs neither embryologically nor histologically to the lens. Embryologically, it forms part of the tertiary vitreous, because the mucopolysaccharides and the collagen bundles develop at the same time, after the atrophy of the Druault fasciculus. Histologically, it is impossible, in the adult, to separate the zonular fibres from the mucopolysaccharides in which they are included. The examination of flat mountings shows that there are two annular bundles of fibres, one of them anterior and the other posterior, extending from the anterior or posterior pre-equatorial zones of the lens toward the anterior part of the ciliary body. At the phase-contrast microscope, the thickness of the fibres is variable. At the polarisation microscope, it is seen that their birefringence is the same as that of adult collagen. At the electron microscope, it is observed that each zonular fibre consists of several delicate collagen fibrils of fairly uniform diameter, whose periodicity is that of adult collagen. When the zonular bundles reach the capsule, they arrange themselves in lamellae, which constitute the superficial peripheral layer of the capsule.
II. Histopathology of the cataractous lens Examination o f flat mounting The epithelium of the cataractous lens displays the following changes: 1. The intercellular spaces are much enlarged, and contain foamy deposits (fig. 3). 2. Between the cells there are giant vacuoles, which may attain 20 to 40 ~m and force the adjacent cells toward the periphery (fig. 4). 3. There are also cells whose cytoplasm contains large irregularly distributed clots of high optical density. 4. Even the ceils that appear to be more or less normal are arranged in irregular mosaics. 5. In some places, the cells are destroyed and replaced by a foamy deposit. These various abnormal cells may occupy more or less extensive areas,
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Fig. 3, Flat mounting of the anterior lens epithelium in a case of cortical cataract. Foamy intercellular deposits are seen (obj. x 50).
Fig. 4. Flat mounting of the cataractous lens epithelium. Large intercellular vacuole (obj. x 50).
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which may lie alongside normal areas. The more or less large proportion of pathological areas depends upon the extent of the cataract.
Electron microscopy Examination at the electron microscope shows the following changes (figs.5, 6 and 7): 1. The ceUular destruction in the cataractous lens stimulates the cellular migration, with the result that nucleated cells are found at the level of the posterior cortex. 2. The intercellular spaces are first dilated. They have a fusiform shape and afterwards a more rounded form. These vesicular dilations measure from 5 to 50 #m. Their walls are constituted by the cellular membranes of the neighbouring cells. These vesicles are empty or may contain membranous debris. 3. Many of the cells contain small particles of the lysosomal type, measuring between 0.2 and 0.5 /~m. Their electron density is average, and their appearance is smooth. 4. The endoplasmic reticulum is well developed. Its membranes display several folds and contain some ribosomes. 5. The Golgi's apparatus is as developed as in a normal cell. 6. In many of the cells, the nucleus is destroyed, the cellular membrane ruptured and the structure disorganized. 7. More or less large areas of destroyed fibres are observed. These necrotic areas are produced by the neighbouring lysosomes. 8. There are swollen mitochondria in the course of destruction alongside normal mitochondria. The most remarkable characteristic of the cataractous lens is the fact that there are foci of developing cells in the posterior cortex and that cytoplasmic organelles are found in all the areas, as indicated in the topographical diagram (Fig. 1).
IlL Cytology of the epithelium of normal and cataractous lenses in tissue culture There are two types of lens cultures: 1. The organotypic culture, whereby the lens is cultivated in render possible physiological studies on its metabolism and studies on its transparence. 2. The histotypic culture, whereby the proliferation of the outside the organ, which renders possible cytological studies
to, so as to biophysical cells occurs on the bio-
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Fig. 5. Electron-microscopy. Cortical cataract at the posterior pole. Important development of lysosome-like particles and cytolysis (x 20.000).
synthesis and catabolism of the cell material, as well as on the cellular respiration and metabolism. The importance of lens cultures was stressed by Nordmann (1954, 1962),
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Fig. 6. Electron-microscopy. Cortical cataract at the anterior intermediary region. Macro- and microvacuolisation. Membranous debris in some vacuoles (x 30.000).
Pirie and Van Heyningen (1956), Lucas (1965), Francois and VictoriaTroncoso (1976). Our studies on lens cultures, which increase the activity of the cells and
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Fig. 7. Electron-microscopy. Anterior epithelium of cortical cataract. Cellular destruction and presence of numerous vacuolar spaces (x 30.000).
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accelerate the pathological process of the cells of the cataractous lens, have a triple objective: 1. The microscopic and histochemical study of the cataractous lens, compared with that of the normal lens. 2. The study of the growth of the cultures, relating to the anterior, equatorial or posterior topography of the lens tissue. 3. The study of the vacuolar and lysosomal systems in cataracts.
Growth o f the lens in tissue culture 1. Normal lens epithelium. It is easy to obtain a growth of the anterior and peripheral epithelium. As might be foreseen, the posterior capsule does not grow. Growth occurs in the specimen after the first twenty-four hours. The cells are seen to take on a rounded shape and their cytoplasm to develop. After between forty-eight and seventy-two hours, they multiply outside the specimen in a centrifugal pattern and follow a very precise direction. They form an irregular network around the specimen, the cell prolongations tending to form very elongated fibres, which frequently lie alongside and adhere to the neighbouring fibres. 2. Pathological lens epithelium. We placed ten cataractous lenses in tissue culture. The growth was slow and irregular. During the first forty-eight hours, rounded cells which burst were seen in the specimen, whereas others migrated in a very anarchic fashion toward the edge of the specimen. It was toward the fourth day that a few isolated cells outside the specimen were to be seen, but sometimes that phenomenon did not occur until about the eighth day. Unlike what was ob~ served for normal epithelial cells, the anterior epithelium grew only rarely, whereas the posterior capsule grew very frequently. The peripheral epithelium grew irregularly; one half might grow and the other half not.
Characteristics or fresh epithelial cells examined at the phase-contrast microscope 1. Normal epithelium. Essentially two types of epithelial cells are found, having numerous intermediate forms:
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a) Elongated or fibrillar cells. These measure from 60 to 100 pm. Generally, they group themselves in twos or threes, seeking to form systems of fibres. They contain large numbers of very fine granulations, as well as mitochondria. b) Cells with abundant cytoplasm. These are generally polygonal and occasionally star-shaped. They measure from 30 to 70 #m. They also contain large numbers of very fine granulations, as well as regularly star-shaped mitochondria. 2. Pathological epithelium. Anisomorphism and irregular distribution are characteristics of these cells, which remain isolated, with the exception of the elongated cells, Which tend to behave as the elongated cells of the normal epithelium. Four types of cells can be distinguished: elongated cells, rounded cells, rectangular or starshaped cells and irregular cells.
a) Elongated cells. These have characteristics similar to those of the elongated cells of the normal epithelium, and they also have a tendency to form fibres. Some of them nevertheless display small vacuoles of 0 . 5 - 1 . 5 pm.
b) Rounded cells. These are highly pathological cells, which contain little cytoplasm. The cytoplasm is above all constituted of granules, which move rapidly. The nucleus is globular. These ceils inevitably separate from the lamella of the culture and move. c) Rectangular or star-shaped cells. The cytoplasm is rather abundant. The branches of the star-shaped forms are short. Large vacuoles of from 1 to 3 p m are observed, in many cases arranged in chains and optically empty. There are also small granulations and irregularly distributed mitochondria, being pressed back by the vacuoles.
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d) Irregular cells. A large variety of cells may be observed, all having different sizes: fusiform, hammer-shaped, club-shaped, geographical form, etc. The nucleus is generally displaced toward one extremity of the cell.
Microcinematography of pathological epithelial cells At the beginning of the growth within the specimen, the cells are grouped in irregular masses. Their cytoplasm contains granules in rapid motion. Later on, some of the cells become isolated in order to elongate and to attempt to form fibres, but because their adherence to the surface of the glass is weak, many cells become detached, whereas others take up a polygonal or irregular shape. Although some of the cells can become elongated, they nevertheless in many cases remain cut off from one part of their cytoplasm. These are the cells which become rounded, detach and die. Whereas, in the cultures of normal epithelium, the migration is centrifugal, we observe, in the cultures of pathological epithelium, some cells which move successively in different directions, their movements becoming thus anarchic. Vacuoles develop inside the cells. Small vacuoles can merge with one another. These vacuoles are very mobile and change their shapes when the cell moves. The intracytoplasmic granules move rapidly, although their displacement is rather small. Pinocytosis phenomena are observed in many cases. DISCUSSION The histological study of normal and pathological lenses has made it possible to establish the following facts. By comparison with the normal lens, the characteristics of the cataractous lens are (figs 8, 9 and 10): 1. Anomalies in the distribution of the cells, which display an irregular migration and are found in the posterior cortex, where normally they are absent. 2. There are cytoplasmic organelles in all the cortical areas indicated in the topographical diagram, whereas in the normal lens, they are only found at the level of the equator. 3. At the electron microscope, enlargement of the intercellular spaces is observed, and large intercellular vacuoles with storage of a foamy material are found in the flat mountings. It is most probably a matter of deterioration of the polyanionic surface of the intercellular glycoproteins. The inter-
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Fig. 8. Electron microscopy. Tissue culture of the posterior epithelium in a case of cortical cataract. Development of lysosomes in the cell fibres (x 24.000). cellular transport must be very slow at that level. These intercellular vacuoles correspond without doubt to the clear cortical slits and to the vacuoles that can be observed at the biomicroscope. 4. Intracellular vacuoles and deposits exist, which indicates an irreversible deterioration of the vacuolar system. That vacuolisation is in relation with
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Fig. 9. Electron microscopy. Tissue culture of cortical cataract. Development of multivesicular bodies (x 24.000). the important development of the lysosomal system and is responsible for the observed cytolytic phenomena. These severely damaged cells are nevertheless alive, as demonstrated by microcinematography (figs 8 and 9). 5. There is a serious deterioration of the cellular membranes of the lens epithelium. 6. The great development of the endoplasmic reticulum indicates an intense biosynthetic activity. 7. Cytolysis with destruction of the mitochondria and nuclei, as well as cell proliferation, are observed. As regards Zinn's zonule, it has to be stressed that it is a mesodermic structure, associated embryologically and histologically with the vitreous. The zonule consists of adult collagen, arranged in bundles, which are immersed in the mucopolysaccharides of the vitreous. As regards the lens in tissue culture, we were able to demonstrate histochemically, in the normal epithelium. 1. The presence of glycoproteins in the lysosomal matrix. 2. The presence of a large number of lysosomes containing acid phosphatases. 3. The presence of glucose-6-phosphate dehydrogenase and lacto-dehydrogenase in the mitochondria.
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Fig. 10. Scanning microscopy. Tissue culture of a normal lens. t"lat aspect of the cells, which show very long cytoplasmic extensions (x 3000).
We compared the normal epithelium and the epithelium of senile cataracts with the help of seriated photographs and microcinematography. We may say that: 1. There is a deterioration of the cell tropism in the cultures of pathological epithelium, the movements of the cells becoming anarchic. 2. There is anisomorphism and anisometry not found in the cultures of normal epithelium. 3. There is a deterioration of the vacuolar system with a tendency to the formation of large vacuoles. This phenomenon is commonly observed in storage diseases and is associated with a deterioration of the lysosomal system. 4. A tendency is observed to form fibres in the most normal cells, but not in the vacuolised cells. The formation of fibres may be considered as a sign of cellular differentiation, whereas the formation of vacuoles is a sign of degeneration and of dedifferentiation. These phenomena ought to be found
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in the epithelium in situ, because they occur during the initial phase of the culture, that is to say, at the time when the cells are still in contact with their natural support, the capsule. 5. The large vacuoles indicate the presence of stored products and that storage is progressive. There is also, in senile cataract, an abnormal migration of cells toward places where they are not found normally, that is to say, at the level of the posterior capsule. Finally, there is a close relationship between the degree of clinical evolution of the cataract and the degree of cellular deterioration. The principal interest of this research is to demonstrate that the culture of normal or pathologic lens epithelium gives constant results, making a cytological study possible. The deteriorations of the lens epithelium in senile cataract continue to occur in tissue culture, with the consequence that we have thus an ideal experimental method of determining whether the cataract is a purely epithelial disease and of stating which cellular organelles are responsible for it. SUMMARY Cortical cataracts are essentially characterized by a cellular deterioration of the lens epithelium with hypertrophy of the vacuolar system, cytolysis and cell proliferation, as well as by an intercellular storage of foamy material and the presence of intracellular deposits. REFERENCI~S Francois J. Les cataractes cong6nitales. Ed. Masson, Paris, 1959. Francois J. & Goes F. L'ultrasonographie dans le diagnostic des affections oculaires. Possibilit6s et limitation de la technique. Bull. Soc. Belge Ophtal. 150:600-614 (1968). Francois, J. & Victoria-Troncoso V. Epithelium of the adult lens in tissue culture. In: Progress of lens biochemistry research, pp. 39-46. Doc. Ophthal. Proc. Series, Junk, The Hague, pp. 39-46, 1976. Hogan M.J., Alvarado A.B. & Weddell J.E. Histology of the human eye, Ed. W.B. Saunders, Philadelphia, 1971. Lucas D.R. Special cytology of the eye. In: Cells and tissues in culture. Methods, Biology and Physiology, Ed. E.N. Willmer, vol. II, Academic Press, London-N.Y., 1965. Nordmann J. Biologie du cristallin. Masson, Paris, 1954. Nordmann J. Acquisitions r~centes dans le domaine de la biologie du cristallin. Adv. OphthaL, Karger, Basel 59: I 47, (1962). Pirie A. and Van Heyningen R. Biochemistry of the eye. Blackwell, Oxford, 1956. Author's address: Ophthalmological Clinic University of Ghent Ghent, Belgium
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