Paper - The development of the eyelids of the albino rat, until the completion of disjunction (1921)

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Addison WHF. and How HW. The development of the eyelids of the albino rat, until the completion of disjunction. (1921) Amer. J Anat. 29: 1-32.

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This historic 1921 paper by Addison and How describes albino rat eyelid development.



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The Development of the Eyelids of the Albino Rat, Until the Completion of Disjunction

William H. F. Addison And Harold W. How

Anatomical Laboratory, University of Pennsylvania, and The Wistar Institute of

Anatomy

Three Text Figures And Two Plates (Thirteen Figures)


When the eyes are first formed no lids have yet appeared. Then, following the formation of the lids, there is a definite period when they are fused together by their epithelial margins. This period varies with the different mammalian species, but is more or less constant for the individuals of any one species. In all, the time of formation and fusion occurs during fetal life, while the subsequent separation or disjunction of the lids occurs, in some species, during a later stage of fetal development, and in others it does not occur until after birth. The time of disjunction is related, in general, to the state of development of the entire organism, and in particular to the development of the retina.


In the form we have studied, the albino rat, the gestation period averages twenty-two days, and it is during the eighteenth fetal day that the eyelids fuse. After birth the lids remain closed usually for fourteen to fifteen days, and sometimes for one to two days longer. In our stiidy, we have followed the histological changes involved in the fusion and disjunction of the lids, and have attempted to correlate the time period of this process with the development of related organs.


Material and Methods

For the histological study sections were prepared of the eye regions of fetuses from the seventeenth to the twenty-first days of gestation, and of young animals from birth to fifteen days, at one-day intervals. For stages up to one week old parafRn embedding was satisfactory, but after the appearance of the hairs double embedding in celloidin and paraffin gave better results. In preparing for embedding the material to show the later stages, especially from seven days onward, sometimes the lens was removed, and in some stages only the eyelids were embedded. For routine staining hematoxylin and eosin were used.

Seventeen-day fetus

At this stage there are protruding ridges in the position where the lids are about to develop, but one cannot say, as yet, that the lids have been formed. As a consequence, the eye is uncovered (fig. 1) and shows plainly on inspection. In sections across the eye region the ridges are seen, as shown in figure 2. When examined under the microscope, both the epithelial cells of the skin of the head and the epithelial cells covering the ridges above and below the eye are found to be two or three rows in thickness, but the epithelium at the margins of the ridges is thicker than the epithelium elsewhere. This is because the cells of the basal row have here a greater height than elsewhere, and are of a distinctly tall columnar shape, while the cells of the basal row in regions near by are of a lower columnar type. The cells of the superficial row or rows everywhere are seen to be flattened in form. Of course, at this stage growth is active everywhere and mitoses are abundant, but at the time under consideration the epithelium at the margin of the ridges has the appearance of being the site of relatively greater activity than in the skin near by. The margin of the ridge constitutes, in fact, an apical line of growth.

Eighteen-day fetus

Inspection of fetuses at this stage (fig. 3) shows that the eyelids have developed rapidly in the preceding twenty-four hours and now cover the eyeball completely. Examination of sections through the eye region (fig. 4) usually shows that the two lids have already fused together by their epithelial margins.

This was found to be the condition in sections of fetuses from three different litters, except in the case of two individuals. In one of these (fig. 5) the development of the Uds was less advanced and fusion had not everywhere taken place. The illustration is taken from the middle region of the eyelids, and laterally to this section the epithelial margins are in much closer approximation. Evidently in the albino rat, as in other species, the middle region of the lids is the last to completely fuse. As the figures show, the two lids are of somewhat different proportions, the upper being thicker and shorter and the lower more compressed and longer.

Microscopic examination of sections, from which figure 5 was taken, shows that the skin epithelium now has four or five layers of cells, the basal row being columnar in type, the remaining ones flattened. On the margin of the eyelid the epithelium is much thicker than elsewhere, and forms, in section, a conicalshaped projection about IOOaj in length. The basal row of cells is taller than on the neighboring skin and there are many more rows of superficial , cells. These superficial cells are rounded or polyhedral for the most part, arranged with their longer axes at right angles to the margin of the lids, and are stained lightly. On the external surface of the epithelial projection, however, the cells are more flattened and are darkly staining, as in the superficial cells of the epithelium of the skin.

In another series of sections, from a fetus about eighteen days of age, where the lids were in practically the same stage of development as in this, the part played by the epithelial portion of the eyelid in the primary closure is seen still more distinctly. In the middle of the lid the epithelial margins are still separated by a short space, but immediately adjacent to this region the epithelium forms a thin narrow connecting membrane 350^t in extent, between the mesenchymal portions of the two lids. The plane of section cuts the lids shghtly obliquely, but in this case, as in the preceding, the evidence is conclusive that the epithelium grows out actively beyond the mesenchymatous portions of the lid, to bridge the gap between the two approaching lids. After the primary union is established, the framework structures of the lids continue to grow and to approximate until the epithelium forms only a narrow band between the two lids. As the two lids come together, it appears that the greater part of the intervening epithelium is pressed aside. This is to be seen as little irregular groups of epithelial cells attached to the junction epithelium, either on the outer side or on the conjunctival side (figs. 4 and 6). In figure 4 the epithelium between the two lids measures 63^. Here the basal row of epithelium on each lid margin is again columnar, while the intervening cells are rounded and vesicular. The length of the band of epithelium measured in the direction of the line of fusion is about 90ac.

At this period, as before, the vibrissae buds of the face are distinct, but now for the first time other hair buds are beginning to develop in the skin, and occasionally one is found on the surface of the lid itself. The muscle fibers in the lid are plainly to be seen and the mesenchyme is distinctly more condensed on the conjunctival side of the lid.

Nineteen-day fetus

At this stage the proportions of the band of epithelial cells forming the junction have already become somewhat altered (fig. 6). By the growth of the opposing mesenchymatous structures the junction epithelium now measures only about 50/x from the basement membrane of one lid to that of the other. While in the direction from the conjunctival surface to the outer surface it has doubled in extent and measures about 180/x. This junction epithelium consists of a basal layer of columnar cells resting on the mesenchymatous portion of each lid, and three or four irregularly disposed rows of pale-staining, rounded, vesicular cells intervening. Both on the inner and outer sides of the junction epithelium are seen clumps of epithelial cells (fig. 6, ep) which have been pressed aside by the opposing growth of the two lids.

Twenty-day fetus

The cells of the epithelium on the outer surface of the lids are now arranged in three strata. The basal layer is columnar, deep-staining, and shows abundant mitoses. The intermediate layer is composed of one or two rows of pale, vesicular cells, while on the surface are two or three rows of flattened cells showing the beginning formation of keratohyalin granules. The junction epithelium consists of the same three varieties of cells, but arranged in the following manner. On the basement membrane of each lid margin is a layer of columnar epithelium, and, filling the intervening space between these two layers, are rounded cells of a pale-staining, vesicular appearance. While on the outer surface of the junction epithelium are two or three layers of flattened, dark-staining cells, containing keratohyalin granules, and continuous with the similar cells of the adjacent skin surface. The progress of the keratinization process in the superficial cells of the epithelial junction will constitute one of the chief features to observe in watching the changes leading up to the final disjunction of the lids.

At the outer margin of the lids are found buds of hair follicles, but as yet no distinct proliferation of epithelium to form the tarsal glands has appeared. The muscle tissue of the lids is now more apparent and the mesenchyme is becoming more differentiated, so that it has a denser texture immediately beneath the epithelium both of the skin and of the conjunctiva with a looser texture intervening. Examination with the binocular microscope shows that a few of the vibrissae of the snout region have already advanced through the epidermis and are now apparent on the outside of the skin.

Twenty-one day fetus

At this age the beginning of the tarsal glands is shown by the proliferations of epithelial cells at the inner angle of the margin of the eyelid (fig. 7, t). Thus the tarsal glands follow closely, in point of time, the appearance at the outer angle of the eyelid of the hair-follicle buds which are noted at the twenty-day stage. This was also the order of appearance observed by Ewetsky (79) in fetuses of domestic cattle. He saw the anlage of the cilia at the 6-cm. stage and of the tarsal glands at the 8.5-cm. stage.


The hair-follicle buds are increasing in number and are seen in various early stages of development. The oldest ones have a papilla at their basal ends, but none yet show the hair shaft within. Many of the vibrissae follicles, however, are completely differentiated and contain a well-defined hair shaft. Examination of the outside of the head region shows that many vibrissae are protruding above the surface of the skin and that some of them are nearly 1 mm. in length.

Measurements of the junction epithelium, both at this and the preceding stage, show that the adjoining parts of the lids have continued to grow and that there is an increase in the dimensions of the junction epithelium as compared with the nineteen-day stage. Measured from the basement membrane of one lid to that of the other, it was 54/i, while in the conjunctivaskin direction it was 240At.

Newborn rat

In the newborn rat (fig. 8) the epidermis shows a differentiation into stratum germinativum, stratum granulosum, and stratum corneum. The stratum germinativum is composed of a basal layer of cells with deeply staining nuclei and of several rows of paler staining cells. The stratum granulosum is very distinct, by reason of keratohyalin granules in the cytoplasm of the cells (fig. 8, k), while the stratum corneum is composed of flat scale-like cells. At the junction the epithelium consists of cells similar, for the most part, to those in the stratum germinativum, i.e., a basal layer of cells with dark staining nuclei on each basement membrane and polyhedral vesicular cells intervening. On the outer surface of the junction, however, the stratum granulosum and the stratum corneum of the surface epithelium continue uninterruptedly.

The dimensions of the junction epithelium are found to be less than those at the preceding stage. This is to be associated, probably, with the change of environment at birth. The length from the conjunctival surface to the outer limit of the stratum granulosum measures 180/x, while at the narrowest point between the two lids it measures 40/z.

Inspection of figure 8 shows that there is a slight indentation of the outer surface of the junction epitheUum, a feature which becomes progressively more marked at later stages. In the skin are more hair follicles than before. These are of various ages, some just beginning, others already differentiated and containing a hair shaft (fig. 8, h). But none of the hair shafts can be traced beyond the epidermis. The vibrissae on adjoining parts of the head are already well-developed structures, projecting as much as 3 mm. beyond the surface of the skin. The hair follicles of the outer angle of the lid, however, are mostly in a young condition, and only in the largest ones can be seen a developing shaft. The longest follicles measure 175^. The tarsal glands at the inner angle are still short, solid, flask-shaped epithelial buds, measuring about 70yu in length. The voluntary muscle fibers in the lids are growing in size and now have their nuclei peripherally placed.

Two-day rat

Considerable advance is seen in the structures within the lids as compared with the condition in the newborn. The hair follicles are more numerous and the older ones have increased both in diameter and in length. Many of the largest follicles contain a hair shaft, and an occasional one of these begins to protrude through the epidermal surface. Those in the lids nearest the junction are, however, not so far advanced as those farther away from the junction, and while some of the former show beginning differentiation of the hair shaft, they are not yet fully formed. Midway between the follicles at the external angle of the lid, and the tarsal glands at the internal angle are seen very small buds of hair follicles which are just beginning to develop from the junction epithelium.

The junction epithelium now measures 250m in length from the conjunctival surface to the outer epidermal surface, as compared with ISOfjL at birth. The diameter of the junction epithelium at its narrowest point is about 30^. The indentation on the outer surface of the junction epithelium is slightly more pronounced than before and both the stratum granulosum and the stratum corneum are a little thicker at that point than in the adjacent skin epidermis.

Three-day rat

Although sections show that hair follicles are now a prominent feature of the skin of the lid and that many of them contain definite hair shafts, very few of the latter have yet appeared above the surface of ths skin. Coincident with the differentiation of the hair shaft within the basal part of the follicle there is a keratinization process going on within the outer part of the follicle where it is continuous with the surface epithelium. This process results in the formation of a canal or outlet to the follicles, through which the developing hair shaft will grow. At this period the process is only partly completed in many foUicles.

The most evident change in the junction epithelium is the slight deepening of the outer groove. Its measurements are 250m in the conjunctival-epidermal length and about 40^ at its narrowest part between the two lids. The hair-follicles at the outer angle of the margin of the lids are in several stages of development. The longest, measuring 200 to 250ai, show a distinct papilla at the base and a growing shaft within the root sheaths. Near the mouth of the follicles are small oval outbuddings of epithehal cells to form sebaceous glands. Where these longest follicles are connected with the outer part of the junction epithelium, the cells in the line of future progression of the hair shaft have become more cornified and their nuclei are no longer apparent. These cells show the situation of the mouth of the follicle, and the stratum granulosum of the epidermis is seen to be dipping in to form the boundary of the mouth of the follicle. It is thus easy to be seen that the formation of the mouths of these follicles has a distinct effect upon the outer part of the junction epithelium in the way of tunnels being formed through it in preparation for the hair shafts growing out.

The tarsal glands now measure 175/^ in length, whereas at birth they measured only 70/jl.

Four-day rat

Hairs are beginning to show on the outside of the Hd epidermis as well as on the other parts of the head and body, but these are barely visible on casual inspection with the naked eye, and in consequence the pink color of the skin is very slightly affected by the white color of the sparse short hair. In sections, however, hairs are readily found projecting from the longest follicles (fig. 9). At the margin of the lids it is noticeable that the follicles are not as far advanced as in other parts of the Uds. In figure 9, h, are shown hair follicles still in an early stage of development from the junction epithelium. In some of the follicles near the margins of the lids, as at the three-day period, there is a beginning differentiation to form the shaft at the basal end, and there is a keratinization process going on in the outer ends of the follicles leading to their canalization. Where this cornification is seen there the stratum granulosum of the epidermis dips inward and the stratum corneum of the epidermis forms a narrow strand or plug where the outlet of the follicles will appear. As we follow the further development of the young hair folhcles, which are now attached to the junction epithelium (fig. 9, h), it will be seen that when they reach this stage of differentiation, just described for follicles on the epidermal surface, the expansion and separation of the lids will have brought their points of attachment to the free margins of the lids (figs. 12 and 13). So that when their hair shafts begin to form and to grow out, they will find a ready passage to the exterior. The junction epithelium is shghtly increased in its longest diameter, now measuring 270 to 288//, while the shortest distance between the two lids is practically the same as at the three-day stage, viz., 40^.

Five-day rat

With the continued growth of the lid margins a slight change in the form of the junction epithelium is now seen. In addition to the deepening groove on the outer stirface there is now distinctly seen a slight concavity of the conjunctival surface of the junction epithelium. The actual dimensions measured from the conjunctival concavity to the epidermal groove are, however, practically the same as at the four-day stage, viz., 270 to 288m by 40m. The conjunctival groove which is now first becoming definite is due to the expansion of the substance of the lid, while the junction epithelium remains stationary. This difference in the rate of growth is entirely responsible for the concavity on the conjunctival surface of the junction epithelium, and is partly responsible for the groove on the epidermal surface. In the latter case, the gradual keratinization of the epithelium is the other factor. This keratinization proceeds in the same manner as in ordinary surface epithelium by the preliminary formation of keratohyalin granules and final cornification of the cells, and has two loci. One is in the midline of the junction epithelium, forming a groove, and the other is in the mouths of the hair follicles attached to the junction epithelium when they are beginning to form hair shafts. The progress of the keratinization in these two loci combines to effect the major part of the disjunction process. The first, in the midline of the junction epithelium, is a continuous process, depending probably on the distance the surface epithelium is from the source of nourishment derived from the vessels of the tunica propria, and the second, in the marginal hair follicles, is associated with their arriving at a certain definite stage in differentiation. The resultant effect — the splitting of the epithelium — appears to be due to the same keratinization process manifesting itself jointly in these two situations. The time relations of the process in the two situations are closely interrelated, with the canalization process in the hair follicles usually slightly in advance. As a marginal hair follicle approaches the condition where it is about to become canalized, its point of attachment to the junction epithelium is found near the outer margin of the lid. In consequence, the line of canalization of the epithelium is usually through the outermost part of the junction epithelium and often ending in the outer groove. The presence of these little canals in the outer part of the junction epithelium seems to lead the way for the deepening of the outer groove, and they serve as numerous little independent centers from which the cornification process extends into the adjoining junction epithelium.

Six-day rat

At this stage the epithelial proliferation at the margins of the eyelids appears to have reached its maximum effect in the production of the junction epithelium. This is shown by the dimensions of the junction epitheHum, which now measures 288At X 40/i. From this time onward it will be seen that the actual measurement of the junction epithelium diminishes in its conjunctival-epidermal dimension, and, conversely, from the eighth day onward, the interpalpebral dimension increases. Henceforth, although epithelial proliferation and growth continue at the margins of the hds, no epithelium is added to the length of the midline of the junction epithelium. Since, however, the epidermal and follicular keratinization continues, the deepening of the epidermal groove continues, and the result is the beginning of the disjunction process.

Seven-day rat

The conjunctival-epidermal length of the junction epithelium is now slightly diminished as compared with that found at six days, while the interpalpebral length remains the same. At the outer angle of the lids are long hair follicles, some containing distinct hair shafts. The tarsal glands have grown greatly in length and measure 540^, but are still soHd structures. But between the long well-developed follicles at the outer angle and the tarsal glands at the inner angle are again seen small follicles in early stages of development growing from the junction epithelium. This area of the lid is evidently still growing and differentiating and is still at an earher stage of its developmental history than either the epidermal or conjunctival part of the lid. As the succeeding stages are studied, it will be seen that the disjunction process is concurrent with the further differentiation and growth of this part of the lid.

Eight-day rat

At this stage the process of separation of the hds may be regarded as definitely beginning, inasmuch as the form and measurements of the junction epithehum are distinctly altered (fig. 10). The shapes of the Hds themselves are also modified at their margins. Under low-power magnification, it is seen that the width of the lid nearest the junction is distinctly thinner than the rest of the lid and that each lid tapers to its narrowest point at the place of junction with the opposing lid. The form of the junction epithelium is altered chiefly by reason of the deepening of the outer groove. The measurements are now 21 Cm X 50 n, and if these be compared with the preceding stage, it is found that the conjunctival-epidermal length has decreased and the interpalpebral distance has increased. Thus it is seen that while the epithelial proliferation at the margins of the lids continues and new cells are being constantly added, the effect now is to thicken the marginal epithelium on each lid, and not to increase the conjunctival-epidermal dimension, as measured in its midline. There are still a few marginal fol icles attached to the junction epithelium which are solid cylinders of cells, and consequently have not advanced to the stage of canalization and do not contain hair shafts (fig. 10, h). The rest of the folHcles at the margins which are attached to the free epithelium are conspicuously long structures, containing hair shafts which project beyond the epidermis. It is noticeable that the cells of the midline of the junction epithelium are very pale staining, as compared with cells in a similar position in the preceding stage, and this change in appearance indicates that they are about to become keratinized.

Nine-day rat

The length of the junction epithelium in the direction of the line of fission is only about half what it was at eight days, now measuring 108-126/x (fig. 11). This difference is principally by reason of the increased depth of the epidermal groove. The interpalpebral distance at the narrowest region of the epithelium is practically the same as before, 50m. The ducts of the tarsal glands show a well-defined lumen in the region near where the duct epithelium is continuous with the junction epithelium, but they have as yet no outlet to the outside. The alveolar out-pouchings of the glands are becoming well developed and their glandular cells begin to show their characteristic pale-staining mature appearance (fig. 11, t). By this time all the immature hair follicles which were seen at seven days attached to the junction epithelium now show hair shafts beginning to grow out. In the interval since seven days, however, the epidermal groove has also deepened so that the outlet of the hair follicles now open into it, and the hair shafts grow through the epithelium of the outer margin of the lid, not into the junction epithelium. There is, in a way, a process of eversion of the growing margin of each lid, which goes on all through the stage of separation of the lids.

Ten-day rat

Both the inner conjunctival furrow and the outer epidermal groove have become deepened, and in consequence the junction epithelium as measured between the two depressions is now reduced to a thickness of 90m. In the epithelial cells lining the conjunctival furrow are now seen for the first time keratohyalin granules. These are in the form of small deep-staining particles, arranged in a linear fashion in the flattened, superficial, four or five rows of cells. The deepest layers of these cells at the bottom of the furrow come into a close relation with similar cells of the epidermal groove. The result is that keratohyalin granules are seen practically all through the midHne of the junction epithelium. This represents a definite stage of advance in the disjunction process, inasmuch as it shows active progress in the keratinization of these cells. The interpalpebral distance is about 75fJL at the narrowest region, and this is a considerable increase as compared with the nine-day measurement of 50;u. Although the lumina of the ducts of the tarsal glands are widely open even up to the marginal epithelium, no patent outlets were seen. The hair follicles at the margin of the lids have all attained a mature condition, containing hair shafts and having outlets through the surface epithelium (fig. 12, h).

At this ten-day stage some variation in the condition of the junction epithelium was noted in animals from different Utters. The description as given above was taken from the most advanced specimen and from a region of the lid about midway between the inner and outer canthi. In other specimens which were not quite so far advanced, the junction epithelium as measured from the inner to outer groove was greater in extent, and no keratohyalin granules were seen in the epithelial cells of the conjunctival furrow.

Eleven-day rat

Along the midline of the junction epithelium some of the cells, which contained the keratohyalin granules in the preceding stage, have now advanced to the stage of complete cornification. The connecting epithehal cells constitute a narrow eosin-staining band, bounded on each side by flattened cells, containing numerous keratohyalin granules. The granules are large and few in number within each of the cells of the one or two rows nearest the cornified zone and are smaller and more numerous in the cells of the succeeding two or three rows.

Twelve-day rat

The narrow keratinized band in the junction epithelium is becoming more distinct (fig. 13). This is the result of more of the cells containing keratohyalin granules becoming cornified. The dimension measured from conjunctival to epidermal side is lessened, and at the midpoint of the lid is about 75ij.. Laterally to the middle region of the Ud, this measurement is considerably greater. As the process of disjunction approaches an end, a shght but appreciable difference is seen in the appearance and dimensions of the keratinized band of epithelium in different parts of the same hd. At the middle of the eyelids the process of disjunction is more advanced, as compared with the condition laterally. This is to be correlated with the observation that the first opening between the two palpebral margins will appear about the median portion of the lids. From here the opening process continues toward both the inner and outer canthus, usually reaching the former first. Outlets of some of the tarsal glands were open at this time, while in other cases a plug of cornified epithelium still occluded the mouths of the ducts.


Fig. A Photograph of section through margins of lids of albino rat thirteen days old, showing the lids connected by only cornified epithelium, and hence nearly ready to separate. Hair follicles occupy the entire thickness of the lids, except for a narrow zone on the conjunctival side where the tarsal glands (t) are situated, x marks position of outlet of duct of tarsal gland. X 50.


Thirteen-day rat

The loosely arranged keratinized epithelium still connects the two lids (fig. A), but forms an extremely thin membrane. Its texture is less compact than at the preceding stage, its elements being curled and irregular and spread apart. The outlets of the tarsal glands are distinctly open. The hairs and hair follicles of the marginal region of the lids are large and numerous. The whole ajipearance suggests the completed eyelids.

Fourteen-day rat

In some animals the final separation takes place on this day; in others, on the fifteenth day, and in a relatively small number the process is delayed until the sixteenth or seventeenth day. As mentioned above, the first o])ening usually a]:)pears in the middle region of the lids and proceeds in both directions, but usually reaches the inner canthus first. For some hours after the margins have separated at the inner canthus the lateral fourth of the lids may still be fused. The time necessary for complete opening after the first slit appears is often twelve hours. The lids on the tw^o sides of the head of an animal do not necessarih' separate at the same time. One may be entirely open before the first slit appears in the other. According to the observations of Dr. H. D. King, it often happens that in the same litter the eyelids of the females open before the eyelids of the males (Donaldson, '15). This circumstance is in line with the general precocity of the female at this period, as compared with the male. It sometimes happens that the eyelids are only partially separated for days after the usual time, and this gives them the appearance of being 'small-eyed' animals. In such cases, according to Dr. J. M. Stotsenburg, of The Wistar Institute, the separation may be easily completed by gentle pressure upon the lids with the tips of one's fingers.

Structure of retina at time of fusion and of opening of lids

At the time of fusion of the lids in the rat, on the eighteenth fetal day, the retina shows an early stage of development. The cells derived from the inner layer of the optic cup still form one continuous layer, but arranged in two zones (fig. B). This is also the condition present in the human retina at the time of fusion of the lids (Bach and Seef elder, '14). Of the two strata, the outer one is composed of closely arranged deep-staining cells, and the inner one has its cells more palely staining and not so compactly arranged. No definitive visual cells are differentiated and no rods have yet developed.


Pigs. B and C Two drawings to show comparison of states of development of retina at time of formation of lids and at time of their opening. In both the internal margin of the retina is shown toward the top of the page. X 250.

Fig. B Retina of eighteen-day albino rat fetus at time of formation and fusion of lids, showing a very early stage of development and consisting principally of two broad zones of epithelial cells.

Fig. C Retina of fourteen-day albino rat at time of disjunction of lids, showing all the definitive layers of the functional retina. The proportions of the various layers, however, change with the further growth of the entire eyeball.


During the period of the rat's development, when the lids are joined together, the retina undergoes its histogenesis. By the time of opening of the lid all the layers of the mature retina are clearly delimited (fig. C), though subsequent growth changes alter their relative proportions somewhat.

Thus it is seen that there is a close correlation between the time of opening of the lids and the arrival of the retina at a functionally active condition. Though the mechanism by which the lids form and separate is entirely apart from the visual organ, yet the processes of development in the lids and in the retina are so coordinated that their definitive end-results are contemporaneous.


Discussion of Observations

In the albino rat two noticeable features in the formation of the eyelids are the late period in gestation at which the eyelids develop and the rapidity of their formation. The gestation period averages twenty-two days, and not until the eighteenth fetal day are the eyes covered by the lids (fig. 3). At the seventeenth fetal day the lids are represented only by ridges above and below the eyeball (fig. 2), but so rapidly does the process of formation proceed that in the space of a day the Hds have grown across the eye, and have met and fused by their epithelial margins (fig. 4). As the hds approach one another in their growth, the epithelium of the margin of the developing hds is the site of especially active proliferation. In consequence of this, it grows out noticeably beyond the mesenchymatous portions of the lids (fig. 5), and first bridges the space between the approximating hds as a thin epithelial membrane. As the mesenchymatous portions of the hds continue to grow toward each other, this thin epithelial membrane is pressed together and portions of it are pressed aside as groups of epithehal ceUs (figs. 4 and 6), which are soon lost.

After fusion occurs, the junction, epithelium continues to increase in the conjunctiva-skin dimension until about four days after birth. There is a temporary diminution in this measurement in the newborn, and this may be associated with the drying of the epidermis and the more rapid keratinization of the superficial epidermal layers of cells then beginning. After the fourth day the junction epithelium remains stationary until the sixth or seventh day. During this time the measurement is 270-288m. Then, due to the deepening of the epidermal groove, the length of this line of fusion becomes progressively less, and at the twelfth day is only 75m . On the thirteenth and fourteenth days only cornified epithelium connects the two lids in the region midway between the inner and outer canthi.

The measurements of the junction epithelium in the interpalpebral direction at the various stages are in a general way the reverse of those in the conjunctiva-skin direction. Immediately after fusion at eighteen days, the interpalpebral measurements is 63m, and as the mesenchymatous portions of the lids press together, this becomes less, so that at two days after birth it is only 30 to 38//. It remains about the same until the seventh day, and then gradually increases, so that at ten days it is 73^, and at twelve days 90m . As the two lids continue to separate through the cornification of the epithelium in the midhne of the junction epithehum, half of the epithelium belongs to each Hd, constituting its surface layer.

Thus during the period of attachment of the lids, there are three stages, as indicated by the measurements of the junction epithelium: 1) Increase in length of line of fusion; 2) stationary attachment; 3) gradual separation. The first stage lasts until four days after birth, the second until the sixth or seventh day, and the third until final disjunction ensues at fourteen to seventeen days.

The period of attachment of the hds is related in time with especially two other developmental processes, the maturing a) of the skin and b) of the retina.

Being specialized integumentary structures, the lids in their developmental history follow the same general course as the skin. Hair folhcles are developed both from the epithehum of the skin and from the junction epithehum. By following the development of the hair folhcles which grow from the latter situation, one gets a sure guide to the time when the separation process is going on. For, as has been shown already, by the time the hair shafts form in these folhcles, the sphtting of the junction epithelium has proceeded far enough for the hair shafts to grow out directly through the epidermis of the margin of the lid. It is found that new hair folhcles continue to arise from the conjoined epithelium for several days after most of the follicles have begun in adjoining parts of lid and head. As a result, one finds the hair shafts first showing in these youngest follicles several days later than in the follicles elsewhere. Now in the albino rat the ordinary hair follicles begin on the lids during the eighteenth, nineteenth, and twentieth days, and the largest of these first show shafts within at birth. These begin to protrude through the skin at two and three days, and by the end of a week after birth there is a complete covering of short hairs. In comparison with this, we find that hair folhcles not yet showing hair shafts within are found in connection with the conjoined epithelium as late as eight days after birth. At nine days these, too, begin to show differentiation of their internal cells, and by twelve days the hair shafts begin to appear above the epidermal surface. At this period a narrow band of keratinized epithelium has developed in the midline of the junction epithelium, and the final disjunction of the lids soon after occurs by the splitting of this keratinized epithelium. So the time of eruption of the youngest hairs at the margins of the lids is closely connected with the time of keratinization of the middle of the last remaining junction epithelium, which is the prelude to final disjunction.

From this survey it is seen that the period of time from union to disjunction of the lids may be correlated with a fairly definite period of development of the general integument of the body. Thus, the time of formation of the lids is just prior to the beginning of the epithelial proliferations which develop into hair follicles of the ordinary small hairs of the skin. One has to make an exception of the special large types of hairs, such as the vibrissae of rodents and the hairs of the eyebrows, upper lip, and chin of man. For in both rats and rabbits the vibrissae follicles make their appearance just before the time of formation of the eyehds. The above general statement holds true for man also, for while we find that both the lids and the hair folhcles of the ordinary lanugo have their beginning during the third month of fetal life, the former practically always antedate the latter. But an exception has to be made of the follicles of the larger types of hairs of the eyebrows, upper lip, and chin, which begin toward the end of the second month.

After the lids are fused they remain in this state until after the arrival of the skin at the stage of cornification of its superficial epithelial layers, the formation of the hair shafts, and their appearance above the surface of the skin. In the rat this is about seven days after birth and ends the stage of stationary attachment.

The final stage, that of gradual separation, is associated with the maturing of the hair folHcles which have originated from the junction epithelium. When the hair shafts of those which are nearest to the tarsal glands have erupted at about 12 days, the midHne of the persisting junction epithelium has become cornified, and often in two days more final separation occurs. The duration of this separation process in common with other developmental processes is widely different in different species. Thus, in the rat, where the animal attains a complete coat of hair at about a week after birth, the separation process is completed at the expiration of another week or ten days. But in man, where the lanugo appears during the fifth fetal month and completely invests the body during the sixth month, disjunction usually takes place at the end of the sixth (Contino, '07) or the beginning of the seventh fetal month (Ask, '08).

The period of attachment of the lids is related in time also with the maturing of the retina. Functionally, the lids are a protective part of the ocular apparatus, and their separation does not take place until the retina has passed through its histogenesis. Thus, in the rat, at the time of formation and fusion of the lids, the wall of the optic cup has a relatively simple structure, with its cells arranged in two broad bands. During the period of attachment of the lids differentiation proceeds, so that at the time of disjunction, at about fourteen days, all the layers of the mature retina are recognizable. The same holds true for man, where at the time of fusion of the lids, about seventy days (Contino, '07, and Ask, '08), the retina has a simple structure corresponding to that of the rat at the eighteenth fetal day (Bach and Seef elder, '14). Separation of the lids occurs at the end of the sixth month or beginning of the seventh, and by that time the retina has structurally all the elements and layers of the mature retina. After this time there is continued growth of the eyeball which alters the proportions of the several layers, but one can at least say that the retina at the time of disjunction of the lids is histologically mature.

The chief single factor in the separation of the lids is the keratinization process which appears in the surface cells of the epidermal side of the junction epithelium, as in epidermal cells everywhere (figs. 7 and 8). For a time the process does not manifest itself more actively here than elsewhere (fig. 9), but at a certain period, which we may associate with a definite stage in the maturing of the hair follicles of the skin, this process continues to progress inward in the midhne of the junction epithelium (figs. 10 to 13), until it reaches the conjunctival side.

The mechanism of cornification in the junction epitheHum is probably comparable to that taking place elsewhere over the integument. The history of the junction epithelium is, however, somewhat different from that of the ordinary epidermis, inasmuch as for some days after the lids fuse it becomes progressively thinner in the interpalpebral direction, then has a stationary thickness until the seventh day, and finally gradually increases. It is only when the last stage is reached that keratinization proceeds rapidly in the epidermal groove. This accounts for the delay in the process within the junction epithelium as compared with the general surface epidermis. Connected with these circumstances, causing delay, is also the fact that the margins of the lids are in a younger developmental condition than the remainder of the Hds. At least, one may infer this from the fact young stages of hair folHcles are still seen there when the adjoining follicles of lids and general skin show welldeveloped shafts. The increase in thickness in the junction epithelium goes slowly, probably because of the concurrent expansion of the marginal area of the lids. There is rapid proliferation of the epithelium, but many of the new-formed cells are necessary to keep up with the expanding marginal surface of the lid, and are distributed laterally. This is the condition up to six or seven days, by which time most of the hair follicles of the adjoining part of the lid are mature and their hair shafts are visible on the outside. As is well recognized, hair folhcles are the seat of numerous mitoses, and they are evidently now able to help supply the necessary cells for expansion and growth, each in its own local area. At any rate, it is at this time that the junction epithehum begins to increase in its interpalpebral dimension, and it is apparently this increase in the mass of epitheUal cells which removes the cells of the midline of the junction epithelium farther from the source of nourishment in the mesenchymatous portion of the lids, and so favors the progress of the keratinizing process.

The tarsal glands are also considered to take a part in the opening of the Hds, but they are found to participate to a comparatively shght degree in the albino rat. These glands have their beginning in the twenty-first fetal day, as epithelial proliferations from the conjoined epithelium, near the inner angle of the eyehd (fig. 7, t). At birth they measure 70m m length and at seven days have increased to 540m, but are still sohd throughout. At nine days the ducts show a distinct lumen, but it is not until the twelfth day that some of the ducts are found to have a direct opening to the outside, while most of the duct mouths are still obstructed by plugs of cornified epithelium. It is just before this last stage that the changes in the developing duct outlet may play a part in aiding the separation of the lids. For at this time the terminal part of each duct where it is continuous with the junction epithehum is still a sohd mass of cells, although the greater extent of the duct is canahzed. In the cells lining the duct immediately adjoining the unopened part some keratinization is going on, and as this process extends out to meet the similarly changing cells of the junction epithelium, the cornification of the conjunctival side of the raphe is accelerated. Schweiger-Seidel ('66), who was one of the first to study the process of lid separation in man, emphasized the formation of secretion in the sebaceous and tarsal glands as assisting in the opening of the hds, but this does not appear so important in the rat.

It is seen by comparing figures 7, 11, and 13 that the points ot attachment of the tarsal glands to the surface epithelium remain constant at the inner angle of the Ud margin. By this comparison one may judge that the inner groove in the junction epitheHum, which is so marked at late stages (figs. 12 and 13), is mostly the result of the expansion of the lid itself.

It is true that there is some formation of keratohyalin granules in the superficial cells of the groove, but the changes do not usually go on to complete cornification before the epidermal keratinizing process reaches them, and so can help but little in the separation of the lids.

Previous investigations of the developing eyelids have been made principally on human material, and of these the most complete are the studies of Contino ('07) and of Ask ('08). These contain many references to previous literature, beginning with Bonders ('58) who was apparently the first to picture a thin section across the fused lids of the human fetus.

The observations on the eyelids of mammals, aside from man, are comparatively few, and are for the most part incidental to other studies on the developing eye. Ewetsky ('79) found in fetuses of domestic cattle that the fusion of the lids took place in fetuses of 5.5 to 6 cm. long, and that they separated in fetuses of 40 to 47 cm. In his figures of the approximating lids he shows the epithelium on the margins of the lids, growing out as a ridge in advance of the mesenchymatous part of the lid, as is shown in our figure 5.

Seller ('90), who studied the development of the conjunctival sac, also examined the eyelids of young puppies at one, four, eight, and nine days after birth, as well as Talpa embryos. In the puppies he observed the formation of a broad cornified cell plate between the lids, leading to their separation.

Nussbaum ('08), in the course of his resume of the process in man, refers to having also examined mice of two stages — at two days and at ten days after birth. In the two-day animal the stratum corneum of the epidermis showed no depression over the junction epithelium, but at the ten-day stage the process of cornification had advanced into the lid fissure, forming a groove which was funnel shaped in cross-section.

Schweiger-Seidl's observations on the process of separation in human fetuses were long quoted, and the figure illustrating the fused lid margins, which was pubhshed in 1866, is still m use in text-books of embryology. He regarded as the chief agencies in the separation, 1) the keratinization within the hair folhcles developing from the conjoined epithelium and, 2) the formation of spaces between the cells of the junction epithelium by the secretion from the sebaceous and tarsal glands.

Contino ('07) studied a larger series of human fetuses, and considered that the separation of the corners of the hd margins was due to the secretion from the sebaceous and tarsal glands and that the separation of the intermarginal epithelium was due to the cornification of the central layers of cells.

Ask ('08), in addition to human fetuses, examined also a series of kittens, of which, however, he gives no description or figures, but says he found the conditions in both practically the same. He places the cornification process in the junction epithelium as the most important factor in separation. Cornification also proceeds from the hair folhcles into the junction epithelium, and, thirdly, there is an independent cornifying process in the posterior part of the junction epithelium.

In our study of a consecutive series of rats of known ages, both fetal and postnatal, we have made measurements of the junction epithelium, and thereby have been able to attempt an analysis of the growth stages of the junction epithehum, and we have also correlated the period of fusion of the hds with certain stages of development of the skin and hair and with the histogenesis of the retina.

Summary

The time of formation and fusion of the eyehds in the albino rat is a definite one, occurring during the eighteenth day of fetal hfe; hence observation of the presence or absence of the fused hds in a fetal rat will determine whether the fetus is older or younger than eighteen days.

The formation of the hds in this animal is a relatively rapid process; at the seventeenth day of fetal hfe there are only slight ridges indicating the beginning of the Hds, but in the succeeding twenty-four hours the Hds have grown over the eyeball and have fused by their epithelial margins.

As the lids approach each other the epithelium at the margin of each lid grows actively in advance of the mesenchymatous portion.

By measurements of the conjoined epithelium, three stages are observed during the period of attachment: 1) increase in length of fusion; 2) stationary attachment, and, 3) gradual separation. The first stage lasts until four days after birth, the second until the sixth or seventh day, and the third until final disjunction at fourteen to seventeen days.


The period from union to disjunction of the lids may be correlated with a fairly definite period of development of the skin and hair. Thus the time of formation of the lids is slightly in advance of the first appearance of hair follicles of the ordinary small hairs. Then the lids remain completely fused until after the arrival of the skin at the stage of cornification of its superficial epithelial layers, the formation of the hair shafts, and their appearance above the surface of the skin (about seven days after birth). Finally, the stage of gradual separation is associated with the maturing of the hair follicles, which have originated from the junction epithelium.


The chief factor in the splitting of the junction epitheUum during stage 3 is the process of keratinization, which continues inward from the surface epidermal cells of the raphe until it reaches the conjunctival side of the junction epithelium. During stage 3 the interpalpebral dimension of this junction epithelium gradually increases, and it is apparently this increase in the mass of epithelial cells which brings the cells of the midhneof the raphe farther and farther away from the source of nourishment, and so favors the progress of the keratinization process within them. The inception of increase in the interpalpebral dimension is correlated in time with the arrival of the skin of the lid at its mature condition, while the margins of the lid are still in a younger developmental condition. By the time the folhcles of the latter have developed hair shafts, the keratinization process has penetrated the entire thickness of the junction epitheUum, and disjunction soon follows.


The separation is also aided by the process of keratinization manifesting itself in, 1) the developing hair folhcles derived from the junction epithelium, and to a less degree, in, 2) the ducts of the tarsal glands, and, 3) the cells lining the conjunctival furrow, just prior to disjunction.


Functionally, the period of attachment of the lids is related to the maturing of the retina. At the time of formation and fusion of the lids the retina has a very simple structure, consisting principally of two broad bands of cells, while at the time of disjunction all the layers of the mature retina are recognizable.

Literature Cited

Ask, F. 1908 Ueber die Entwicklung der Lidrander, etc. Anat. Hefte, Bd. 36, S. 189-279.

Bach, L., und Seefelder, R. 1914 Atlas zur Entwicklungsgeschichte des menschlichen Auges. Wilhelm Engelmann, Leipzig and Berlin.

CoNTiNO, A. 1907 Ueber Bau und Entwickelung des Lidrandes beim Menschen. Arch. f. Ophthalmologie, Bd. 66, S. 505-577.

Donaldson, H. H. 1915 The rat. Data and reference tables for the albino rat (Mus norvegicus albinus) and the Norway rat (Mus norvegicus). Memoirs of The Wistar Institute of Anatomy and Biology, no. 6, Philadelphia.

EwETSKY, Th. 1879 Beitrage zur Entwickelungsgeschichte des Auges. Arch. f. Augenheilkunde, Bd. 8, S. 305-356.

NtrssBAUM, M. 1908 Entwickelungsgeschichte des menschlichen Auges, Graefe Samisch Handbuch der Gesammten Augenheilkunde, 2nd edition, vol. 2, part 1, pp. 52-53, Leipzig.

Schweiger-Seidl 1866 Ueber die Vorgange bei Losung der miteinander ver klebten Augenlider des Foetus. Virchow's Archiv. f. Path. Anat. u. Phys., Bd. 37, S. 228-230.

Seiler, H. 1890 Zur Entwickelung des Conjunctivalsackes. Archiv. f. Anat. u. Entwickelungsgeschichte, S. 236-249. (Anat. Abth. of Arch. f. Anat. u. Phys.)

Plates

Plate 1

Explanation Of Figures

1 Seventeen-day albino rat fetus, showing eye not yet covered by lids. Orth's fixation; photographed in 80 per cent alcohol. X 3.

2 Section across eye region of seventeen-day albino rat fetus, showing eyelids represented by ridges above and below. Wistar Institute collection, no. 15370. X 30.

3 Eighteen-day albino rat fetus, showing eye covered by lids. Fixation in 10 per cent formaldehyde solution; photographed in same. X 3.

4 Sections across eye region of eighteen-day albino rat fetus, showing lids covering eye and fused together by their epithelial margins. Wistar Institute Collection, no. 15371. X 30.

5 Section across eye region of another eighteen-day albino rat fetus, showing lids not yet in continuity, but their epithelial margins extending out as narrow ridges and closely approximated. The upper lid is thicker and shorter, while the lower is thinner and longer. X 30.


Plate 2

Explanation Of Figures


Drawings of series of sections across junction epithelium of eyelids of albino rats at various ages, from nineteen days fetal stage until twelve days after birth. In all, the skin surface of the lids is shown toward the top of the page. X 160.

6 Nineteen-day fetus. Eyelids fused together by their epithelial margins; (cp) groups of epithelial cells, which formed the projecting marginal ridges of epithelium in the approximating lids (fig. 5), and which have been turned aside by the lids pressing close together; (/t) beginning development of hair follicle at outer angle of lid.

7 Twenty-one-day fetus. Increased thickness of junction epithelium in conjunctiva-skin direction; hair follicles (h) developing at outer angle of lid and on skin surface; (t) epithelium at inner angle of lid beginning to proliferate to form tarsal gland.

8 At birth. Junction epithelium somewhat decreased in longest diameter; hair follicles (h) in different stages; (k) keratohyalin granules very distinct in cells at outer surface of junction epithelium.

9 Four-day. Junction epithelium increased in length; (h) hair follicle buds beginning from middle of junction epithelium; a hair shaft is seen projecting through skin of lid; granules (A:) of stratum granulosum very evident.

10 Eight-day. A distinct groove on outer surface where lids join; (/) cells of alveoli of tarsal glands are differentiating, but duct is still solid cord of cells; (h) young hair follicles developing from epithelium near outer angle of lid; (s) sebaceous glands of older hair follicles, which open on the skin surface.

11 Nine-day. The epidermal interpalpebral groove has deepened; {t) ducts of the tarsal glands are canalized.

12 Ten-day. A groove has appeared also on conjunctival side of junction epithelium; (/i) hair follicles and developing sebaceous glands; (t) alveoli of tarsal glands; keratohyalin granules are seen not only in cells of epidermal groove, but also in junction epithelium and in cells lining conjunctival groove.

13 Twelve-day. Area of epithelial junction is much lessened and shows keratohyalin granules throughout; point of attachment of ducts of tarsal glands (0 is now opposite last remaining conjoined epithelium; owing to this diminution of the junction epithelium, the hair follicles which developed from it now open upon the epidermal surface of the lid.



Cite this page: Hill, M.A. (2020, August 6) Embryology Paper - The development of the eyelids of the albino rat, until the completion of disjunction (1921). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_development_of_the_eyelids_of_the_albino_rat,_until_the_completion_of_disjunction_(1921)

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