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=The Cytological Processes Involved In The Formation Of The Scalae Of The Internal Ear= 

James O. Foley  

Departments of Anatomy, Tulane Iniversity and The Unioerstty of Alabama  

Two Plates (Twenty-Three figures)   

==Introduction==

Recent experimental work on the development of the auditory apparatus has given an impetus to the study of the formation of the internal ear. One of the most interesting approaches to this problem is that of Fell (’28) in which normal parts of the fowl ear were allowed to develop in vitro, using explants of the otocyst. As a. result of this work, Fell is of the opinion that any possible influences exercised by the nervous system, vascular system, or associated organs may be eliminated. This would seem to indicate that the influences which control the development of the various parts of the internal ear emanate alone from the ectodermal anlage.


However, before positive conclusions can be reached from experimental work of this kind, all the normal developmental processes must be thoroughly understood. Among the most interesting and the least comprehended portions of the ear are the periotic spaces. Very little work has been done on the detail of the process of origin of these cavities. The only pertinent article with which the writer is familiar is that of Streeter (’17) dealing with the development of the scalae of the human embryo. He found that these spaces which are hollow and lined with flat epithelium in the adult develop from compact mesenchyma by an enlargement and coalescence of existing mesenchymal spaces. As so aptly pointed out by this author, the formation of these normal spaces or scalae is not a capricious phenomenon, but is a precise and orderly process of development. Although Streeter was not primarily concerned with the cytological features of their development, he is of the opinion that, in the human embryo, the enlargement and coalescence of spaces during the formation of the scalae is essentially a process of retraction and rearrangement of the mesenchymal elements and that this tissue is not lost in the process, but actively participates in it.


The present investigation was undertaken in the hope of throwing more light on the nature of these processes and particularly of ascertaining whether or not the mesenchymal syncytium is used up in the formation of the scalae. The writer is indebted to Doctor Hardesty not only for suggesting this problem, but also for his most generous assistance.


==Materials and Methods==

Some forty embryos and fetuses ranging from 2 to 14 cm. (crown-rump) were sectioned for study. The adult cochlea was also used for making measurements. In some cases the whole embryo or fetus was fixed after the brain had been removed from the cranium. In such specimens measurements were made following fixation. « In other instances measurements were made first; after which the head was sectioned sagittally, the brain removed from the cranium, and the two halves of the head were submerged in Held’s fluid. This latter method gave slightly better fixation.


Both the parafiin and celloidin methods of embedding were employed. The results obtained by the use of the paraffin method were practically worthless because of the shrinkage; accordingly, this method was discarded in favor of the celloidin technique. In those fetuses in which the cartilaginous (prebony) capsule was present better preparations were obtained by leaving some of the tissue around the capsule, for the pressure that was sufficient to clear away all the pericapsular tissue seemed to injure the softer internal tissues. A dissection laying bare the cochlea previous to embedding was not attempted in specimens under 5 cm. DEVELOPMENT OF AUDITORY SCALAE 3  Considerable difficulty was experienced in securing a satis— factory cytological stain. After much experimenting with various formulae, the following technique was developed. Celloidin sections 13 to 15 u in thickness were mordanted from three to six hours in a modified Benda’s solution. This was prepared by taking 25 cc. of Benda’s mordant (Lee, ’28) and adding 70 cc. of 95 per cent alcohol. Following mordanting, the sections were washed thoroughly in 70 per cent alcohol and stained for twelve to twenty—four hours in a 0.5 per cent solution of hematoxylin in 70 per cent alcohol. The only object in using alcoholic solutions was to avoid unnecessary injury from passage of sections through the low grades of alcohol to Water and then back through the higher alcohols. After the stained sections were differentiated in the mordant,  thoroughly washed in 70 per cent alcohol, and passed into‘  95 per cent alcohol, they were counterstained in a 0.5 per cent solution of erythrosin in beechwood creosote. Then the excess erythrosin was removed by rinsing in pure creosote and the sections were passed into oil of cedar wood, from which they were mounted in balsam. This technique gives an excellent differentiation of nuclear and cytoplasmic constituents. The celloidin is not colored by the mordant and the erythrosin persists as a permanent and brilliant dye in the syncytial cytoplasm, the finer processes of which are very delicate and usually invisible with other techniques.

==Order of Appearance of the Periotic Tissue Spaces==

In the human embryo (Streeter, ’17) the periotic cistern of the vestibule develops first, then the scala tympani, and finally the scala vestibuli. But in the pig embryo, as observed in these studies, the scala vestibuli develops before the scala tympani. The first space to appear, however, is the vestibular cistern, which is just visible in 4-cm. embryos. By the 4.5—cm. stage it is well developed. Thus the process of space formation, beginning in the vestibule, progresses from vestibule to cochlea and in the cochlea from base to apex. 

In 6-cm. fetuses the first indication of the scala vestibuli may be seen in axial sections of the basal whorl of the cochlea. Almost immediately afterward, the scala tympani becomes visible (fig. 18). As space formation progresses from base to apex, the scala vestibuli of a given whorl always keeps definitely in advance of the corresponding tympani (see fig. 21, an axial section of an 8.5-cm. fetus).

==The Cytology of the Developing Scalae==

Fetuses of 8.5 cm. are most favorable for this study. In specimens of this length the scalae have not appeared in the most apical whorl. Coils basal to it show all gradations in their development. The scalae begin their formation on the sides of the spiral ganglion. In these localities the undifferentiated periotic reticulum is composed in the early stages of close-meshed, delicate strands of cytoplasm (figs. 1, 22). The first indication of space formation, as also reported by Streeter for the human embryo, is an enlargement of the meshes of the reticulum (A, fig. 2). This is followed by attenuation, parting, and retraction of the delicate mesenchymal strands. At this early period the retra.ction of the strands is the only observable feature. But as development proceeds a new process appears which is destined to become more and more conspicuous. This is the detachment and subsequent degeneration of individual mesenchymal cells. The cytoplasm of such cells undergoes granular degeneration and the nucleus usually undergoes karyolysis. In some instances retrograde changes in the nucleus are of a pycnotic character (fig. 10). There is the possibility that the retraction phenomenon, associated with liquefaction, is in itself the initial step in degeneration, as suggested by the recently shown moving pictures of rat sarcoma, in which degeneration following irradiation is preceded by the withdrawal and rearrangement of the cytoplasmic processes}  ‘ In considering the part that liquefaction plays in this process, it is instructive to recall the work of Thomson (’19) on the formation of the fluid of the human graafian follicle. He asserts that it is formed by disintegration and liquefaction (plasmorrhexis) of the granulosa cells, liquefaction being preceded by granula once the spaces have developed to the size of those shown in B, figure 2, evidence of degeneration is of frequent occurrence. Two or more such spaces then unite to form a larger one, following degeneration of a portion of the limiting wall. Cellular degeneration is characterized, first, by granulation of the cytoplasm (figs. 4, 9, 14, 19, 20, 23) and then by karyolysis (figs. 4, 5, 15, 17, 23). The latter is often preceded by a  massing of the chromatin to one side of the nucleus (figs. 4, 14, 17 ).


In the later phases of space formation the process of‘  degeneration is characterized by the appearance of cells that have been interpreted as connective—tissue macrophages (figs. 6, 7, 11, 12, 23). The cytoplasm of these cells is highly vacuolated and often contains one or more ingested, degenerating nuclei (figs. 7, 12). figures 6 and 11 show cells of this type which are devoid of ingested nuclei, but which contain a granular vacuolated cytoplasm. The evidence thus indicates that the products of degeneration, other than those which may undergo liquefaction, are removed by phagocytes.


As development proceeds, larger and larger spaces coalesce, following granular degeneration of the syncytial connecting strands, until the scalae are completed. For a while stiff, blunt granular processes may be seen projecting into the spaces (fig. 16). These later undergo degeneration (figs. 5, 17). As the process continues a limiting Wall may undergo degeneration at more than one locus, resulting in the detachment of a portion of the reticulum. Most of these fragments immediately degenerate, but occasionally they round up into plasmodia (fig. 13), the ultimate fate of which has not been  tion of the cytoplasm. In a similar way disintegration of the cytoplasm of the periotic mesenchyma presumably results in accumulation of fluid and withdrawal of_connecting strands. The fact that the macrophages contain very little granular material in their vacuoles suggests that much of the degenerating cytoplasm undergoes liquefaction. Once the accumulation of fluid is started, further enlargement of the spaces might proceed by pressure atrophy as well as by plasmorrhexis. Somewhat more problematical is the extent to which infiltration of lymph from surrounding tissues may aid in the enlargement of the periotic spaces. Certainly it is difficult to understand how such infiltration, if it occurs at all, could initiate the process of space formation  determined. Presumably these masses degenerate with liquefaction or are ingested by phagocytes.


The epithelium which eventually forms the outer limit of the scalae is derived from the cells separating the most peripheral spaces from the unmodified mesenchyma. Before the cells are transformed into the epithelium they are characterized by rounded nuclei and an open-meshed cytoplasm which exhibits processes projecting into the space (similar to those of B, fig. 2). With the enlargement of the scalae the nuclei of the border cells become elongated and flattened and the connecting cytoplasmic strands are pressed back into a continuous border which is smooth on the side next to the cavity (fig. 8). Apparently the epithelium thus formed is similar to the mesenchymal epithelium that lines the cavities of joints and bursae.

==Discussion==


From a consideration of the above facts it is evident that the chief problem in the development of the scalae is the cause of the initial changes in the periotic mesenchyma. Since it was suggested that the early separations of cytoplasmic processes might be due to mechanical stresses in the growth of the whole cochlea, careful measurements of sections of this organ were made throughout the entire period of development. The figures obtained from fourteen specimens, so measured, showed that at the 6- and 7-cm. stages—the period in which the initial formation of the scalae takes place—— there was less growth in the main basal and axial planes of the cochlea than at any subsequent or preceding period. Although the relatively small number of specimens used is insufiicient for statistical study, the preliminary findings suggest that growth stresses play an insignificant role in the early formation of the periotic spaces.


The most significant finding in this investigation, therefore, seems to be the occurrence of degeneration at the site of the formation of the scalae and the simultaneous appearance of connective-tissue macrophages. Such-participation of macrophages in the removal of degenerating tissues is of common occurrence in even younger stages of vertebrate embryos. Boyden (’22, ’18) was among the first to point out their role in the removal of vestiges of gill filaments of chick embryos, and the modeling and attrition of such areas as the anal plate, caudal intestine, and cloaca. The difficult  thing in these instances, as in the periotic spaces, is to deter mine what causes the initial degeneration of epithelial or mesenchymal tissues in well-vascularized portions of the embryo.


This consideration, together with the results obtained by recent experimental work, suggests that the factors that control the development of the mesodermal components of the inner ear reside in the otic vesicle itself. Ogawa (’26) ha.s shown that the cartilaginous capsule of the ear in amphibians does not form following removal of the otic vesicle. filatow (’27) states that following transplanting of the otic vesicle of amphibians to regions of undifferentiated mesenchyma the otic vesicle stimulates the mesenchymal cells to the formation of a new capsule. The results of Fell’s work on the development of the isolated otocyst in vitro have already been mentioned. Altogether, these various findings would therefore seem to indicate that some influence, emanating from the otic vesicle, controls the development of the various parts of the inner ear and presumably initiates the formation of the periotic spaces.

==Summary==

Development of the scalae in pig embryos begins in the basal turn of the cochlea and progresses toward the apex. Contrary to the conditions in the human embryo, the scala vestibuli begins in advance of the scala tympani, and continues to maintain its lead.


Cytologically, the first step in the formation of the scalae is an enlargement of the mesenchymal spaces. Cavities, thus initiated, coalesce to form still larger ones. The initial process seems to consist of liquefaction accompanying attenuation, separation, and retraction of the mesenchymal strands, but this is soon followed by visible degeneration of these strands and the removal of granular débris by connectivetissue macrophages. The scalae thus enlarge through destruction of the mesenchyma, and not merely by a rearrangement of that tissue.


Measurements of the growing cochlea show that during the period in which the initial formation of the scalae takes place there is less growth in the main axial and basal planes than at any subsequent or preceding period. Therefore, it is believed that mechanical growth stresses play an insignificant, if any, part in the early formation of the periotic spaces. Indeed, the evidence obtained by other authors from experimental removal of the otocyst suggests that the degenerative changes that occur at the site of the periotic spaces and that initiate the formation of the scalae—as described in this article—arise in response to factors which reside in the membranous labyrinth.

==Literature Cited==

BOYDEN, E. A. 1918 Vestigial gill filaments in chick embryos, with a note on similar structures in reptiles. Am. Jour. Anat., vol. 23, no. 1. 1922 

The development of the cloaca. in birds with special reference to the origin of the bursa of Fabricius, the formation of a urodaeal sinus, and the regular occurrence of a cloacal fenestra. Am. J our. Anat., vol. 30, no. 2. 

FELL, H. B. 1928 The development in vitro of the isolated otocyst of the embryonic fowl. Arch. exp. Zellforsch., Bd. 7, Heft 1. 

fiLATOVV, D. 1927 Aktivierung des Mesenchymes durch eine Ohrblase und einen Fremdkorper bei Amphibien. Zeitschr. Wiss. Biol. Abt. d. Roux’ Arch. Entwicklungsmech. Org., Bd. 10, Heft 1. 

LEE, A. B. 1928 Microtomist’s vade-mecum.‘ P. B1akiston’s Son & Co., Philadelphia. 

OGAWA, C. 1928 Einigc experimente zur Entwicklungsmechanik der Amphibianhtirbléischen. Folia. Anat. Jap., Bd. 4, Heft 6. 

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THoMsoN, A. 1919 The ripe human graafian follicle, together with some suggestions as to its mode of rupture. Jour. of Anat., vol. 54, pt. 1.

==Plates==

All the figures of plate 1 were drawn at table level with a camera lucida. The figures of plate 2 are photomicrographs of selected areas from the developing scalae of 8.5-cm. pig fetuses. 


===Plate 1===

1 Area of undifferentiated mesenchyma.


2 An early stage in the development of a scale. Note the cytoplasmic portion of a macrophage in space E. The nucleus was not sectioned.


3 Cytoplasmic degeneration of a portion of the periotic reticulum. The nucleus i as yet apparently unaffected.


4 A portion of the wall of a space,‘ showing cytoplasmic degeneration and beginning nuclear dissolution. Note the massing of the chromatin to one side of the nucleus. The nuclear membrane also appears to be thinner in this region.


5 A projecting portion of the reticulum, showing cytoplasmic and nuclear degeneration. One nucleus is apparently normal, but with no surrounding cytoplasm, whereas the other is undergoing disintegration. In this mass- are many isolated, stiff-appearing portions of cytoplasm which have resisted degeneration. Not all of the granular material shown is the product of cytoplasmic granulation; much of it is coagulum of the periotic fluid. .


6 and 7 Drawings of two macrophages. figure 7 shows a macrophage which has ingested two nuc1ei——one, pycnotic and enclosed in a vacuole; the "other, staining very lightly.


8 A portion of the wall of a scala which is almost completed, illustrating the character of the epithelium.


===Plate 2===


9 Granular (legener:x.tion of the cytoplasm.


10 A portion of the syncytium undergoing (legeneration by pycnosis and gra.11u1a.tio11 of the cytoplasm.


11 and 12 I’l1otomic1'ogI'z1pl1s of Inzlcropllages shown in figures 6 and 7 of plate 1.


13 A syncytial mass which beealne detached during the coalescence of spaces and later rounded up.


14 and 15 l\'uelei unalergoiilg dissolution. Attention is directed to the massing of the chromatin in figure 14.


16 A portion of the peripheral wall of a nearly completed scala. Note that only local areas in the wall have broken down, leaving processes of the syncytium projecting into the seala.


17 Nuclei undergoing (lissolution. Note rupture of one of the nuclear men1br:n1es.


18 A11 apical coil which shows the sczrla. vestihuli to he more :1(lv:1.11ee1l in its (lovelopment than the soulii tympani.


19 and 20 Granular degeneration of the cytoplasm.


21 Section of the cochlea of an 8.5—(:m. fetus, showing the stages in the fornrution of the sczllae.


22 An area of undiffentiated I!1CSellCllyIIl2l, in a region of scale formation.


23 An area 1"'1'o1n a developing scula, showing degeneration of cytoplasm and nuclei. Note four degenerating nuclei. At the lower left-hand corner :1 xnzreropllagc may be observed.

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