[ 3 ] SELECTIVE AND HISTOCHEMICAL STAINING OF THE OTOLITHIC MEMBRANES, CUPULAE AND TECTORIAL MEMBRANE OF THE INNER EAR k By GEORGE B. WISLOCKI AND AARON J. LADMAN* Department of Anatomy, Harvard Medical School, Boston, Massachusetts t The present investigation reveals that the tectorial membrane, the gelatinous sub- stance of the otolithic membranes and the cupulae of the internal ear are stained intensely and selectively by the alum-haematoxylin element of Gomori's chrome alum-haematoxylin and phloxine stain and by his aldehyde fuchsin stain. They react strongly also with the periodic acid-Schiff technique following exposure to saliva. These reactions are similar to those shown by several other structures of the central nervous system, namely, the hypophysial Herring substance (Bargmann, 1949; Bargmann & Scharrer, 1951), the subcommissural organ and Reissner's fibre (Wislocki & Leduc, 1952 a, b; Bargmann & Schiebler, 1952) and the ocular ciliary zonula (Wislocki, 1952). Brief mention of the selective staining of the tectorial membrane (Wislocki, 1952) and of the otolithic membranes and cupulae has ap- peared elsewhere (Wislocki & Ladman, 1954). The present paper gives a more com- plete account of the staining of these otic structures by a number of histochemical methods and selective stains and discusses the possible significance of the results. MATERIAL AND METHODS The material consisted of two foetal mouse heads of 17 days of gestation, six heads of newborn mice and the temporal bones of two 34-month-old mice. It was supple- mented by two temporal bones obtained from a fresh human foetus of 17 cm. crown- rump length. The material was fixed according to the prescriptions of the different staining pro- cedures. The temporal bones of the 34-month-old mice were fixed in a saturated solution of mercuric chloride in 10 % formalin and decalcified in 5 % trichloracetic acid before sectioning them. The less densely calcified heads of the foetal specimens and newborn mice proved soft enough to section them without resort to a special decalcifying agent. The heads were embedded in paraffin and sectioned at 5p, either in the horizontal or the sagittal plane. The former proved more favourable for encompassing all the structures in question within a single section. Gomori's chrome alum-haematoxylin and phloxine method (1941), and his alde- hyde fuchsin stain counterstained with orange G and light green as modified by Halmi (1952), were applied to deparaffinized sections of material fixed in mercuric chloride and formalin, Rossman's fluid (a saturated solution of picric acid in absolute alcohol: 90 ml.; formaldehyde: 10 ml.), Bouin's fluid, Orth's fixative and Zenker's acetic acid fixative. * Research Fellow of the American Cancer Society, Inc., upon recommendation of the Com- mittee on Growth of the National Research Council, 1952-4. 1-2 4 George B. Wislocki and Aaron J. Ladman The periodic acid-Schiff method of McManus (1946) and Hotchkiss (1948) was applied to deparaffinized sections of the same blocks. Prior to staining them, the sections were placed in saliva for 2 or 3 hr. to remove glycogen. Other saliva-treated sections were stained with Schiff's reagent without previous oxidation with periodic acid. Pap's ammoniacal silver nitrate method for the demonstration offibrous reticulum, as modified by Mitchell & Wislocki (1944), and Weigert's resorcin-fuchsin stain for elastic tissue were applied to sections from the same blocks. Other deparaffinized sections of internal ears fixed in 4 % basic lead acetate or Zenker's acetic acid fixative were stained in either a 1 % aqueous solution of methy- lene blue or toluidin blue for the demonstration respectively of possible cytoplasmic basophilia and metachromasia. A series of sections of one of the labyrinths of the human foetus listed above was stained in solutions of methylene blue of graded pH as described by Dempsey, Bunting, Singer & Wislocki (1947). Sections of two heads of newborn mice fixed in a solution of 1 % trichloracetic acid in 80 % ethanol and in Zenker's acetic acid fluid were stained by the methods of Barrnett & Seligman (1952, 1954) for protein-bound sulph-hydryl and disulphide groups. OBSERVATIONS Staining with chrome alum-haematoxylin and aldehyde fuchsin. Similar staining results were obtained by both methods at the three stages of development of the mouse and in the human tectorial membrane. Consequently, it is unnecessary to represent each structure at every stage by both methods. PI. 1, fig. 1, illustrates a typical horizontal section through the internal ear of a newborn mouse, revealing a crista ampullaris, the utricle and saccule with their maculae, and a portion of the cochlea with the developing organ of Corti. The sec- tion was stained by the chrome alum-haematoxylin and phloxine method. The three regions of the section contained in rectangles coincide approximately with the areas of figs. 2 and 19, 3 and 14, and 6 and 18 in Pls. 1-3. Reference to PI. 3, figs. 14, 18 and 19, illustrates that the otolithic membrane of the macula of the utricle, the developing tectorial membrane and the cupula of a crista ampullaris are stained selectively blue by the alum-haematoxylin component of Gomori's stain. PI. 1, fig. 4, and PI. 3, fig. 15, of the macula sacculi of a 38-month-old mouse reveal a similar selective staining of the otolithic membrane by the aldehyde fuchsin stain. These representative illustrations will serve to show that the tectorial membrane, cupulae and otolithic membranes are selectively stained. Precisely what elements of these membranes are stained is not so easy to judge. The tectorial membrane is generally accepted as being composed of a fibrillar matrix and a jelly-like ground substance. The otolithic membrane is believed to consist of a layer of a gelatinous substance in the outer part of which there are numerous small bodies, the otoliths or otoconia, which are composed of a mixture of calcium car- bonate and a protein. The cupulae are described as consisting of a jelly-like mass of substance containing pores or canals into which the hairs of the sensory cells of the cristae project. The cupulae are also described under some conditions as possessing a finely striated texture, an appearance which, according to Kolmer (1927). may be Selective and histochemical staining 5 a fixation artifact of the colloids of which they are composed. From examination of PI. 3, figs. 14, 15, 18 and 19, it is apparent that no critical distinctions between the several components of these membranes can be made upon the basis of their staining by means of chrome alum-haematoxylin or aldehyde fuchsin. In the case of the otolithic membranes (PI. 3, figs. 14, 15) it could be the protein of the otoliths that is stained, but it is equally possible that the gelatinous matrix precipitated upon the otoliths could account for the staining. Fibrillar shreds of selectively stained material appear to extend from the otolithic membrane on to the flagellae and the outer surface of the cells of the macula. The same relation of stained material to cells is faintly seen in the case of the cupula and crista (PI. 3, fig. 19). In the developing organ of Corti, quite heavily stained, blue fibrils appear to connect the under surface of the tectorial membrane with the adjacent surface of Corti's organ, and thence bluish strands seem to penetrate between the columnar cells (PI. 3, fig. 18). On the other hand, the bluish fibrils visible quite generally in the subepithelial connective tissue of the labyrinth (PI. 3, figs. 14, 18 and 19) are ap- parently collagenous fibrils which are known to stain variably strongly with chrome alum-haematoxylin. The periodic acid-Schiffreaction. By this procedure the tectorial membrane (P1. 2, fig. 7), the otolithic membranes (PI. 3, fig. 16) and the cupulae (PI. 1, fig. 5; PI. 3, fig. 17) are intensely stained. Since this staining occurs in sections which were ex- posed to saliva, it cannot be attributed to the presence of glycogen. Here, as with the previous stains, a decision cannot be reached in regard to which components of the membranes-whether the gelatinous substance or the fibrillar component, or both-are specifically stained. It is also possible that the protein of the otoliths may be stained. A basement membrane upon which the epithelium of the maculae and cristae rests also stains intensely (PI. 3, figs. 16, 17). Similar staining is observed in the foetal and newborn mouse heads, as well as in the human tectorial membrane. Sections stained with Schiff's reagent without prior oxidation with periodic acid reveal mild staining of the tectorial membrane, but none of the otolithic membranes. Metachromasia and basophitia. For the investigation of these manifestations of staining, sections were used from ears fixed in 4 % basic lead acetate, which is re- commended for the preservation of acid mucopolysaccharides (Holmgren & Wil- ander, 1937; Wislocki, Bunting & Dempsey, 1947), or fixed in Zenker's acetic acid fluid. In sections of ears of newborn mice so fixed and then stained with toluidin blue, no metachromasia was observed in the tectorial and otolithic membranes or in the cupulae. In the ear of a 3+-month-old mouse which was decalcified in 5 % tri- chloracetic acid there was similarly no metachromasia of the structures in question, except in one section in which the staining was allowed to run overnight, when a somewhat reddish blue coloration developed. In all of the sections referred to, car- tilage matrix and the granules of mast cells showed intense metachromasia, attri- butable to their acid mucopolysaccharide content, thus serving as test-objects for comparison with the membranes under investigation. Basophilic staining, carried out on a series of sections stained in solutions of methylene blue of ascending pH (Dempsey et al.