Hearing Research, 52 (1991) 133-146 Q 1991 Elsevier Science Publishers B.V. 0378-5955/91/$03.50

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HEARES 01517

Expression of inte~~diate filament proteins in the mature inner ear of the rat and guinea pig

W. Kuijpers I, E.L.G.M. Tonnaer I, T.A. Peters ‘, F.C.S. Ramaekers 2 * Deportments of Otorhinolaryngologv ’ and Pathology ‘, University Hospital Nijmegen, Ngmegen, The Netherlunch

(Received 10 May 1990; accepted 22 September 1990)

The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, de&n, neurofilament proteins and glial fibrillary acidic protein

(GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins,

suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the

inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and

19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the co&ear and

vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neuro~l~ent proteins.

Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP.

The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates

a close similarity between the different epitopes.

Intermediate filament proteins: Rat; Guinea pig; Inner ear; Adult

IIItdUCtlO~

All eukaryotic vertebrate cells contain an in- tracellular network of protein filaments, consisting of microfilaments, intermediate filaments and mi- crotubules. The intermediate filaments, which can be distinguished by their diameter (7-11 run) from both ~crofil~ents (6 nm) and microtubules (20-25 nm), have received much attention during recent years because of their remarkable tissue specificity (Moll et al., 1982; Ramaekers et al., 1983a).

It has become clear from immunohistochemical and biochemical studies that several different

Correspondence to: W. Kuijpers, Department of Gtorbinolaryn- gology, University of Nijmegen, Ph. van Leydenlaan 15, 6500 HB Nijmegen, The Netherlands.

* Present address. Department of Molecular Cell Biology, University of Limburg, Maastricht, The Netherlands.

classes can be distinguished, most of them distrib- uted in a tissue-specific manner. In general cyto- keratin filament proteins are found in cells of epithelial origin and differentiation, while vimen- tin occurs in mesenchymal-derived cells, desmin in muscle cells, neurofilament proteins in neurons and glial fibrillary acidic protein filaments in the several types of glial cells. (Osbom, 1984; Traub, 1985). Nuclear lamins A, B and C, which are also considered intermediate filament proteins, make up the nuclear lamina in most adult cells (Aebi et al., 1986).

Cytokeratins (CKs), are members of a complex family of intermediate filament proteins that con- sist of at least 19 distinct polypeptides in man.

According to the catalogue composed by Moll et al. (1982), they are numbered 1-19. Cytokeratin 1 has the highest molecular weight (68 kDa) and the highest isoelectric pH, while cytokeratin 19 has the lowest molecular weight of 40 kDa and a low isoelectric pH. From the use of monoclonal

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antibodies, which have been prepared to several of the individual cytokeratin polypeptides it has be- come apparent that the basic pattern of expression is consistent with certain rules by which cytokera- tins polypeptides are co-expressed and co-assem- bled in pairs of acidic and basic constituents. Furthermore, only a limited number of cytokera- tins are expressed in a given epithelial cell type, while specific patterns are characteristic of a par- ticular type of epithelial differentiation.

Moreover, the expression has been found to depend on various extrinsic factors, the functional state of the cell and the pathological conditions (Franke et al., 1981; Tseng et al., 1982; Quinlan et

al., 1985; Sun et al., 1985). The membranous inner ear is composed partly

of highly specialized epithelia which are involved

in signal processing and homeostasis of the endo- lymph. As all these epithelia are derived from the epithelial lining of the otocyst, it would be inter- esting to examine cytokeratin expression patterns in the finally differentiated structures. This can possibly add a new dimension to our insight into

inner ear development and functional cytology. So far, only scarce data are available on the

expression of intermediate filament proteins in the

inner ear. In addition to studies on the developing inner ear (Ann&o et al., 1986, 1987, 7989a,b,c; Raphael et al., 1987; Wikstrom et al., 1988) which show contradictory results, reports on the expres- sion of intermediate filament proteins in the ma- ture inner ear have only appeared incidentally. Schrott et al. (1988) reported on the presence of cytokeratins in the epithelial lining of the cochlear duct of the mouse in paraffin-embedded speci- mens using a polyclonal antibody. Expression of CK 18 and 19 in frozen sections of dissected specimens of the epithelial lining of the cochlear duct in guinea pigs was established by Raphael et al. (1987). In addition, some studies reported the presence of vimentin-type intermediate filaments in distinct parts of the epithelium of the adult inner ear, but the presented data are not uniform (Kasper et al., 1987; Raphael et al., 1987; Schulte and Adams, 1989). One of the main diffic~~es hampering the immunocytochernical demonstra- tion of intermediate filament proteins in the intact

inner ear is the fact that routine fixation and decalcification which are necessary for the pre-

servation of the delicate structures, very often lead to a loss of antigenicity of the intermediate fila- ment constituents.

In a previous study we established that the antigenicity of intermediate filament proteins in the inner ear of two-day-old rats was well pre-

served in cryosections of unfixed specimens which had been decalcified in a medium containing eth- ylene diamino tetra acetic acid (EDTA) and poly-

vinylpyrrolidone for a maximum period of two days (Tonnaer et al., 1990). This method was employed in the present study on the epithelial lining of the mature inner ear of the rat and guinea pig. A large panel of mainly monoclonal antibodies, directed against individual cytokera- tins, neurofilament protein, desmin and glial fibrillary acidic protein (GFAP) was applied in combination with immunohistochemical tech- niques.

Methods

Tissue preparation Throughout this study, Wistar rats (aged be-

tween 15 and 25 days) and young albino guinea pigs (aged 2 days) were used. At these ages the inner ear of both animals has reached morphologi-

cal and functional maturity (Bosher and Warren, 1971; Schmidt and Fernandez, 1963). A total number of 15 rats and 5 guinea pigs were used for immunocytochemistry. The animals were killed by an intracardial injection of Nembutal and there- after decapitated. The inner ear was carefully dis- sected from the skull and quickly transferred to a decalcification solution containing 10% EDTA and 7.5% pol~nylpyrrolidone in 0.1 M Tris-HCl buffer (pH 7.2) (Jonsson et al., 1986; Kasper et al., 1987) at 4°C for two days.

After rinsing in the same solution without

EDTA for 4 h, the specimens were frozen in liquid nitrogen and cryosectioned. Sections of 7 pm were placed on poly-L-lysine coated slides, dried in a cold air stream and stored at - 70” C until re- quired.

Antibodies One polyclonal and 10 monoclonal cytokeratin

antibodies were used in this study. In addition, 1 monoclonal neurofilament antibody, 1 mono-

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TABLE I SPECIFICITY OF THE RELEVANT INTERMEDIATE FILAMENT ANTIBODIES APPLIED IN THIS STUDY

Antibody Antigen protein recognized

Dilution References

pKer RCK 102 RCK 103 ’ RCK 105 RGE 53 CK 18-2 LP2K RD 301 RNF402 pGFAP

Several CKs CKs5+8

CK7 CK 18 CK18 CK 19 desmin 200 kDa neurofilament protein Gtial fibriIIary acidic protein

1:40 undil 1:2

1:3 1:6 undil undil undil undil 1:lOOo

(Ramaekers et al., 1983a) (Ramaekers et al., 1987a) (Ramaekers et al., 1987b) (Ramaekers et al., 1987b) (Ramaekers et al., 1983b) (Ramaekers et al., 1987a) (Lane et al., 1985) (Ramaekers et al., 1987a) (Ramaekers, unpubl.) (Ramaekers et aI., 1983a)

’ not yet fully character&d.

clonal desmin and 1 polyclonal GFAP antibody were applied. The specificity of the relevant anti- bodies is summarized in Table I and has been described previously (see Table I). In addition, the following antibodies directed against cytokeratin polypeptides specific of stratified epithelia were used: lC7 (CKl3), 6BlO (CK4), LLO02 (CK14), RKSE60 (CKlO) (van Muijen et al., 1986; Ramaekers et al., 1987a; Purkis et al., 1990). Al- though these antibodies have mainly been tested on human tissues and the nomenclature used, refers to that of human cytokeratins, similar specificity has been shown in various rat tissues (Herman et al., 1985; Verhagen et al., 1988; Ramaekers et al., 1989) and guinea pig tissues (van den Molengraft et al., 1986).

immunohistochemistty For immunohistochemistry, the frozen sections

were fixed briefly in cold aceton (5 min), rinsed in phosphate buffered saline (PBS) and incubated for 45 min with one of the various antibodies, either as undiluted culture supematant or in an ap- propriate dilution in PBS (Table I).

The sections were subsequently washed in PBS and incubated with horse-radish peroxidase con- jugated rabbit anti-mouse i~unoglobu~n (or swine anti-rabbit for the polyclonal antibodies pK.er and GFAP) diluted in PBS (1: 40) for 30 min. After washing in PBS and Na-acetate buffer (pH 4.9) for 5 min, peroxidase activity was de- tected using a mixture composed of 0.02% 3- amino-9’-ethyl carbazole, 5% dimethylformamide

and 0.01% hydrogenperoxide in Na-acetate buffer (pH 4.9). The sections were counterstained with Mayers’ haemalum and mounted in glycerine jelly. As a control, the primary antibody was omitted.

Apart from frequent distortion in the organ of Corti, especially in the older rats, the quality of the remaining cochlear and vestibular structures was usually good or satisfactory. To improve the morphology without loss of immunoreactivity, various fixation procedures were tested. It was established that only the antibody RCK102 was found to resist short fixation in a fixative modified after McLean and Nakane (1974). For this pur- pose, fresh specimens were fixed for 2 h at 4°C in 0.04 M phosphate buffer (pH 6.2) containing 0.1 M lysine-HCl, 0.01 M metaperiodate and 2% paraformaldehyde, prior to storage in the decalci- fication solution.

Comparison of sections from EDTA-treated specimens of the co&ear duct epithe~um to freshly dissected specimens, revealed that EDTA has no disadvantageous effect on the detectability of intermediate filament proteins; it even showed an enhancing effect as has previously been found in the inner ear of two-day-old rats (Tonnaer et al., 1990).

R4?SUltS

Immunohistochemical staining of the epithelial lining of the endolymphatic space of the rat inner ear using antibodies directed against different cy- tokeratin polypeptides, displayed a large dif-

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Fig. 1. Diagrams of immunohistochemical staining of rat cochlear duct with various cytokeratin antibodies; (A) CKl%2; (B) RGE53; (C) RCK103; (D) RCKlOS. n Strong staining: q moderate staining; q weak staining.

ference in staining pattern of the various cell types. The results obtained for both cochlear and vestibular parts are summarized in Table II. In addition the most characteristic cytokeratin pro- files observed in the cochlea with different anti- bodies are presented diagrammatically in Fig. 1.

With pKer, RCK102, and CK18-2 and LP2K (Fig. 2) all epithelial non-sensory cells of the cochlear duct revealed a positive reaction although staining intensity varied between the different an- tibodies (Table II). With these antibodies a pro- nounced reaction was observed in the stria vas- cularis, in the area of the marginal cells (Figs. 2

and 3). The heavy staining of the marginal cells in

the fresh 7 pm sections with most antibodies seri- ously hinders the demonstration of the exclusive staining of these cells in the micrographs. To show this more clearly a micrograph obtained from a 4 pm section which had been taken from tissue fixed in the periodate-lysine-formaldehyde fixative and stained with the antibody RCKlO2 has been included in Fig. 3. In the lateral wall also the

spiral prominence and external sulcus cells with their marked basal cell projections penetrating

deeply into the spiral ligament revealed distinct staining with these four antibodies (Figs. 2 and 3).

With respect to the epithelial lining of the tympanal wall a strong to moderate staining was

Fig. 2. Immunohistochemical staining of rat cochlear duct with various cytokeratin antibodies. (a) Survey showing staining of all non-sensory cells with the antibody LP2K. (b) Organ of Corti, after previous fixation in periodate-lysine-paraformaldehyde, demonstrating distinct staining of the inner border cells and the basal and apical parts (reticular lamina) of pillar and Deiters’ cells

with the antibody RCKIOZ, whereas the sensory cells do not react. The faint staining areas in the sensory cells represent the counterstained (haemalum) nuclei.

established with all four reagents in the internal sulcus cells, the cells of Claudius and the interde- ntal cells (Table II; Figs. 1, 2 and 4).

In the area of the organ of Corti, staining with the four broadly reacting antibodies was limited to the nonsensory cells: supporting cells of the inner hair cells, pillar cells, cells of Deiter and cells of Hensen (Table II, Fig. 1). Staining was especially marked in the basal parts of the cells and the reticular lamina (Fig. 2). No cytokeratin expres- sion could be established in the sensory cells (Figs. 1, 2). With the antibodies RGE53, RCK103 and RCKlO5, the epithelial lining of the cochlear duct revealed a more heterogeneous staining (Table II,

137

Fig. 1). With RGE53 only, the stria vascularis revealed positive staining, while with RCKlOS, staining was limited to a few transitional cells between the stria vascularis and Reissnefs mem- brane and the stria vascularis and spiral promi- nence. With the antibody RCK 103, these cells were also positive, while the remaining epithelium, except for the stria vascularis and sensory cells, revealed a weak positive reaction (Table II, Figs. 1, 3). Surprisingly, apart from the staining of epithelial cells RCK103 showed distinct staining of a small population of ganglion cells in the spiral ganglion as well as weak staining of the nerve fibers (Fig. 5). No reaction was observed in

TABLE II EXPRESSION OF INTERMEDIATE FILAMENT PROTEINS IN THE MATURE INNER EAR OF THE RAT

Antibodies pKer RCK RCK RCK CK RGE LP2K RNF

Protein several 102 103 ’ 105 18-2 53 19 402

recogn. CK’S CK’S CK CK CK CK Neuro- s+g 7 18 18 fil.

Co&l. portion:

Reissn. membr. ++ ++ f ++ ++

Stria vast.:

Marginal c. ++ ++ -2 -2 ++ ++ ++

Spir. prom. ++ ++ jr + ++

Ext. suicus ++ ++ f + ++

Claudius. c. ++ ++ If: + ++

Organ of Corti:

supp. c. 4-4. ++ f + ++

Sens. c.

Int. sulcus + + f + +

Interd. c. + + f + +

Nerve fibers - * ++

Spir. gangl. +3 - +4

Perineur. tissue - - -

Mesothehum Vestib. portion:

Flat epith. ++ ++ +s +5 ++ t5 ++ Cuboidal cells ++ ++ ++ ++

Dark cells ++ ++ ++ ++ +6 :

Crista epith. ++ ++ * + f ++ Macula epith. ++ ++ * + f ++ Nerve fibers - f ++

Vestib. gangl. - f3 - f4 Perineur. tissue - -

Mesothehum -

+ + strong staining; + moderate staining; f weak staining; - no staining. ’ This antibody is not yet fully characterized, ’ Only positive staining in transitional cells between stria vascularis and spiral

prominence and Reissner’s membrane. 3 Only staining of distinct population of ganglion cells. ‘Strong and weak staining

population of ganglion cells. 5 Heterogeneous staining. 6 Apical staining.

Fig. 3. immunohistochemical staining of the lateral wall of the co&ear duct of the rat with the cytokeratin antibodies: RCKlO2 (a); LPZK (b); CK 18-2 (c); RGE 53 (d); RCK103 (e); RCKIOS (f) Detail of stria vascularis, after previous fixation in periodate-lysine-

paraformaldehyde, stained with LP2K showing exclusive staining of marginal cells (g).

the so-called mesothelial lining of the per- ilymphatic space with any of the cytokeratin anti- bodies applied.

Fig. 4. Immunohistochemical staining of rat interdental cells with the cytokeratin antibody LP2K.

With the neurofilament antibody RNF 402, the ganglion cells of the spiral ganglion and the nerve fibers up to the base of the sensory cells, were positive. The ganglion revealed the presence of a small population of strongly stained and a large population of weakly stained cells (Fig. 5).

Generally, the staining patterns of the inter- mediate filament antibodies applied to the cochlear duct epithelium of the guinea pig, did not differ fundamentally from these observed in the rat. In the stria vascularis staining was most pronounced

Fig. 5. Immunohistochemical staining of neurons of the rat inner ear. (a) Spiral ganglion with neurofilament antibody RNF 402. (b) Spiral ganglion with cytokeratin antibody RCK103. (c) Vestibular ganglion with neurofilament antibody RNF402. Note the

differential staining of the ganglion cells and the numerous staining fibers at the base of the inner hair cells.

at the apical surface, while in contrast to the rat, the antibody LP2K did not stain the stria vas- cularis (Fig. 6).

In the vestibular partition of the rat, the whole epithelial lining of both the otolithic and ampullar organs, including the semicircular canals displayed strong positive homogeneous staining with the an- tibodies pKer, RCKlO2 and CK18-2. In the cris- tae and maculae, staining was found to extend between the basal lamina and lamina reticularis into the area of the supporting cells and was most pronounced at the base and on the apical surface (Table II, Figs. 7 and 8). Both sensory and non- sensory areas were also found to react strongly with the antibody LPZK. However LPZK failed to react in distinct areas of the non-sensory epi- thelium adjacent to the sensory areas, composed

of cuboidal/columnar cells, whereas the dark cell epithelium only stained apically (Figs. 7 and 8).

Both cell populations revealed very heavy stain- ing with the antibodies pKer, RCK102 and CK18- 2 and also with RGE53, but RCK103 and RCK105 showed no reaction (Table II, Figs. 7 and 8).

The remaining part of the non-sensory epi- thelium, including the semicircular canals, re- vealed heterogeneous staining patterns with the antibodies RGE53, RCK103 and RCK105, which only partly coincided. Weak staining of the sensory areas with the antibodies RGE53 and RCK103 was mainly seen at the apical surface, but there was no reaction with RCK105 (Table II, Figs. 7 and 8). No cytokeratin expression was found in the mesothelial lining of the perilymphatic space (Fig. 9).

Fig. 6. I~uno~st~h iemical staining of the lateral wall of the cochlear duct of the guinea pig with various q (a) RCKf02; (b) LF2K; (c) CK 18-2; d: RGE53; (e) RCK103; (f) RCKlOS.

rtokeratin antibodies.

In the different compartments of the vestibular partition of the guinea pig inner ear, the staining profiles obtained with the various antibodies di- rected against cytokeratins did not fundamentally differ from those observed in the rat. However, with the antibody LP2K staining was not only absent in the cuboidal columnar ceils adjacent to the sensory areas but also in the dark cells. Some typical examples are shown in Figs. 8 and 10.

The nerve fibers of the different vestibular nerves of both species displayed positive staining with the neurofilament antibody RNF402, reveal- ing the characteristic calyx-formed nerve endings at the base of the sensory cells (Fig. 11). The vestibular ganglion showed the presence of weakly and strongly stained ganglion cells (Fig. 5).

In both animal species no reaction could be established with the cytokeratin antibodies lC7, 6B10, RKSE60 and LLO02, or with the antibodies directed against GFAP or desrnin.

Discussion

The present study illustrates the complex ex- pression patterns of cytokeratin filament proteins in the epithelial lining of the mature inner ear.

The results obtained are similar to what is seen for cytokeratin expression in other simple epi- thelia, which are characterized by the presence of cytokeratins 7, 8, 18, and 19 (Sun et al., 1985; Tseng et al., 1982; Quinlan et al., 1985; Franke et al., 1981). No expression of cytokeratins 4 (6BlO), 13 (lC7), 14 (LLO02) and 10 (RKSE60), character- istic of stratified epithelia, was established. In contrast to the observations made by Ann&o et al. (1987, 1989~) in the developing human inner ear no cytokeratin expression was found in the extra- cellular structures, like tectorial membrane and gelatinous maculae. The resemblance of the ex- pression patterns observed in the rat and guinea pig inner ears, indicates a close similarity of the different antigenic epitopes.

The different types of epithelial cells in both the cochlear and vestibular portions, originate from the primitive ectoderm of the otic vesicle. Despite this common origin their cytokeratin pro- files reveal distinct regional differences which seem to be related to the specialized functions of the cells involved. With regard to the non-sensory epithelium of the cochlear duct, the marginal cells of the stria vascularis behave differently from the remaining epithelium. As the other types of non-

Fig. 7. I~uno~~h~~ staining of the epithelial lining of the ampulla of the rat with various cytokeratin antibodies: (a) RCK102; (b) CKlS-2; (c) LP21(; (d): Hae~toxy~~n stained section to identify the cu~i~/~i~~ cells and the dark celi

area (dc); (e) RGES3; (f) RCK103; (g) RCKlOS; (h) detail of(c)-

sensory cells, these cells show staining with the monoclonal antibodies RCKlO2 (CKs 5 + 8), CK18-2 (CK18) and LIQK (CK19), but the stria vascularis is the only structure which reacts with RGE53 (CKB), and where staining with RCK 103 is absent. Staining with RCK105 (CK 7) is lacking in all co&ear cell types, except for a small population of cells at the margins of the stria vascularis, also reacting with RCK103 which could not be further defined. The cytokeratin profile in

the various types of epithelial cells lining the co&Lear duct of the guinea pig was very similar to that observed in the rat, although in this species expression of CK19 (LP2K) was absent in the stria vascularis. The expression of CKs 18 and 19 appeared to be consistent with the observations made by Raphael et al (1987) on whole mount specimens of the adult coeblear duct, although they did not mention the absence of CK19 expres- sion in the stria vascularis.

Fig. 8. I~uno~st~he~~ staining of the epithelial lining of the utricle of the rat (a-f) and guinea pig (g,h) with various

cytokeratin antibodies. (a) RCK 102; (b): CK18-2; (c) LPZK; (d) RGE53; (e) RCK103; (f) RCKlOS; (g) RCKlOZ; (h) LPZK. (i)

Detail of utricular macula of the rat stained with the cytokeratin antibody RCK102, showing distinct staining of supporting cells. (j) Detail of (c) showing absence of staining with LPZK in the cuboidal/columnar cells.

Although both the monoclonal antibodies The existence of such a masking phenomenon has RGE53 and CKlS-2 detect CK18 epitopes, they recently been described by Franke et al. (198’7) displayed different staining patterns. This dif- and Schaafsma et al. (1989). ference can most likely be ascribed to structural The vestibular partition exhibits a comparable differences in the configuration of the two differ- differential cytokeratin staining pattern in the ent epitopes recognized in cytokeratin 18 by these non-sensory epithelium of both species as ob- antibodies in the separate parts of the epithelium. served in the cochlear partition.

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Fig. 9. Immunohistochemical staining of the epithelial lining of the ampulla of the rat with the polyclonal cytokeratin antibody pKer. Note the absence of cytokeratin expression in the

mesothelial layer c: crista.

Comparison of the different cell types in the cochlear and vestibular portions reveals that in the guinea pig the cytokeratin profile of the vestibular dark cells and the population of cuboidal/ columnar cells adjacent to the sensory areas, is similar to that observed in the stria vascularis. A comparable conformity was found in the rat, ex- cept for a partly different expression of cytokera- tin 19 (LP2K) which is absent in the cuboidal/ columnar cells. In common with the marginal cells of the stria vascularis, these cells have a secretory function. The dark cells with morphological fea- tures of the marginal cells are assumed to contrib- ute to the maintenance of the peculiar cation contribution of the endolymph in the vestibular portion as is also the case with the stria vascularis in the cochlear duct (Kimura, 1969; Kuijpers, 1969). The cuboidal/columnar cells bordering the sensory areas and only present in the vestibular

Fig. 10. Immunohistochemical staining of guinea pig crista and adjacent epitbelium (cuboidal/columnar cells and dark 41s) with various cytokeratin antibodies. (a) RCKlOZ; (b) RGE53; (c) Ll??K; (d) RCK103; (e) RCKlOS.

Fig. 11. Immunobistochemical staining of vcstibular sensory epitheiium with neurofilament antibody RNF402, showing calyx-shaped nerve endings. (a) Guinea pig crista; (b) Rat utricular macula.

part have been shown to be involved in the secre- tion of macromolecules (Manni and Kuijpers, 1987). The remaining part of the vestibular non- sensory epithelium, which can be considered as the analogue of Reissner’s membrane in the co&ear duct, showed a more complex cytokeratin profile than Reissner’s membrane. Although both epithelia displayed a homogeneous reaction with the antibodies RCK102, LP2K and CK18-2, only Reissner’s membrane stained homogeneously with RCK103, while the vestibular epithelium revealed heterogeneous staining with RCK103, RCK105 and RGE53, indicating a more complex nature.

A very conspicious finding is the virtual ab- sence of cytokeratins in the sensory cells of the organ of Corti; these cells also failed to stain with the antibody directed against neurofilaments.

A comparable lack of staining of the sensory cells has been reported by Raphael et al (1987) in the mature organ of Corti in the guinea pig using antibodies directed against cytokeratins 18 and 19 and an antibody directed against ne~o~l~ents. In addition, these authors failed to demonstrate the presence of intermediate filaments in these cells electronmicroscopicalIy. This is quite a re- markable observation, because these cells develop together with the non-sensory epithelium from the same primitive ectoderm of the otic placode.

In the vestibular sensory organs the close prox- imity of the sensory and supporting cells interferes with a final decision on the absence of cytokeratin filament proteins in the sensory cells. However, the observation that the cytokeratin profile exactly mimics the anatomical position of the supporting cells-extending from basement membrane to apex-, seems highly suggestive for the existence of a comparable condition as observed in the sensory cells of the organ of Corti. The cytokeratin pro- files of the supporting cells in the vestibular sensory organs and in the organ of Corti appeared to be similar, except for the staining of the vestib- ular supporting cells with RGE53.

The present data on the CK-expression in the sensory cells are at variance with the observations made in the developing inner ear of mouse and man (Anniko et al., 1989b,c). These authors re- ported CK-expression in both human cochlear and vestibular sensory cells, although the profiles differed, while in mouse only the vestibular hair cells were positive. However, in a recent report Arnold and Anniko (1989) failed to find cyto- keratin expression in the sensory cells of the adult human organ of Corti.

The absence of cytokeratin pol~eptides in sensory cells of a comparable origin has only been observed in the sensory cells of the olfactory epi-

145

thelium. The sensory cells in this epithelium de- velop from the ectodermal nasal placode and dif- ferentiate into bipolar neuronal cells, which also lack neurofilament (Vollrath et al., 1985).

With respect to the so-called mesothelial lining of the perilymphatic space, no cytokeratin expres- sion could be detected in these cells. This in fact confirms its origin from the embryonic mesoderm and is in line with immunohistochemical studies on the immature human inner ear (Anniko et al., 1987).

Staining with the neurofilament antibody RNF 402 revealed the presence of different populations of ganglion cells, both in the spiral ganglion and in the vestibular ganglion. These findings are con- sistent with comparable immunohistochemical studies on the ganglion of the mature rat inner ear (Romand et al., 1988; Hafidi and Romand, 1989) and the developing human inner ear (Anniko et al., 1987). The presence of different populations of ganglion cells was also evident from staining reac- tions with RCK103. This antibody has not yet been fully characterized, but the present observa- tions, showing both staining of cytokeratins and neural elements, agree with earlier reports (Feitz et al., 1986; Verhagen et al., 1988). The differen- tial staining of the inner ear ganglia supports morphological observations which demonstrate the presence of different populations of ganglion cells (Rosenbluth 1962; Spassova, 1982; Schwartz, 1986).

Summarizing, we can conclude that monoclonal antibodies to individual intermediate filament cy- tokeratin and neurofilament proteins are valuable markers for the detection and charaterization of the different cell populations in the inner ear. They add a new dimension to our knowledge of inner ear cytology and may be of help in the study of inner ear development.

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