Intermediate filament proteins in the inner ear

INTERMEDIATE FILAMENT PROTEINS IN THE INNER EAR

Wim Kuijpers and Frans C.S. Ramaekers

I. 11. III. IV.

General Introduction Anatomy of the Inner Ear Immunohistochemistry of the Inner Ear: Technical Aspects Expressionof Intermediate Filament Proteins in the Inner Ear A. Man B. Rat C. Guinea Pig D. Gerbil E. Mouse F. Discussion V. Expression oflFP in the Stato-acoustic Nerve A. Neurofilaments B. Vimentin C. Discussion References

The Cytoskeleton, Volume 3, pages 159-183. Copyright © 1996 by JA! Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-689-4 159

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I. GENERAL INTRODUCTION Among the different components of the cytoskeleton, much attention has been focused on the intermediate filament proteins (IFP) because of their cell-lineagespecific pattern of expression. They can be subdivided into six groups (Ramaekers et al., 1992). Type I and II IFP contain the cytokeratins (CKs), which are generally specific for epithelia. Type III IFP consists of vimentin which is specific for mesenchymal cells, desmin which is specific for muscle cells, glial fibrillary acidic protein (GFAP) which is expressed in glial cells and astrocytes, and peripherin, which is present in peripheral neurons. Type IV are the neurofilament proteins expressed in nerve cells. Type V, the lamins, constitute the fibrillary network of the nuclear lamina and type VI IFP comprise nestin, which is specific for CNS stem cells. The largest group of IFP is the CKs. They are distributed in specific combinations depending on the type of epithelium. For example, simple epithelial cells express CKs 7, 8, 18, and 19. Mixed epithelia express CKs 5, 14, and 17 in their basal cell layers and CKs 8 and 18 in their luminal cells. Stratified epithelia express CKs 5 and 14 in their basal cell layer. In non-comifying epithelia CKs 4 and 13 are expressed in the suprabasal cell layers, whereas comifying epithelia express CKs 1, 2, 10, and 11. Hyperproliferative epithelia express CKs 6 and 16. Antibodies to IFP are widely used in immunohistochemical studies on tissue differentiation and tissue development. This study deals with the expression of IFP in the membranous inner ear. The cytoarchitecture of this organ shows a remarkable multitude of morphologically and functionally different epithelial cell types. These partly highly specialized epithelia, involved in signal processing and homeostasis of endolymph, are derived from the uniform epithelium of the otocyst. Because of their tissue specific and developmentally regulated expression, IFP are excellent candidates in the study of the relationship between the different tissues which originate from the otocyst. In the first part of this chapter, a brief introduction is given on the complicated anatomy and morphology of the membranous inner ear and on the difficulties encountered in the immunohistochemical approach to this organ. The second part deals with the expression patterns of IFP in the inner ear of various mammalian species.

II. ANATOMY OF THE INNER EAR The membranous inner ear consists of a series of interconnected compartments (Figure I a), which can be divided into (1) a cochlear part, comprising the cochlear duct concerned with the perception of sound, and (2) a vestibular part, consisting of the otolithic organs (saccule and utricle) and the ampullar organs concerned with the function of equilibrium and the endolymphatic sac (Bast and Anson, 1949). These compartments are lined by a large variety of structurally and functionally different cell types and are filled with endolymph. Endolymph is a peculiar extracellular fluid with an intracellular cation composition (Johnstone et al., 1963;

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Figure h Diagrams showing the various compartments of the membranous inner ear (A), the sensory areas of the cochlear duct (organ of CortI) (C) and of the vestibular compartments (D) and micrograph of the cochlear duct of the rat showing various types of epithelial cells (B). C: semicircular canals; CO: cochlear duct; S: saccule, SE: endolymphatic sac; U: utricle. The black areas represent the sensory epithelium. 1. Outer hair cells; 2. Inner hair cells; 3. Cells of Hensen; 4. Cells of Deiters; 5. Pillar cells; 6. Inner border cells; 7. Nerve fibers; 8. Tectorial membrane; 9. Mesothelium; 10. Vestibular sensory cells; 11. Vestibular supporting cells, c: Cells of Claudius; e: Endolymph; id: Interdental cells; is: Inner sulcus; o: Organ of Corti; os: outer sulcus; p: Perilymph; r: Reissner's membrane; s: Stria vascularis. Bar in B indicates 40 jum.

Bosher and Warren, 1971). All these compartments contain an area of neurosensory epithelium (black areas in Figure la), that is, (1) the organ of Corti in the cochlear duct (Figures la and lb), (2) the maculae sacculi and utriculi (Figure la) in the saccule and utricle, respectively, and (3) the cristae ampullares in the ampullar organs (Figure la). The areas of sensory epithelium comprise two types of cells: sensory cells and supporting cells. The sensory cells transduce the appropriate energy into neural activity w^hile the supporting cells provide structural support for the sensory cells. The organ of Corti shows a fairly complicated construction with inner and outer

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hair cells and various types of supporting cell as shown in Figure Ic. In the sensory areas, concerned with the function of equilibrium, type I and type II hair cells and supporting cells are closely packed together (Figure Id). The epithelium outside the sensory areas consists of different types of epithelial cells, some of which have been shown to be actively involved in the maintenance of the intracellular cation composition of the endolymph. Various structures play a prominent role in this process, such as the stria vascularis (Kuijpers, 1969; Kuijpers and Bonting, 1969) which is situated in the lateral wall of the cochlear duct (Figure lb), the so-called dark cells (Kimura, 1969; Kuijpers, 1969) situated in the vestibular duct system and presumably also the endolymphatic sac (Figure la) (Lundquist, 1965; Kimura, 1976; Rask-Andersen et al., 1981; Friberg et al., 1985; Manni and Kuijpers, 1987). The stria vascularis is composed of three cell layers: (I) a layer of epithelial (marginal) cells which border the endolymphatic space, (2) a layer of intermediate cells, and (3) a layer of basal cells (Rodrigues-Echandia and Burgos, 1965). The basal cell wall of the marginal cells shows extensive infoldings which interdigitate with the cytoplasmic processes of the intermediate cells. These two cell layers are separated from the underlying stroma by the basal cells. The dark cells reveal some features of the marginal cells (Kimura, 1969), but there are no intermediate and basal cells in the dark cell area. The endolymphatic sac is lined by different types of epithelial cells which differ in their content of cytoplasmic organelles and infoldings of the basolateral cell membrane (Lundquist, 1965; Bagger-Sjoback et al., 1986). The epithelium of the membranous inner ear is derived from the otic placode. This patch of ectoderm flanks the rhombencephalon of the early embryo and thickens and invaginates to form the otic vesicle. The spherical otic vesicle develops into a series of interconnected compartments in the cochlear and vestibular partitions, because of differential growth (Bast and Anson, 1949). In the adult, the membranous inner ear is separated from the surrounding bone by a fluid-filled periotic labyrinth, referred to as the perilymphatic space (Figures I a and lb), which is lined by so-called mesothelium, which originates from the embryonic mesoderm.

III. IMMUNOHISTOCHEMISTRY OF THE INNER EAR: TECHNICAL ASPECTS The results of many studies have shown that no uniform fixation and processing protocol can be followed if cells and tissues are to be examined by means of immunohistochemical staining procedures (Curran and Gregory, 1980; Altmannsberger et al., 1981). Depending on the nature and structure of the antigens, appropriate protocols are required to prevent alteration, destruction, or masking of the antigenic epitopes. Many monoclonal antibodies and polyclonal antisera to the different IFF subclasses do not cross-react with paraffin embedded tissues fixed in formaldehyde, due to masking or destruction of the antigenic sites (Altmannsberger et al., 1981). These difficulties can be avoided by using frozen secfions prepared

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from fresh tissues or tissues fixed in ethanol, methanol, or acetone. In contrast to the cross-Hnking fixatives, these coagulating fixatives frequently show better immunostaining results, since the IFP apparently undergo only minor damage or cross-linking. Alternatively, a digestion step with a protease, applied before incubation with the primary antibody, can have an unmasking effect in formalin fixed tissue. Apart from the effects of fixation, additional difficulties must be overcome to localize IFP in tissues which contain bone, as in the case of the inner ear epithelia. To circumvent these problems various tissue treatment protocols have been introduced. Bauwens et al. (1991a) developed a dissection procedure which enabled the investigation of frozen sections of human inner ear structures fixed in acetone. For this purpose, human temporal bones were perfused with acetone for 10 minutes within three hours after death, followed by perfusion with PBS. Acareful dissection procedure freed the membranous inner ear from the adhering bone of the otic capsule. The dissected tissues were embedded in Tissue-Tek and prepared for cryosectioning. An ahernative method has been introduced by Arnold (1988) and Anniko and Arnold (1990a). They perfused human temporal bones with paraformaldehyde within 6-8 hours after death, followed by storage in a solution containing paraformaldehyde and HgCl2. After decalcification in EDTA, the temporal bones were embedded in paraplast at 54C and sectioned. Although excellent structural preservation of the inner ear epithelia was obtained with this method, this procedure appears to destroy the immunogenic properties of IFP, because the majority of the antibodies applied failed to show any reaction (Anniko and Arnold, 1990a). Analysis of the effects of fixation and decalcification on the immunoreactivity of the membranous inner ear tissues of the rat with monoclonal antibodies directed against various classes of IFP has been performed by Tonnaer et al. (1990). As the inner ear of the rat is extremely small and dissection of individual structures from the bony capsule causes loss of or serious damage to the epithelia, controlled decalcification with EDTA was applied. This chelating agent has been shown to preserve surface antigens and antigens in the extracellular matrix of bone (Kalervo Vaananan and Kalevi Korhonen, 1984; Jonsson et al., 1986). It was established that the epitopes recognized by the various CK and vimentin antibodies were usually destroyed by fixation in media which contained paraformaldehyde or glutaraldehyde (Tonnaer et al., 1990; Kuijpers et al., 1992). Exposure of freshly dissected inner ears to a solution containing 10% EDTA and 7.5% polyvinylpyrrolidone at 4°C, did not affect the epitopes, as long as the exposure time did not exceed two days. Exposure to EDTA even appeared to enhance the staining intensity in comparison with fresh untreated specimens (Figure 2), probably caused by unmasking of the antigenic epitopes. In addition, structural integrity of the epithelial lining of the inner ear was fairly well preserved.

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Figure 2. Effect of preincubation with EDTA on the detectability of CK 18 in the epithelium of the cochlear duct of a two-day-old rat. Note the increased immunostaining in EDTA treated tissue (B) in comparison to untreated tissue (A). Bar indicates 25 fam.

IV. EXPRESSION OF INTERMEDIATE FILAMENT PROTEINS IN THE INNER EAR Expression of IFP has been investigated in the inner ear of man and some small rodents. The rat inner ear has been studied most comprehensively throughout the whole developmental period up to maturity. Therefore, special attention is paid to the IFP expression pattern in the inner ear of this animal. A. Man In man, IFP expression has been studied in the fetal and adult inner ear. Fetal Inner Ear

Studies on the human fetal inner ear have been performed on cryosections of fresh inner ear specimens of 14-21-week-old fetuses (Anniko et al., 1987,1989a). At that age the development of the inner ear is comparable with that in the newborn rat and mouse. Both monospecific and polyspecific antibodies directed against various CKs of simple epithelia were used. With part of the chain-specific antibodies directed against CKs 8 and 18 the whole epithelial lining of both the cochlear and vestibular partition showed a positive reaction, but not all of the antibodies applied reacted with these cells. Staining with polyspecific antibodies recognizing CKs 8, 18, and 19 or CKs 7, 17, and 19, was limited to the non-sensory epithelium. Expression of CK 7 was limited to the stria vascularis, spiral prominence, dark cells, and sqme cell populations in the non-sensory epithelium of the vestibular partition. The epithelial lining of the endolympha-

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tic sac showed expression of CKs 7, 8, 18, and remarkably also of CK 10. The presence of CK 10 is puzzling, because so far this CK has only been found in keratinizing epithelia. Vimentin expression was observed in the mesothelial lining of the perilymphatic space, in the non-sensory epithelium of the vestibular compartments, and in the supporting and sensory cells. In the cochlea, vimentin expression was observed in nearly all epithelial cell types with the exception of the stria vascularis. Adult Inner Ear IFP expression in the human adult inner ear has been studied using cryosections of acetone-fixed inner ear tissues (Bauwens et al., 1991a, 1991b, 1992) and paraplast sections of tissue fixed in paraforitialdehyde-HgCl2 and subsequently decalcified in EDTA (Arnold and Anniko, 1989,1990; Annikoetal., 1990; Anniko and Arnold, 1990b). Cryosections. Immunohistochemistry on acetone-fixed cryosections of the inner ear tissues was performed with a broad panel of antibodies recognizing individual CKs and other IFP (Bauwens et al, 1991a, 1991b, 1992). In the cochlear partition no expression of CKs was observed in the sensory cells of the organ of Corti, but various types of supporting cells (pillar cells and Deiters' cells) showed the expression of CKs 8, 18, and 19, and failed to stain with the CK 7 antibodies. The epithelium outside the organ of Corti showed the expression of CKs 7, 8, 18, and 19, except for the marginal cells of the stria vascularis which failed to stain with the CK 7 antibody. In the vestibular partition, the sensory areas expressed CKs 8, 18, and 19, but it could not be established whether immunostaining was confined to either the sensory or supporting cells or was present in both cell types. The same pattern of expression was observed in the so-called transitional epithelial cells and in the dark cells. The flat epithelium of the vestibulum expressed CKs 8 and 19 and local CK 7 reactivity. Surprisingly, in the wall of the semicircular canals only CK 19 was found. No reaction was observed with antibodies directed against CKs specific for complex or stratified epithelia, except for the epithelium of the endolymphatic sac (Bauwens et al., 1991a, 1991b, 1992). These cells showed the homogeneous expression of CKs 7,8,18,19, and vimentin and heterogeneous expression of CKs 14 and 17 (Bauwens et al, 1991 b), which are markers of the basal cell compartment of complex and stratified epithelia. In a comparable immunohistochemical study on the extradural part of the endolymphatic sac Altermatt et al. (1990) showed the expression of CKs 13, 18, 19, and vimentin in the epithelial lining. The presence of CKs which are typical of complex and stratified epithelia in the endolymphatic sac seems to be in accordance with the morphological observations made by

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Zechner and Altmann (1969), who reported the presence of areas of pseudostratified epithelium in this structure. In frozen sections of the the cochlear duct, the expression of vimentin was found in Reissner's membrane, supporting cells of the organ of Corti, outer sulcus cells, spiral prominence, and in the intermediate and basal cells of the stria vascularis (Bauwens et al., 1991a). The whole epithelial lining of the vestibular partition and the endolymphatic sac also showed vimentin expression (Bauwens et al., 1992). Paraformaldehyde fixed sections. In the immunohistochemical studies on human inner ear tissues which were fixed in paraformaldehyde and embedded in paraplast, only a limited number of the antibodies directed against CKs of simple epithelia were found to react with the appropriate epitopes (Anniko and Arnold, 1990a, 1990b; Anniko et al., 1990; Arnold and Anniko, 1989,1990). The majority of antibodies, which have previously been found to immunostain unfixed, immature human inner ear epithelia, failed to react. Furthermore, only part of the various epithelial cell types showed a differing positive staining with the reacting monospecfic CK 8 and polyspecific antibodies. The sensory cells failed to stain with any of the antibodies applied. In these formaldehyde fixed tissues vimentin expression was found in the pillar cells of the organ of Corti, outer sulcus cells, interdental cells, Reissner's membrane, spiral prominence, intermediate cells of the stria vascularis, and supporting cells in the vestibular sensory areas. In general it can be stated that in comparison with the studies on frozen sections fewer cells were stained in formaldehyde fixed paraffin embedded sections. This may be due to both the fixation and decalcification procedures used. Even when antigenic epitopes resist fixation and decalcificafion, the staining intensity has been shown to depend on the exposure time to these agents (Tonnaer et al., 1990). B. Rat

IFP expression in the rat inner ear has been studied from day 12 postconception up to maturity. Cryosections of fresh inner ear specimens and specimens treated with EDTA were exposed to a broad panel of antibodies directed against individual CKs and vimentin (Kuijpers et al., 1991a, 1991b, 1992). Most of the antibodies used were raised against human IFP and they showed a comparable specificity in the rat as compared to that seen in the human system. The results are summarized diagrammatically in Figure 3 and illustrated in the micrographs in Figures 4-9. At 12 days postconception (dpc), shortly after separation of the otocyst from the primitive ectoderm, the pseudostratified epithelium of the otocyst showed homogeneous expression of CKs 8, 18, and 19 (Figure 3a). Vimentin expression was present in the surrounding mesenchyme and also in the epithelium of the otocyst (Figure 3c). At 15 dpc, the otocyst had differentiated into a cochlear and a vestibular duct system. The whole epithelial lining of these ducts showed homoge-

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12 dpc

15 dpc

18 dpc

Figure 3. Diagram of IFP expression during development of the cochlear duct of the rat. A: CK 8, 18, and 19; B: CK 7; C: Vimentin; dpc: days post conception; dab: days after birth, c: Cells of Claudius; g: Greater epithelial ridge; i: Intermediate cells; id: Interdental cells; is: Inner sulcus; I: Lesser epithelial ridge; m: Mesothellum; o. Organ of CortI; os: outer sulcus; r: Relssner's membrane; s: Stria vascularis. Arrow indicates spiral prominence.

neous expression of CKs 8,18, and 19 (Figure 3a), although the expression pattern in the presumptive sensory areas differed from those in the non-sensory epithelium. CK 7 was first expressed in part of the non-sensory epithelium of the vestibular partition. Vimentin expression was present in the whole epithelial lining (Figure 3c). Cochlear Duct At 18 dpc, the delineation of different cell types present in the adult is morphologically visible. The stria vascularis and Reissner's membrane can be distinguished

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Figure 4, Expression of CK 18 (B) and vimentin (C) in the cochlear duct of the rat at 18 dpc. (A) Haematoxylin-eosin stain, e: Endolymph; g: Greater epithelial ridge; I: Lesser epithelial ridge; m: Mesothelium; o: Organ of Corti; os: outer sulcus; p: Perilymph; r: Reissner's membrane; s: Stria vascularis. Bar indicates 25 |im.

in the anterior wall. The posterior wall shows the developing organ of Corti, the greater and lesser epithelial ridges, and the outer sulcus (Figure 4a). All the epithelial cell types expressed CKs 8,18, and 19, but the expression pattern differed between the various epithelia (Figures 3a and 4b). The expression of CK 7 was limited to the developing stria vascularis and the adjacent part of the outer sulcus (Figure 3b).

Figure 5. Expression of CK 18 (A, C) and vimentin (B, D) in the cochlear duct of the rat at 1 dab (A, B) and at 5 dab (C, D). E and F details of organ of Corti at 5 dab. E. shoves expression of CK 18 in supporting cells, but not in in inner (o) and outer (*) sensory cells. F. shows vimentin expression in supporting cells and in the infranuclear area of both the inner (o) and outer (*) sensory cells. G and H (tangential section) details of the organ of Corti in the adult inner ear showing vimentin expression in both sensory cells (o,*) and supporting cells, c: Cells of Claudius; dc: Cells of Deiters; e: Endolymph; g: Greater epithelial ridge; i: Intermediate cells; id: Interdental cells; is: Inner sulcus; I: Lesser epithelial ridge; m: Mesothelium; o: Organ of Corti; os: outer sulcus; p: Perilymph; r: Reissner's membrane; s: Stria vascularis. Bar indicates 25 jam (A-D); 8 ^ m (E-H).

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Apart from the surrounding mesenchyme and the developing mesothelial lining of the perilymphatic space, the whole epithelium displayed vimentin expression (Figures 3c and 4c). At 1 day after birth (dab) the expression of the various CK polypeptides was similar to that seen at 18 dpc (Figures 3a, 3b, and 5a). Vimentin expression was still present in the major part of the epithelia (Figures 3c and 5b). An accumulation of vimentin-positive cells was present below the epithelial (marginal) cells of the stria vascularis (Figures 3c, 5b, 6e, and 6f). The expression of CKs and vimentin in the developing organ of Corti was mainly found in the basal part (Figures 5a and 5b), but it could not be established whether the expression of IFF was limited to the supporting cells or whether it was also present in the developing sensory cells. During further development of the various cell types, major changes took place in the expression of IFF. At 5 dab, when the supporting cells and sensory cells in the organ of Corti can be clearly distinguished, the sensory cells appear to be devoid of CKs (Figures 5c and 5e), but they display vimentin expression in the infranuclear area (Figures 5d and 5f). The supporting cells displayed the expression of CKs 8, 18, 19, and vimentin (Figures 3a, 3c, and 5c-5f). All the cell types outside the organ of Corti displayed the expression of CKs 8,18, and 19 (Figures 3a and 5c). The expression of CK 7 was limited to the marginal cells of the stria vascularis and outer sulcus cells (Figure 3b) but was decreased in comparison with I dab. These epithelia showed basal cell extensions (Figures 5c, 6a, and 6b). At this stage, the accumulation of vimentin-positive cells, which represent the developing intermediate cells of the stria vascularis, was more pronounced than seen at I dab (Figure 5d). Nearly the whole epithelium, except for the marginal cells, still showed vimentin expression (Figures 3c and 5d). During final maturation up to 40 dab, the expression of CK 7 in the cochlear duct gradually disappeared from the stria vascularis and outer sulcus (Figure 3b). In the mature cochlear duct CK 7 was only expressed in a few transitional cells at the margins of the stria vascularis (Figures 3b and 6d). The expression of CKs 8, 18, and 19 persisted in all the non-sensory cell types, including the stria vascularis (Figure 6c) and the supporting cells of the organ of Corti, that is, Deiters' cells, pillar cells, inner border cells and Hensen's cells (Figure 3a).

Figure 6, Expression of CKs (B-D) and vimentin (F-H) in the stria vascularis (marginal cells (mc) and intermediate cells (i)) and outer sulcus (os) during postnatal maturation. A: toluidin blue stain at 5 dab; B: CK 19 at 5 dab; C: CK 19 in adult inner ear. D: CK 7 in adult inner ear. E: toluidin blue stain at 1 dab; F: vimentin at 1 dab; G: vimentin at 7 dab; H: vimentin in adult inner ear. Note differential expression of cytokeratin and vimentin in intermediate (i) and marginal cells (mc), the coexpression of CK 19 and vimentin in outer sulcus as well as the absence of CK 7 in the marginal cells in the adult ear. Arrow indicates spiral prominence. Bar indicates 12 |am.

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In the stria vascularis, an increasing number of vimentin-positive capillaries and cell processes became visible (Figures 6g and 6h). They belong to the developing intermediate cells which have penetrated between the marginal cells. Vimentin expression disappeared from the major part of the epithelial lining, but it persists up to maturity in the supporting cells of the organ of Corti and in the outer sulcus cells (Figures 3c, 6g, and 6h). In the sensory cells, which lacked CK expression, vimentin expression remained present as a small infranuclear spot (Figures 5g and 5h). Vestibular Duct

At 18 dpc, the vestibular duct system is subdivided into the saccule, utricle, and ampullar organs with the semicircular canals and the endolymphatic sac. The various areas of sensory epithelium can be clearly distinguished morphologically. At this stage, the whole epithelial lining displayed the expression of CKs 8, 18, and 19 (Figure 7a). CK 7 was heterogeneously expressed in the epithelium outside these areas and in the endolymphatic sac. Vimentin expression was present in the whole epithelial lining (Figure 7b). At this age, no definitive decision could be made about the IFP expression in the sensory cells. During further maturation, part of the epithelium outside the sensory areas differentiates into transitional cells adjacent to the maculae and cristae, and into an area of dark cells in the utricle. At 5 dab the epithelium of the vestibular duct system reaches morphological maturation. At this age and in the adult, the sensory areas revealed the homogeneous expression of CKs 8,18, and 19 (Figures 8a, 8b, 8d, and 9a) and a minute population expressed CK 7. CK expression appeared to be confined to the supporting cells (Figure 9a). CKs 8, 18, and 19 were homogeneously expressed in nearly the whole epithelial lining outside the sensory areas (Figure 8a), except for CK 19 which was absent in the transitional cells (arrows in Figures 8b and 8d). Also, CK 7 was absent

A

m-

Figure 7. Expression of CK 18 (A) and vimentin (B) in the vestibular partition (saccule) of the rat at 18 dpc. e: endolymph; p: Perilymph; sa: Sensory area. Bar indicates 36 }im.

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D Figure 8. Expression of CKs 18 (A) and 19 (B, D) and vimentin (C) in the vestibular partition (utricle) of the rat at 5 dab. Note the absence of CK 19 expression in the transitional epithelium (arrows) and at higher magnification in D. Bar indicates 35 |im (A-C);15|am(D).

in the transitional cells as well as in the dark cells. The remaining part of the non-sensory epithelium, which is mainly composed of flat cells, showed weak heterogeneous expression of this CK. Vimentin expression was especially marked in the supporting cells (Figures 8c and 9b), but the staining profiles differed at various sites in the sensory areas. At some sites, all the supporting cells stain with vimentin antibodies, while at other sites only the basal part is immunoreactive (Figure 9c). A proportion of the sensory cells showed a vimentin spot close to the nucleus (Figure 9c). The epithelium outside the sensory areas displayed the heterogeneous expression of vimentin. During postnatal development, the CK profile of the endolymphatic sac did not change significantly. The epithelium displayed the homogeneous expression of CKs 8, 18, and 19, but CK 7 expression appeared to be limited to a distinct population of cells. Using enzyme histochemistry it was possible to demonstrate a high concentration of succinic dehydrogenase and cytochrome oxidase in these cells (Kuijpers et al., unpublished observations) which indicates an exceptional position of these cells. Vimentin expression was found in the major part of the epithelium.

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Figure 9. Expression of CK 19 (A) and of vimentin (B-D) in the adult sensory areas of the vestibular partition of rat (A-C) and guinea pig (D). Both CK 19 and vimentin expression (A, B) mirrors the position of the supporting cells. Vimentin expression in the sensory cells indicated by (X) of the rat is less pronounced than in the guinea pig. Bar indicates 6 fim.

Throughout the whole developmental period no expression of CKs specific for complex or stratified epithelia was seen in any of the inner ear epithelia. C.

G u i n e a Pig

The guinea pig has been the subject of several studies on IFP expression. In most reports, however, only a limited part of the inner ear epithelium was investigated using a small number of antibodies. In a developmental study on the guinea pig cochlear duct, Raphael et al. (1987) demonstrated the expression of CKs 8, 18, and 19 in the whole epithelial lining from the otocyst up to maturity. In the adult organ of Corti, the hair cells appeared to be devoid of CKs. Regarding the expression of CKs 8 and 18, comparable observations were made by Kuijpers et al. (1991b) in the mature inner ear of the guinea pig, but CKs 7 and 19 were absent in the stria vascularis. The absence of CK 7 was also observed in rat (Kuijpers et al., 1991a, 1991b) and man (Bauwens et al., 1991a). In the vestibular partition of the mature guinea pig inner ear (Kuijpers et al., unpublished observations), comparable CK and vimentin expression profiles have been established similar to those seen in the mature rat inner ear. In the vestibular

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partition of the guinea pig vimentin expression was more pronounced in the sensory cells (Figure 9d) and in some cell types outside the sensory areas. In a comparable study on formaldehyde fixed, Spurr embedded specimens, Oesterle et al. (1990) observed less pronounced vimentin expression. Only the outer sulcus cells and a proportion of the supporting cells of the organ of Corti were positive, whereas no vimentin expression was observed in the intermediate cells of the stria vascularis. D. Gerbil Immunohistochemical studies on the expression of IFP in the adult gerbil have been confined to the expression of vimentin (Schulte and Adams, 1989), which was observed in the supporting cells of the organ of Corti, the external sulcus cells, and the intermediate and basal cells of the stria vascularis. In the vestibular partition, vimentin expression was observed in the sensory cells and the supporting cells, but the expression differed at the different sites of the sensory areas as also observed in the rat (Kuijpers et al., 1992). These studies were performed on tissue specimens fixed in formaldehyde or Camoy's solution. It was established that fixation in Carnoy's solution provided excellent preservation of vimentin antigens. However, paraformaldehyde appeared to block immunostaining at all sites, except for those showing high vimentin concentrations. These effects of fixation are in accordance with those observed by us in the rat inner ear (Kuijpers et al., 1992) and might explain the finding that fewer cells in the inner ear of some other species show vimentin expression, when paraformaldehyde fixation is applied as compared to unfixed tissue or tissue fixed in Camoy's solution. E. Mouse

Studies on the expression of IFP in the mouse have been mainly confined to the late gestational period and the newborn mouse, when the inner ear is still immature (Anniko et al., 1986, 1989b, 1989c; Berggren et al., 1990). Different sets of monospecific and poly specific CK antibodies were applied to cryosections of fresh tissues. Widely varying expression patterns were obtained with monospecific antibodies directed against CK 8 and CK 18, and with polyspecific antibodies recognizing CKs 8, 18, 19 or CKs 7, 17, and 19. Most of the antibodies showed a positive reaction in the epithelium of the endolymphatic sac. In the remaining part of the epithelial lining of the inner ear, staining was either absent or limited to part of the cochlear or vestibular partition, but a considerable variation was seen in the staining pattern of antibodies recognizing the same CK polypeptide. In the immature mouse inner ear vimentin was expressed in the surrounding mesenchyme, both the sensory and supporting cells of the cristae, and in part of the non-sensory epithelium. These observations on the expression of CKs differ greatly from those made in other species. It is likely that the antibodies applied were responsible for the

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discrepancies since they were raised against human antigens and might not recognize the appropriate mouse antigens. In addition, the use of mouse monoclonal antibodies on frozen mouse tissue sections can lead to non-specific reactions because of the presence of mouse immunoglobulins (Ig), which will be detected by the secondary conjugated anti-mouse Ig antibodies used in the immunohistochemical procedure. F. Discussion

The above-mentioned studies on the expression of IFP in the inner ear demonstrate clearly that many pitfalls can be encountered when immunohistochemical staining techniques are applied to this organ. Careful analysis of the effects of tissue preparation (fixation and decalcification) on the various epitopes and the specificity of monoclonal antibodies is obligatory in order to avoid misinterpretation of the data obtained. Even if a positive reaction is seen with a particular antibody in fixed or decalcified tissues, comparison of the results with those obtained in untreated, frozen specimens is necessary to exclude any possible adverse effects of tissue processing on the results of immunostaining. Generally, the data reported in the various studies revealed a fairly uniform expression pattern of IFP in the inner ear epithelia of certain mammalian species. Any discrepancies could be ascribed to differences between species and between the techniques applied. The major part of the epithelial lining in the adult inner ear only revealed the presence of CKs which are typical for simple epithelia. CKs typical for complex and stratified epithelia were only present in the human endolymphatic sac. Not all the CKs were expressed equally in the various cell types. During development modulation of CK expression takes place dependent on differentiation. CKs 8, 18, and 19 are already present in the undifferentiated epithelium of the otocyst from which all the different epithelial cell types of the adult inner ear are derived. Only the expression of CK 7 appears to be delayed and limited to part of the epithelium. The sensory cells of both the cochlear duct and the vestibular duct system apparently become devoid of CKs during the final stages of maturation. This absence of CKs in epithelial cells is quite remarkable and has so far only been reported for a very limited number of epithelial cell types, including the sensory cells of the olfactory epithelium (Volrath et al., 1985). These cells develop in a comparable way from the embryonal ectoderm, the nasal placode, as the cells of the otocyst form the otic placode. In contrast, the olfactory cells differentiate into bipolar nerve cells. During the final stages of maturation of the different cell types outside the sensory areas, CK 7 disappeared from the marginal cells of the stria vascularis and from the outer sulcus cells. This coincided with the morphological maturation of the intermediate cells, which penetrate between infoldings of the marginal cells of the stria vascularis. In the vestibular duct system, CK 7 disappears from the dark cells, which

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Ml

are functionally comparable to the marginal cells of the stria vascularis (Kimura, 1969; Kuijpers, 1969). The transitional cells showed disappearance of both CK 7 and 19. Comparison with biochemical and physiological investigations shows that these changes coincide with the functional maturation of the inner ear, as indicated by the typical intracellular cation composition of the endolymph (Bosher and Warren, 1971), Na-K-ATPase activity (Kuijpers, 1974) and electrical activity (Curthoys, 1979; Romand, 1983; Ryback et al., 1992). It is therefore tempting to speculate on a possible relationship between the functional condition of these highly specialized cell types and their IFP composition. The expression of vimentin in the mesenchyme and the mesenchyme-derived structures, including the mesothelial lining of the perilymphatic space, is in accordance with the pattern of vimentin expression normally observed in these types of tissues. Vimentin expression in the mature intermediate cells and their precursor cells which accumulate underneath the marginal cells during development, seems to be in accordance with their suggested origin from melanocyte precursors in the neural crest (Schrott and Spoendlin, 1987; Schrott et al., 1988). It has been shown that melanocytes only express vimentin-type IFP (Ramaekers et al., 1983). The co-expression of CKs and vimentin is a fairly common, but often transient phenomenon, related to epithelial differentiation. During final stages of differentiation, vimentin expression normally disminishes. This is also the case in the major part of the inner ear epithelium. However, vimentin persists in the adult in some highly specialized cells such as the sensory supporting cells of both the organ of Corti and the vestibular partition, the outer sulcus cells, and the epithelial cells of the endolymphatic sac. In the rat, vimentin also remains present in the sensory cells of both the cochlear duct and the vestibular partition. One can only speculate on a relationship between the functional aspects of these different cell types and IFP (co)expression. In the cochlear duct, the specific composition of the cytoskeleton of the supporting cells might be related to the special properties of the organ of Corti regarding signal reception and transduction. During acoustic stimulation, the whole organ of Corti moves as one rigid structure with respect to the tectorial membrane, which results in deflection of the sensory hairs. As a result, the organ needs a rigid tissue structure, which might be provided by its IFP network of the supporting cells. The sensory cells, however, exhibit active contraction during depolarization (Zwicker 1979; Zenner et al., 1985) and a less rigid cytoskeleton as reflected by the absence of CKs or the presence of only vimentin might be related to this cell flexibility. A similar correlation might exist in the sensory areas of the vestibular partition. The coexpression of vimentin and CKs in part of the non-sensory epithelium might indicate a specialized function of these cells in endolymph homeostasis. Future studies will have to reveal whether this assumption can be supported by cell biological and physiological data.

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V. EXPRESSION OF IFP IN THE STATO-ACOUSTIC NERVE A.

Neurofilaments

The afferent innervation of the sensory epithelium of both the cochlear duct and the vestibular partition originates from the acustico-vestibular ganglion. The latter is derived from epithelial cells which migrate out off the otic vesicle, while the neural crest cells are also assumed to contribute to this structure. During development, this ganglion is split into (1) the spiral ganglion that supplies the sensory cells of the organ of Corti in the cochlea, and (2) the ganglion of Scarpa that supplies the various sensory areas of the vestibular partition (Neely et al., 1986; Ryu, 1986). Neurofilaments are the IFP of neurons and are normally in the adult composed of a triplet of neurofilament proteins with molecular weights of approximately 70 kD, 150 kD, and 200 kD, referred to as NFL, NFM, and NFH, respectively (Liem et al., 1978; Schlaepfer and Freeman, 1978). Both the 150 and 200 kD neurofilament proteins are highly phosphorylated, whereas the expression of the different subtypes is developmentally regulated. During development the axonal cytoskeleton undergoes a process of maturation, which involves the successive expression of the NFL and NFM, followed by the NFH. Thereafter, phosphorylation of the NFM and NFH types takes place (Dahl and Bignami, 1986; Dahl, 1988; Garden et al., 1987). Many studies have been performed on the expression of neurofilament proteins in the stato-acoustic nerve, but a proportion of these studies have not specified the different subsets of neurofilaments expressed (Anniko et al., 1987; Bauwens et al, 1991a, 1992; Kuijpers et al., 1991b; Pirvola et al., 1991). Studies using antibodies directed against the individual subsets of neurofilament proteins have been focused mainly on the ganglion cells of both the cochlear and vestibular nerves. Very little attention has been paid to the axonal and dendritic processes. Studies on the adult ganglion of Scarpa of the rat (Hafidi and Romand, 1989a; Ylikoski et al., 1993) and guinea pig (Usami et al., 1993), showed that approximately one-third of the cells, which represented the larger cells, stained intensely with antibodies directed against the different subsets. The remaining cells failed to stain or stained only weakly for neurofilament proteins. This observation was made irrespective of the antibody used. In the human ganglion, however, Anniko et al. (1993) observed that 5% of the cells displayed a positive reaction with an antibody to NFH, while all the cells stained homogeneously after exposure to a NFL antibody. In the adult spiral ganglion of various species two populations of ganglion cells have been observed. Ylikosky et al. (1993), Romand et al. (1988), and Hafidi et al. (1990) found that in the rat 6-9% of the ganglion cells, that is, the smaller type II cells, stained intensely with all the antibodies directed against the different subsets of neurofilament proteins. The remaining, larger type I cells, did not stain or stained only weakly.

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Berglund and Ryugo (1991) observed selective staining of type II ganglion cells in the rat and some other species only with NFH antibodies. With antibodies directed against NFL and NFM subunits, both types I and II stained equally. In addition, a population of type I cells was observed in the basal part of the cochlea, which stained intensely with the NFH antibody. After treatment with alkaline phosphatase the population of type II cells no longer stained with the NFH antibody, while no effect was observed on the staining pattern with the NFL and NFM antibodies. This suggests that only the type II population of cells, which is assumed to innervate only the outer hair cells (Berglund and Ryugo, 1987; Brown, 1987), contains highly phosphorylated NFH. The presence of non-phosphorylated filaments in type I cells has been suggested by Romand et al. (1988). In a developmental study on the rat inner ear, Hafidi et al. (1989b) observed the first occurrence of staining of the neuronal cell bodies with the NFL and NFM antibodies at 16 dpc. Staining with the NFH antibody occurred only after birth, when differential staining of the type I and II ganglion cells became apparent. Although neurofilaments are intermediate filaments specifically linked to neurons, a remarkable observation was made by Hasko et al. (1990). They observed the transient expression of neurofilaments in the cochlear sensory cells between five and 20 days afi;er birth. B. Vimentin

In addition to neurofilaments, other IFF can be expressed in neurons of the stato-acoustic nerve. In the human fetal inner ear, vimentin expression was found in all neurons, including the ganglion cells and Schwann cells (Anniko et al., 1987). In the human adult inner ear, Bauwens et al. (1991a, 1992) failed to find vimentin expression in the nerve fibers, while Wikstrom et al. (1988) observed the presence of vimentin in the stato-acoustic ganglion at the otocyst stage and in the newborn mouse. Anniko et al. (1986) and Berggren et al. (1990) reported vimentin expression in the mouse around birth not only in the Schwann cells but also in the neurons. Remarkably, these latter authors also found a positive reaction in the neurons with a broadly reacting CK antibody A comparable finding was reported in both the human fetal and adult inner ear (Anniko et al., 1987, 1990). It was suggested that this coexpression of CKs, vimentin, and neurofilament proteins might be related to the dual origin of the stato-acousfic ganglion, which is composed of cells from both the wall of the otocyst and cells from the neural crest (Berggren et al., 1990). In a developmental study on the rat, vimentin expression was limited to the Schwann cells, but during final maturation, vimentin expression became apparent in some of the peripheral nerve endings in the vestibular sensory areas (Kuijpers et al., 1992).

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Discussion

The most conspicuous finding in these studies on neurofilament proteins is that two populations of ganglion cells are present in both the spiral ganglion and the ganglion of Scarpa. Both ganglia differ, however, with regard to their expression of the various subsets of neurofilament proteins. In the cochlear duct, the population of ganglion cells can be subdivided into cells which constitute the afferent innervation of the inner sensory cells and cells which form the innervation of the outer sensory cells. This suggests that neurofilament protein expression is functionallyrelated because the outer and inner sensory cells have different functions in signal processing. So far, no such functional relationship has been found for the vestibular neurons. In the sensory areas, two different types of sensory cell can be distinguished which possess anatomically different afferent nerve endings (Figure Id). It is tempting to assume that the differences in neurofilament protein expression might be related to these different types of nerve endings. Further studies on the expression of the various subsets of neurofilaments in the dendritic processes are required to confirm this suggestion. REFERENCES Altermatt, H.J., Gibbers, J.O., Arnold, W, & Laissue, J.A. (1990). The epithelium of the human endolymphatic sac: Immunohistochemical characterization. O.R.L. 52, 113-126. Altmannsberger, M., Osbom, M., Schauer, A., & Weber, K. (1981). Antibodies to different intermediate filament proteins. Cell type-specific markers on paraffin-embedded human tissues. Lab. Invest. 45, 427-434. Anniko, M., Thomell, L.E., Gustavsson, H., & Virtanen, I. (1986). Intermediate filament proteins in the newborn inner ear of the mouse. O.R.L. 48, 98-106. Anniko, M., Thomell, L.E., & Virtanen, I. (1987). Cytoskeletal organization of the human inner ear. Acta Otolaryngol. Suppl. 437, 1-76. Anniko, M., Thomell, L.E., Ramaekers, F.C.S., & Stigbrand, T. (1989a). Cyto-keratin diversity in epithelia of the human inner ear. Acta Otolaryngol. 108, 385-396. Anniko, M., Sjostrom, B., Thomell, L.E., & Virtanen, I. (1989b). Cytoskeletal identification of intermediate filaments in the inner ear of the Jerker mouse mutant. Acta Otolaryngol. 107, 191-201. Anniko, M., Thomell, L.E., Hultcrantz, M., Virtanen, I., Ramaekers, F.C.S., & Stigbrand, T. (1989c). Prenatal low-dose gamma irradiation of the inner ear induces changes in the expression of intermediate filaments. Acta Otolaryngol. 108, 206-216. Anniko, M., & Amold, W. (1990a). Methods for cellular and subcellular visualization of intermediate filament proteins in the human inner ear. Acta Otolaryngol. Suppl. 470, 13—22. Anniko, M., & Amold, W. (1990b). Cytoskeletal network of intermediate filament proteins in the adult human vestibular labyrinth. Acta Otolaryngol. Suppl. 470, 40-50. Anniko, M., Amold, W., Thomell, L.E., Ramaekers, F.C.S., & Pfaltz, C.R. (1990). Regional variations in the expression of cytokeratin proteins in the adult human cochlea. Eur. Arch. Otorhinolaryngol.sofar 247, 182-188. Anniko, M., Amold, W., & Stigbrand, T. (1993). Protein pattem in vestibular ganglion cells and hair cells with functional interpretations. Acta Otolaryngol. Suppl. 503, 136-142.

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