Histochemical analysis of glycoconjugates in gelatinous membranes of the gerbil's inner ear.

The gelatinous membranes of the gerbil inner ear were analyzed histochemically for glycoconjugates with a battery of twenty horseradish peroxidase-conjugated lectins. Glycoconjugates with mannose (Man) and/or glucose (Glc), galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (NeuAc) were detected in the tectorial and otolithic membranes and cupula. Differences in lectin reactivity were observed between tectorial and vestibular membranes and also among zones and between the medial and lateral regions of the middle zone of the tectorial membrane. The distribution of staining differed markedly for several lectins that bind specifically to GalNAc or to GlcNAc but vary in affinity for oligosaccharides containing these sugars in different sequences or linkages. The findings suggest presence of the terminal disaccharides GalNAc alpha 1,3Gal in tectorial membrane and Gal beta 1,3GalNAc in vestibular membranes. Lectin binding profiles provided evidence that the limbal zone's fibrous and attachment layers contain mainly O-glycosidically linked oligosaccharides whereas the middle zone's medial fibrous layer contains both O- and N-linked chains. The remaining regions of the tectorial membrane contain mainly N-linked oligosaccharides with bisected biantennary type chains predominating. Additionally, the marginal band and the middle zone's basal layer contain abundant N-linked oligosaccharides with a triantennary structure.     

Effects of perinatal hypothyroidism in the carbohydrate composition of cochlear tectorial membrane

The presence of carbohydrates in the cochlear tectorial membrane (TM) of normal and hypothyroid rats was analyzed using fluorescent lectin probes. SBA and WGA lectins exhibited a similar reactivity in both normal and hypothyroid TMs. DBA, RCA1, UEA1 and Con A lectins were also reactive, although they showed a different distribution pattern between normal and hypothyroid TMs. Lastly, one of the lectins, PNA, was only labeled in hypothyroid TMs. These findings suggest that carbohydrate chains containing residues of N-acetyl-D-galactosamine (GalNAc) and N-acetyl-D-glucosamine, are similarly distributed in normal and hypothyroid TMs. Other carbohydrate residues as GalNAc alpha 1,3 GalNAc, D-galactose (Gal), L-fucose and D-mannose, are present, but are abnormally distributed in hypothyroid TMs. The Gal beta 1,3GalNAc residues, recognized by PNA, could be present only in the hypothyroid TMs. Alterations in glycosylation of the glycoproteins in the hypothyroid TM could be responsible for the abnormal distribution pattern of carbohydrate residues here described, and for the distorted shape of the hypothyroid TM.     

Glycoconjugates of the tectorial membrane

The type and quantity of carbohydrate present in the tectorial membrane (TM) was analysed using gas-liquid chromatography and lectin staining of TM protein subunits previously separated by electrophoresis. A relatively large amount of carbohydrate was found, and glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose and N-acetylneuraminic acid were detected. The presence of mannose and the reaction of many of the protein bands with lectins suggest that at least part of the carbohydrate present is in the form of glycoprotein. The reaction of the main protein band with the lectins RCA1 and ConA is consistent with the suggestion [Thalmann et al. (1985) J. Acoust. Soc. Am. Suppl. 1, Vol. 78, S66] that this band is similar to collagen type II. The failure to detect any uronic acid in these experiments indicates that the more common proteoglycans are probably not a major component of the TM (although keratan sulphate might be present).     

Expression of glycoconjugates in the human fetal cochlea.

The distribution of glycoconjugates in the human fetal cochlea was analyzed using six biotinylated lectins: wheat germ agglutinin (WGA), abrus precatorius agglutinin (APA), ulex europaeus agglutinin I (UEA-I), ricinus communis agglutinin 120 (RCA120), helix pomatia agglutinin (HPA), concanavalin A (ConA). The tectorial membrane (TM) in the 11- and 15-week-old human fetuses was labelled with WGA, APA, RCA120 and ConA, but not with UEA-I and HPA. In the 19-week-old fetuses, the reaction of the TM decreased. In the 11-, 15- and 19-week-old fetuses, the surface of the greater and the lesser epithelial ridges were respectively labelled with WGA, APA and RCA120. Reissner's membrane was labelled with WGA, ConA, APA, RCA120 and HPA. WGA, RCA120 and APA strongly stained the stria vascularis, especially in the 15-week-old fetuses. HPA did not stain the 11-week-old fetal cochleas at all, while it stained the apical surface of the hair cells, Reissner's membrane, the cells within the stria vascularis and spiral osseous lamina in the 15-week-old fetuses. In the 19-week-old fetuses, the fluorescent reaction with HPA became decreased and the apical surface of the hair cells was not labelled with HPA at all. This result suggests that HPA reactive glycoconjugates may be related to the molecule responsible for stereociliary adhesion only during development.     

Localization of anionic sulfate groups in the tectorial membrane.

Colloidal iron hydroxide (CIH) staining demonstrates the existence of anionic sulfate groups of glycoconjugates associated with several constituents of the tectorial membrane (TM). In the adult animal, labelling in the main body of the TM appears as long, electron-dense patches surrounding type A fibrils which show alternating stained and unstained zones. On the other hand, labelling of the fibrils of the matrix of the TM appears as single, CIH particles with no special arrangement. Some of the structurally distinct regions of the TM are also labelled (limbal zone, Hensen's stripe and inner portions of the cover net), while others are not (marginal band and outer portions of the cover net). Staining of type A fibrils in the major TM is already present in newborn animals; while, both the outermost region of the TM closest to the cells of the organ of K?lliker and the minor TM are not labelled. The implications of these distributions of sulfated glycoconjugates for the electrochemical properties of the TM are discussed.     

Lectin-binding patterns and cell kinetics of head and neck squamous cell carcinomas

In order to elucidate the cell characteristics of head and neck squamous cell carcinomas, the cell kinetics and lectin binding patterns were compared with the histological classification and staging of the tumors, using surgically resected materials (maxillary sinus 10, oral cavity 21, pharynx 8, larynx 11). Eight biotinylated lectins (WGA, 1-PHA, ConA, UEA1, RCA1, SBA, DBA, PNA) were applied to the paraffin-embedded sections, and were visualized histochemically by the streptavidin-alkaline phosphatase method. The DNA contents of the isolated carcinoma cells obtained from the adjacent thick sections were evaluated using an epi-illumination cytofluorometer after propidium iodide staining. On lectin histochemistry, the binding pattern of WGA lectin was similar between carcinoma tissues and normal tissues, but the binding was more intense in well differentiated than less differentiated carcinomas. Lymph node metastasis was found to be related to the presence of cells with poor WGA-binding. In the binding patterns of the other lectins, RCA1, SBA and ConA were related to the differentiation of carcinomas, but they were not related to the TNM-classification. DNA cytofluorometry exhibited marked polyploidization, which progressed with the advancement of the clinical and pathological staging of carcinomas. However, the DNA ploidy pattern was not associated with the cell characteristics such as the degree of histological differentiation and the lectin-binding pattern, except that the appearance of aneuploidy had some relationship with the binding-patterns of UEA1 and 1-PHA.     

Lectins demonstrate the presence of carbohydrates in the tectorial membrane of mammalian cochlea

Histological sections of rat and guinea pig cochleas were exposed to lectins to identify the carbohydrates present in the tectorial membranes. N-Acetylglucosamine, galactose, mannose and fucose were found to be present in both rats and guinea pigs, but N-acetylgalactosamine was not detected. In addition, two control experiments were performed. In the first, each lectin was preincubated with its specific inhibitory sugar. In the second, the unfixed tectorial membranes were exposed to lectins. Radioautographic studies confirmed the presence of glucosamine and fucose in the tectorial membrane of 1-day-old rats.     

Expression of glycoconjugates in the mouse inner ear after prenatal irradiation

Summary Irradiation of the murine fetal inner ear is known to produce damage both to the vestibular and cochlear parts in the adult mouse. Fluorescein-labelled lectins were used to reveal possible differences in the glycoconjugate content between normal and irradiated inner ears. In the vestibular part, the otoconia showed the highest uptake of labelled sugars. This uptake was weaker after irradiation when compared to non-irradiated specimens. The type I hair cells in the ampulla and in the utricle showed a weaker uptake, but no labelling was demonstrated in the type II hair cells compared to the non-irradiated controls. In the cochlear part of the inner ear almost no uptake of fluorescent-binding lectins could be demonstrated in the irradiated groups except for in the tectorial membrane. In the endolymphatic sac no uptake was shown after prenatal irradiation. These findings are discussed and correlated to the already known damage of the inner ear following prenatal irradiation.     

Ultrastructure of the normal human organ of corti. new anatomical findings in surgical specimens

CONCLUSION: A thorough scanning electron microscopy (SEM) investigation of immediately fixed human adult cochleae obtained during surgery for petro-clival meningiomas conveyed new information about morphology. OBJECTIVE: To investigate the ultrastructure of human adult cochleae using SEM. MATERIAL AND METHODS: Two human cochleae were decalcified, fixed with glutaraldehyde and osmium and prepared for SEM. RESULTS: The excellently preserved morphology showed the pathways of nerve fibres through the organ of Corti. Undulating lateral cell membranes of Hensen and Claudius cells created an enlarged surface that may be important for homoeostasis. The distal attachment of the tectorial membrane to the reticular lamina was present in the shape of a marginal net, which was extended through marginal pillars. Stereocilia imprints extended as far as the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cells attached to the marginal pillars raises questions about differences in the excitation of the last row of outer hair cells as a result of differences in the composition of the tectorial membrane.     

The protein composition of the avian tectorial membrane.

Gel electrophoretic analysis of the avian tectorial membrane under non-reducing conditions reveals the presence of 2 major proteins with apparent molecular masses of 195 and 41 kDa on 8.25% gels. Under reducing conditions, 6 polypeptides with apparent molecular masses of 146, 60, 56, 43, 35 and 31 kDa are consistently observed. None of these six polypeptides observed under reducing conditions are sensitive to digestion with collagenase, and all, except for the 43 kDa component, are degraded by treatment with cold acidic pepsin. The 60, 56 and 43 kDa polypeptides bind the peroxidase conjugated lectins from Canavalia ensiformis and Triticum vulgaris, indicating the presence of mannose, N-acetyl glucosamine and/or sialic acid. The 146, 60 and 56 kDa bands undergo a shift in electrophoretic mobility after treatment of native tectorial membranes with the enzyme neuroaminidase. Fibronectin and Type II collagen cannot be detected in the avian tectorial membrane by either immunoblotting or immunofluorescence techniques. Polyclonal antisera raised against the different polypeptides after partial purification by one dimensional gel electrophoresis confirm that these proteins are all components of the tectorial membrane, and show that they are restricted to the otolithic and tectorial membranes within the inner ear. Analysis of a wide variety of other tissue types indicates that the 60, 43 and 35 kDa components can only be detected within the inner ear, and that the antisera recognising the 146 and 31 kDa components only show cross-reactivity within the head, with the anti-146 kDa antibodies staining the mucus ducts supplying the olfactory epithelium and the anti-31 kDa antibodies staining granular elements in the cells of the respiratory epithelium. The results suggest that certain of the tectorial membrane components may be novel matrix molecules unique to the inner ear, and that some of the other proteins may be antigenically related to mucins.     

Ultrastructure of the normal human organ of Corti. New anatomical findings in surgical specimens

Conclusion A thorough scanning electron microscopy (SEM) investigation of immediately fixed human adult cochleae obtained during surgery for petro-clival meningiomas conveyed new information about morphology.

Objective To investigate the ultrastructure of human adult cochleae using SEM.

Material and methods Two human cochleae were decalcified, fixed with glutaraldehyde and osmium and prepared for SEM.

Results The excellently preserved morphology showed the pathways of nerve fibres through the organ of Corti. Undulating lateral cell membranes of Hensen and Claudius cells created an enlarged surface that may be important for homoeostasis. The distal attachment of the tectorial membrane to the reticular lamina was present in the shape of a marginal net, which was extended through marginal pillars. Stereocilia imprints extended as far as the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cells attached to the marginal pillars raises questions about differences in the excitation of the last row of outer hair cells as a result of differences in the composition of the tectorial membrane.     

Glycoconjugates in the chinchilla tubotympanum.

Various biotinylated lectins were used to characterize and semiquantitate glycoconjugate residues in the tubotympanum. Epithelial goblet cells were stained predominantly by WGA, LFA, SNA, RCA-I, Con-A, LCA, SBA, PHA-E, and UEA; this finding suggests they contain alpha-neuraminic acid, beta-galactose, alpha-mannose, N-acetyl alpha-galactosamine, and alpha-fucose. Glandular mucous cells were stained predominantly by WGA, LFA, SNA, and RCA-I; this finding suggests that they contain alpha-neuraminic acid and beta-galactose. The glandular serous cells were stained predominantly by Con-A, WGA, and LFA; this finding suggests that they produced alpha-mannose and alpha-neuraminic acid that represented serum-type glycoprotein. The positive staining of epithelial goblet cells and glandular mucous cells with PNA after neuraminidase digestion suggests that they produced mucin-type glycoproteins. The staining of the mucous blanket by WGA, LFA, SNA, RCA-I, LCA, PNA, SBA, PHA-E, and UEA suggests the presence of alpha-neuraminic acid, beta-galactose, N-acetyl alpha-galactosamine, and alpha-fucose. The epithelial cell (nonsecretory) surface was stained largely by WGA, LFA, SNA, RCA-I, Con-A, and LCA; this finding suggests the presence of alpha-neuraminic acid, beta-galactose, and alpha-mannose.     

High resolution scanning electron microscopy of the human organ of Corti. A study using freshly fixed surgical specimens

Scanning electron microscopy on immediately fixed human cochleae obtained during surgery for life-threatening petro-clival meningioma showed excellently preserved morphology. We compared the morphological findings with those from transmission electron microscopic sections of well preserved human and animal tissue. The characteristics of neural innervation, the pathways of the nerves through the organ of Corti and the intimate relation of nerves to supporting cells along their route could be studied in detail. The lateral membranes of Hensen and Claudius cells were folded creating a surface enlargement. Marginal pillars extended the distal end of the tectorial membrane and correspond to the marginal net or "randfasernetz" described earlier. Stereocilia imprints at the undersurface of the tectorial membrane go as far as to the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cell, attached to the marginal pillars, raises questions about differences in the excitation of the last row of outer hair cells. The complex nature of many supporting cells, stria vascularis and Reissner's membrane, intracellular complexities as well as surface features are described. Supernumerary inner hair cells were observed and the different arrangement of outer spiral fibres in contrast to findings in animals and variations of nerve fibres within the organ of Corti between apex and base are discussed.     

Expression von Lektinbindungsstellen als prognostischer Parameter bei Mundschleimhautkarzinomen

BACKGROUND: Normal oral mucosa and invasive squamous cell carcinoma of the oral mucosa and the lips were examined immunoenzymatically for different lectin-binding sites and evaluated semiquantitatively. In 110 patients suffering from invasive squamous cell carcinoma of the oral mucosa or the lips, the course of the disease was examined retrospectively with regard to metastases, recurrence and survival time over a minimum of 60 months and was correlated with cell surface binding to lectins (PNA, WGA, UEA I) of the primary tumor. Normal oral mucosa shows intensive cell surface binding to the lectins PNA, WGA and UEA I, but in more than half of the cases the PNA-defined epitopes are blocked by sialic acid. RESULTS: The comparison of the cumulated survival rates showed a much better prognosis for primary tumors with lectin-binding sites than for those without. Strong and medium WGA-binding pT2 tumors showed significantly better survival rates than those pT2 tumors with poor WGA-binding sites. Carcinomas without lectin binding showed the highest recurrence risk in the course of the disease. In squamous cell carcinomas without WGA and UEA I bindings, the period of time before a relapse occurred was significantly shortened. Carcinomas expressing UEA I-binding sites showed a considerably lower metastasis rate than UEA I-negative tumors. Squamous cell carcinomas with complete sialic acid-blocked PNA-binding sites had the same lymph node metastasis rate as those without expression of PNA-specific disaccharides. A significantly lower metastasis rate than both these groups was exhibited by tumors without or with only partially blocked PNA-binding sites. DISCUSSION: In addition to aiding routine grading, the study of PNA-, WGA- and UEA I-binding patterns can therefore provide further information in cases of oral squamous cell carcinoma regarding the potential for metastasis and tumor prognosis.     

Localization of the marginal zone of the tectorial membrane in situ, unfixed, and with in vivo-like ionic milieu.

In order to determine the normal positional relationship between the tectorial membrane and the organ of Corti, a preparation method was developed which made it possible to study the unfixed tectorial membrane in its normal position in relation to the cochlea and with in vivo-like ionic conditions. With this method, post-mortem changes visible with the light microscope were detectable after 60 to 90 min instead of the normal 30 min. When endolymph of artifical endolymph are present in the scala media, the marginal zone lies in close contact with the surface of the organ of Corti. If the endolymph is replaced by artificial perilymph, first the marginal zone of the tectorial membrane, and later the whole membrane shrinks. At this stage, latex particles suspended in the perilymph are free to enter the subtectorial space.     

Mechanical properties of the tectorial membrane in situ

According to the classical model of cochlear hair-cell stimulation, the tectorial membrane moves in cross-section like a stiff beam, rotating around the lip of the spiral limbus. This produces a shearing motion against the reticular lamina and, as a result, a radial deflection of the hair-cell stereocilia. The deflection can be effectively produced by the tectorial membrane only if its stiffness in the radial direction is greater than that of the stereocilia. We were able to manipulate the tectorial membrane through a scala-media access in live Mongolian gerbils and to measure its transverse and radial stiffness. We found the membrane to behave like a rubber band and to be much less stiff than the stereocilia. This is incompatible with the classical model. The tectorial membrane must act on the stereocilia as a mass load rather than a stiff anchor.     

Localization of the marginal zone of the tectorial membrane in situ,unfixed, and with in vivo-like ionic milieu

Summary In order to determine the normal positional relationship between the tectorial membrane and the organ of Corti, a preparation method was developed which made it possible to study the unfixed tectorial membrane in its normal position in relation to the cochlea and with in vivo-like ionic conditions. With this method, post-mortem changes visible with the light microscope were detectable after 60 to 90 min instead of the normal 30 min.When endolymph or artificial endolymph are present in the scala media, the marginal zone lies in close contact with the surface of the organ of Corti. If the endolymph is replaced by artificial perilymph, first the marginal zone of the tectorial membrane, and later the whole membrane shrinks. At this stage, latex particles suspended in the perilymph are free to enter the subtectorial space.This work was carried out in the Sonderforschungsbereich 50 Kybernetik München, which is supported by the Deutsche Forschungsgemeinschaft.     

Tectorial membrane. II: Stiffness measurements in vivo

The tectorial membrane is assumed to play a crucial role in the stimulation of the cochlear hair cells and was thought for decades to serve as a stiff anchor for the tips of the hair-cell stereocilia, particularly those belonging to the OHCs. Yet, its stiffness has never been measured under conditions approximating its normal environment in live animals. We have developed a method for doing this. The tectorial membrane is approached through the lateral wall of scala media. The bony cochlear capsule is removed along scala media over somewhat less than 1/4 turn, and the underlying spiral ligament and stria vascularis are carefully reflected. With the help of a three axial hydraulic manipulator, a flexible micropipette filled with isotonic KCl is inserted into the tectorial membrane at one of two different angles and moved either transversally, away from the basilar membrane, or radially, toward or away from the modiolus. This causes the tectorial membrane to be deformed and the micropipette to bend. The micropipette stiffness is calibrated on an instrument of a new kind, so as to convert the bend into force. The calibration allows us to determine the point stiffness of the tectorial membrane from the amount of micropipette bend. The stiffness of the tectorial membrane per unit length has been calculated from the point stiffness with the help of the deformation pattern. Transversal and radial stiffness magnitudes have been determined in the second cochlear turn in Mongolian gerbils. Both are smaller by almost an order of magnitude than the corresponding aggregate stiffness of the OHC stereocilia. As a consequence, the tectorial membrane cannot act as a stiff anchor for the stereocilia but only as a mass load, except at relatively low sound frequencies where mass effects are negligible. This means that the classical model of shear motion between the tectorial membrane and the reticular lamina must be replaced.     

Lectin detection of carbohydrates in the endolymphatic sac

The carbohydrate contents of the guinea pig endolymphatic sac were investigated by the use of lectins. The lumen of the endolymphatic sac was filled with stainable precipitate containing N-acetyl glucosamine, mannose, glucose, galactose and fucose. N-Acetyl galactosamine was also detected but in minute amounts. This composition corresponded to other areas in the inner ear, such as the cupula, the otolithic membrane and the tectorial membrane. The function of these carbohydrates may play an important role in preventing the lumen of the endolymphatic sac from collapsing as well as in regulating transepithelial fluid transport.