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.     

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.     

Lectin detection of carbohydrates in the endolymphatic sac

Summary 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.This study was supported by a grant from the Swedish Medical Research Council (no. 00720 X)     

Morphological changes in the tectorial and basilar membranes of aged rats

Using both light and transmission electron microscopy presbycusic degeneration of the cochlea was observed in particular in the tectorial and basilar membranes, in naturally aged rats. These animals showed a descending auditory pattern as determined by auditory brainstem response. Ultrastructurally, the number of collagen fibers in the tectorial membrane was reduced and straight type A fibers were increased relative to branched, coiled type B fibers. The basilar membrane in the basal turn was also thickened by an increased homogeneous ground substance. These findings indicate that the specificity of vibration of the tectorial and basal membranes is very different in aged and young rats.     

Acoustic overstimulation alters the morphology of the tectorial membrane

After a permanent threshold shift was induced by exposing guinea pigs to a 1 kHz pure tone at 105 dB(A) for 72 h, light microscopic observations of freshly dissected and stained tectorial membranes showed an increased waviness and clumping of the fibers of the middle zone. Hensen's stripe was not seen as a continuous dense structure running through the middle zone but was at times discontinuous and curved. As measured from cross-sections of the cochlea, the thickness of the tectorial membrane was decreased after acoustic overstimulation. The stereocilia of the inner and outer hair cells lie directly under the middle zone. Visual detection levels of threshold of tectorial membrane movement was determined by stimulating the marginal zone of the tectorial membrane of isolated cochlear coils by an oscillating water jet. After acoustic overstimulation the tectorial membrane became more compliant. The tectorial membrane abnormalities were restricted to the regions of the cochlea that demonstrated a 40–50 dB hearing loss.     

豚鼠耳蜗盖膜下面的超微结构

目的 利用扫描电镜技术详细地观察豚鼠盖膜下的超微结构,为耳蜗的感音机制提供新的认识.方法 用S-4800型超高分辨率扫描电子显微镜观察了6只豚鼠12只耳蜗的盖膜.结果 (1)耳蜗各圈盖膜下面均可观察到外毛细胞静纤毛压迹,这种压迹多半与静纤毛最高排的形态一致,第一圈呈“W”型,由基底圈到顶圈,逐渐由“W”型渐变为“V”型及不规则的簇状.压迹为一排圆形的小凹,将盖膜下表面表层辐射状纤维断开,只有最高排的静纤毛才与盖膜接触形成压迹,压迹排列呈串珠一样.(2)盖膜下内毛细胞相应的位置有一条较深的波浪状沟槽,呈线性结构,沟槽比外毛细胞静纤毛的压迹浅而宽,由耳蜗的基底圈到顶圈这种压迹逐渐呈带状.(3)盖膜下的纤维由盖膜的外缘到盖膜与内毛细胞静纤毛压迹之间以蜗轴呈辐射状排列,但在外毛细胞静纤毛压迹的位置被这些压迹所阻断,而总的辐射方向和纤维走行并没有改变,纤维非常的精细,每根纤维之间又相互交错相连.内、外毛细胞压迹之间,柱细胞顶部的盖膜纤维很细,均匀呈细丝状.而内毛细胞与螺旋缘之间,即内沟上方的盖膜下方的纤维更加细长.结论 通过对盖膜超微结构的观察,盖膜下内外毛细胞静纤毛的压迹充分说明毛细胞静纤毛与盖膜的接触.盖膜下的纤维由盖膜的外缘到柱细胞和柱细胞到盖膜下内毛细胞静纤毛带状压迹之间也就是内、外毛细胞压迹之间,柱细胞顶部的盖膜的纤维与内毛细胞顶部盖膜带状结构到螺旋缘之间的纤维的粗细都不一样.这种纤维的分布特点,可能与盖膜在声音调谐方面所起的作用有一定的关系.     

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.     

Displacement of the reticular lamina with and without the tectorial membrane in the guinea pig cochlea

OBJECTIVE: In an attempt to establish the mechanical relationships between the reticular lamina and tectorial membrane, we studied the morphological changes of the reticular lamina on a micrometer scale when an in vitro preparation of guinea pig cochlea with and without tectorial membrane was exposed to a potassium-rich medium. MATERIAL AND METHODS: Using video-enhanced differential interference contrast microscopy, the radial displacement of the inner hair cells (IHCs) and outer hair cells (OHCs) in the reticular lamina was measured in real time after exposure to the potassium-rich medium for 3 min. RESULTS: The amplitude of the displacement of the OHCs in preparations with an intact tectorial membrane was half of that observed in those in which the tectorial membrane had been removed. A similar displacement response was also observed for the IHCs, although it was smaller than that for the OHCs. There was no significant difference in the amplitude of the displacement among the three rows of OHCs. CONCLUSIONS: These results suggest that the structure linking the OHCs to the pillar cells is very elastic and that the movement of the OHCs in situ is weakly mechanically coupled to the IHCs. The tectorial membrane provides increased compliance in the motion of the IHCs and OHCs.     

Ultrastructural localization and semiquantitative analysis of glycoconjugates in the tectorial membrane.

The tectorial membrane of the gerbil cochlea was analyzed with lectin-gold cytochemical methods for demonstrating and characterizing glycoconjugates (GCs) in situ. Binding of lectins from Limax flavus (LFA), Lens culinaris (LCA), Datura stramonium (DSA), Ricinus communis (RCA I), Ulex europeus (UEA I) and Phaseolus vulgaris (PHA L) was assayed semiquantitatively on ultrathin sections. Binding occurred throughout the tectorial membrane with all lectins except UEA I but the labelling density with a given lectin differed among substructures. The cover net disclosed the highest level of GC with four lectins whereas the fibrous layer revealed the lowest level. DSA, LCA and PHA L demonstrated considerable similarity between the cover net and the marginal band in content of GC with N-linked oligosaccharide. The cover net differed from the marginal band, however, in containing more RCA I reactive GC with terminal lactosamine. Hensen's stripe, with which inner hair cell stereocilia are thought to interact, differed from other substructures in containing the highest level of PHA L-reactive traintennate N-linked chains and except for the basal layer the lowest concentration of GC with terminal lactosamine. Fucosylated GC detectable with UEA I-gold was present at low levels in all substructures except the cover net and marginal band. Distribution of GCs in the fibrous layer and less consistently in the cover net differed between limbal and middle zones. The differences observed here in the carbohydrate composition among substructures in the tectorial membrane support and extend previous cytochemical observations and imply a role for different classes of GCs in determining the biophysical and physiological properties of the tectorial membrane.     

Prevention of wound complications in salvage pharyngolaryngectomy by the use of well-vascularized flaps

Objective In an attempt to establish the mechanical relationships between the reticular lamina and tectorial membrane, we studied the morphological changes of the reticular lamina on a micrometer scale when an in vitro preparation of guinea pig cochlea with and without tectorial membrane was exposed to a potassium-rich medium.

Material and methods Using video-enhanced differential interference contrast microscopy, the radial displacement of the inner hair cells (IHCs) and outer hair cells (OHCs) in the reticular lamina was measured in real time after exposure to the potassium-rich medium for 3 min.

Results The amplitude of the displacement of the OHCs in preparations with an intact tectorial membrane was half of that observed in those in which the tectorial membrane had been removed. A similar displacement response was also observed for the IHCs, although it was smaller than that for the OHCs. There was no significant difference in the amplitude of the displacement among the three rows of OHCs.

Conclusions These results suggest that the structure linking the OHCs to the pillar cells is very elastic and that the movement of the OHCs in situ is weakly mechanically coupled to the IHCs. The tectorial membrane provides increased compliance in the motion of the IHCs and OHCs.     

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.     

Structural relationships of the unfixed tectorial membrane

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.     

Wheat germ agglutinin binding sites in the organ of Corti as revealed by lectin-gold labeling

The distribution of wheat germ agglutinin(WGA)-binding sites in the organ of Corti of the guinea pig and mongolian gerbil was studied. WGA was conjugated with gold particles and applied on thin sections of the cochlea embedded in Spurr's resin and in Lowicryl K4M. WGA-binding sites were found on the plasma membrane, lysosomes and cytoskeletons of hair and supporting cells as well as on the tectorial and basilar membranes. No distinct difference was discovered between hair cells and supporting cells in terms of WGA-binding activities.     

Cochlear changes after herpes simplex virus infection

After direct inoculation of herpes simplex virus (HSV) into the scala tympani of the guinea pig, the tectorial membrane showed various morphological changes: atrophy, roll-up and dot formation. Immunofluorescent and electronmicroscopic studies revealed that the changes were due to HSV infection. The findings were compared with those observed in the temporal bones of a 77-year-old patient who suffered from sudden deafness. The tectorial membranes of both temporal bones showed various changes identical with those observed in experimental viral labyrinthitis. This supports the view that sudden deafness in this particular patient was of viral origin. In the animal experiment, HSV antigen could be detected from the cochlea of the non-inoculated side, which was morphologically normal. Further study is required to reactivate HSV in the cochlea with latent infection. This animal can probably be used as an animal model for sudden deafness.     

Carbohydrates detected by lectins in the vestibular organ

In the vestibular organ the presence of carbohydrates in the cupula and otoconia of young (6-day-old) and adult rats was investigated using fluorescent lectins. The following sugars have been identified in both young and adult rats: N-acetyl-glucosamine, galactose, mannose and fucose. In contrast, N-acetyl-galactosamine was not detected. In order to demonstrate the specificity of the reaction, control experiments were performed after preincubation of the lectin with its specific inhibitory sugar. The same sugars were identified in calcified (i.e. otoconia) and non-calcified (i.e. cupula) structures. The role of these sugars in the mineralization and fusion process of otoconia is discussed.     

小鸡和豚鼠耳蜗毛细胞β-肌动蛋白分布比较

目的 :比较小鸡基底乳头和豚鼠耳蜗毛细胞 β-肌动蛋白 (β- actin)分布的特点。方法 :应用免疫组织化学方法观察小鸡和豚鼠耳蜗中β- actin免疫反应活性。结果 :鸡基底乳头高、矮毛细胞的静纤毛 ,盖膜根部附着处缘上皮细胞胞浆 ,豚鼠耳蜗三排外毛细胞胞浆 ,内、外支柱细胞胞浆和指状突β- actin免疫反应阳性。结论 :小鸡和豚鼠耳蜗毛细胞具有相同结构蛋白β- actin,但两种动物之间存在明显的分布差异。     

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).     

Changes in the stereocilia and non-monotonic pattern of threshold shift after exposure to impulse noise

The recovery pattern of threshold shift (TS) and 'dynamic' changes in the stereocilia after exposure to high-level impulse noise in guinea pigs were investigated. 33 albino guinea pigs were exposed to 10 impulse noises at the rate of 1/min. The noise had peak level of 166 dB SPL and a duration of 0.1 ms. Thirteen of the exposed animals were used to systematically measure threshold shifts at regular intervals from 0.5 h to 30 days post-exposure by click auditory cortex evoked response (AC-ER). Twelve of the animals who had typical TS at the same intervals were killed for scanning electron microscopic examination. The recovery pattern of threshold shifts was non-monotonic, which was different from that seen with continuous noise. There was an increase in TS after the exposure and the maximum level of TS was found at 8 h post-exposure. Morphological analysis also showed delayed changes in the stereocilia after exposure. The severity of changes in the stereocilia reached a peak at 8 h, at which time complete fusion of hair bundles took place. The tectorial material found on the tips of the stereocilia may be responsible for the sequence of the changes.     

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.     

The surface morphology of the avian tectorial membrane

The structure of the tectorial membrane of the chick was evaluated by scanning electron microscopy (SEM), using standard techniques, and, for the first time, by studying unfixed tectorial membranes with video-enhanced light microscopy techniques (AVEC-DIC). The SEM pictures show a widely varying morphology, ranging from a fully perforated tectorial membrane to a completely closed upper boundary, with a smooth surface. Based on several indicators, it is concluded that the latter presents the more natural state. This was confirmed by the results of the AVEC-DIC technique, which show a highly homogeneous structure. In contrast to the bulk of the tectorial membrane, its lower surface shows discrete structures, especially regularly oriented fibril bundles.