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.     

Glycerol effect on the guinea pig tectorial membrane

Summary The tectorial membrane (Tm) of guinea pigs has been found to have an altered organization of its matrix fibers in response to intravenously administered glycerol. Following treatment, the Tm middle zone shows an increase in waviness and clumping of fibers in nonhydropic and several hydropic ears in contrast to nontreated control ears. Residue of the internal sulcus cells occasionally fills the subtectorial space. In the present study, additional investigations were performed with scanning electron microscopy in order to study the relationship between the Tm and the organ of Corti, as well as the relationship between Hensen's stripe and the inner hair cell. Present findings provide evidence for a connection between the inner hair cell stereocilia and Hensen's stripe which may be the molecular basis for the modulation of hearing during the glycerol test in a patient with Meniere's disease. Correspondence to: A. M. Meyer zum Gottesberge     

Rotated stereociliary bundles and their relationship with the tectorial membrane in the guinea pig cochlea

Hair cells with rotated stereociliary bundles have been observed in the cochleae of control and kanamycin-treated guinea pigs. The affected outer hair cell bundles have a variable degree of rotation, with some being completely reversed. The inner hair cells are more rarely affected, and only small areas of an individual inner hair cell bundle are abnormal. In counts from ten cochleae, the number of outer hair cells with rotated bundles was most commonly between 10% and 20%, with almost 27% of all outer hair cells affected in the most extreme case. The rotated outer hair cell bundles often have distorted outlines but in other respects closely resemble normal bundles. In particular, they have the usual gradation in stereociliary height, intracellular cross-links and intercellular links to adjacent normally-orientated bundles. There are also corresponding imprints in the tectorial membrane which match the pattern of the stereocilia. In kanamycin-treated guinea pigs, imprints of both normal and rotated hair bundles are present, even when the corresponding bundle is absent, and there are frequently remnants of stereocilia inserted in the imprints. These observations suggest that, apart from their abnormal orientation, the rotated bundles are similar to normal bundles in both their organization and association with the tectorial membrane. The implications of this with respect to transduction and cochlear mechanics are discussed.     

Guinea pig tectorial membrane profile in an in vitro cochlear preparation

The guinea pig cochlea was examined under high-magnification light microscopy in an in vitro preparation. After extraction of the otic capsule, the bulla was opened widely and a small hole made into the fourth turn of the scala vestibuli. The organ of Corti was visualized under artificial endolymph at 600 X magnification. Added 1-micron titanium dioxide particles settled on the upper surface of the transparent tectorial membrane. Particle positions showed that much of this upper surface lay in a flat sheet that extended centrifugally almost to the Hensen's cells, giving the impression it was attached there. The sheet extended at least to the level of the inner hair cells, where a tectorial membrane thickness of about 40 micron was reached. Titanium dioxide particles were seen regularly in immediate proximity to the hair cell cilia, indicating that scala media is continuous with the subtectorial space. Upon mechanical manipulation, Hensen's cells proved to be extremely cohesive and elastic. It is suggested that hair cell stereocilia provide major mechanical connections for the tectorial membrane.     

Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma

Damage to the tectorial membrane caused by acoustic trauma was examined with scanning and transmission electron microscopy immediately after exposure and at selected time points over a 10 day recovery period. At 0 h of recovery the structure of the tectorial membrane overlying the region of hair cell damage was severely disrupted and connections between the membrane and the basilar papilla were lost. By 24 h of recovery, regeneration of the tectorial membrane was evident in the secretion of new matrix materials by the supporting cells of the basilar papilla. By 10 days of recovery a new honeycomb-like matrix had replaced the segment of damaged tectorial membrane, re-established connections with hair cell stereocilia and become fused with adjacent regions of undamaged tectorial membrane. However, the regenerated segment included only the honeycomb-like structure of the lower layer of the normal tectorial membrane. The laterally-oriented fibers which form the upper layer of the membrane were not regenerated over the damaged region. These findings indicate that the tectorial membrane is regenerated in parallel with the hair cells during recovery from acoustic trauma but the full extent of this recovery and its effect on cochlear function are not yet clear.     

Hereditary deafness in the cat: An electron-microscopic study of the tectorial membrane

Summary The tectorial membrane is affected at an early stage of the cochlear degeneration in the hereditarily deaf white cat. The membrane first descends towards the organ of Corti with obliteration of the intervening sub-tectorial space in the basal coil during the second post-natal week. Both the microvilli of supporting and Hensen's cells, and the hair cell stereocilia make deep indentations on the under-surface of the membrane. Cells are found insinuated between the tectorial membrane and Corti's organ, and numerous cellular processes occur within the former. A phagocytic function would appear probable for these cells, which seem to originate from the internal sulcus region. The membrane is retracted into the latter around the 2-month stage. At all ages, small spherical structures, which may represent altered interdental cell secretions, are found within the membrane, these becoming calcified in older animals.This investigation has been supported by the Norwegian Research Conncil for Science and the Humanities through grant C.37.59-1     

Factors affecting the onset of inner ear function

The developing inner ear receptors have a very significant influence on the onset of stato-acoustic function and on its evolution. The factors which prevent the stato-acoustic system from functioning are called 'the limiting factors'. At present, it is possible to postulate that these factors are restricted to the inner ear cells and related structures. At least four places are particularly relevant for the onset of function: (1) connections of the apical part of hair cell with the tectorial membrane; (2) the internal structure of hair cell; (3) connections between the base of the hair cell and nerve fibers; (4) the ganglion cell with its processes. Special emphasis is devoted to the apical part of the inner hair cell and its connections to the tectorial membrane which are considered as very important for the onset of the cochlear function. For the labyrinth, it is technically difficult to determine precisely the onset of function because of its early prenatal onset. Nevertheless, it is postulated that the limiting factors for the onset of function are also related to certain components of hair cells.     

Stereociliary bundles reorient during hair cell development and regeneration in the chick cochlea

We have examined changes in the orientation of stereociliary bundles of hair cells in the cochlear sensory epithelium that occur during normal embryonic development and during the regeneration of hair cells that follows acoustic trauma. At the time when hair cell surfaces become recognizable in the embryonic cochlea, the bundles of stereocilia exhibit a range of orientations, as indicated by the position of the kinocilium and later, by the location of the tallest row of stereocilia. With time, the orientations of bundles on neighboring hair cells become more uniform, a condition that is maintained in the adult. Changes in stereocilia orientation are also observed during the regeneration of hair cells after acoustic trauma. When new hair cells first differentiate at sites of trauma in the recovering sensory epithelium, their stereociliary bundles are not uniformly oriented. Then as the cells mature over a period of days, the bundles become aligned both with the neighboring bundles in the region of the previous lesion and with the pre-existing bundles that surround the site of regeneration. We conclude that the stereociliary bundles of hair cells are reorienting as the cells differentiate. A common mechanism may guide reorientation both during embryonic development and during regeneration. Observations in living cochleae indicate that differentiating stereociliary bundles establish asymmetric linkages to the extracellular matrix of the developing tectorial membrane. During the growth of the tectorial membrane, its progressive extension across the surface of the sensory epithelium may exert traction forces through those asymmetric linkages that pull the bundles of the hair cells into uniform alignment.     

Imprints of the inner sensory cell hairs on the human tectorial membrane

Summary Imprints indicating possible direct inner sensory cell hair contact with the tectorial membrane were observed in the cochlea of a 77-year-old woman under a scanning electron microscope (SEM). The imprints were seen in the lower and upper basal cochlear turns but not in the apical and middle turns. The small dot of imprints numbered from a few up to 12 and were arranged in various forms rather than straight lines. Contact between the tectorial membrane and inner and outer sensory cell hairs of the human cochlea was discussed from the SEM findings found in this case.     

Stereociliary bundle morphology in organotypic cultures of the mouse cochlea

Organotypic cultures of the neonatal mouse cochlea have a band of hair cells consisting of 3-5 rows of outer hair cells and a single row of inner hair cells. The outer hair cell stereociliary bundles show progressive differentiation from the apical to the basal ends of the band. Undifferentiated apical bundles have a disk-like array of short stereocilia resembling microvilli. Partially differentiated bundles are hemispherical with poorly organized rows of thickly clustered stereocilia, which gradually increase in height in the direction of the kinocilium. More differentiated bundles remain hemispherical with many microvilli-like stereocilia, but have highly organized rows of sterocilia along the side nearest to the kinocilium, and well-defined height increments between the rows. Highly-differentiated, basal bundles usually have a 'V' or 'W' shape, although some can be almost polygonal. The basal bundles have 4-5 regular rows of stereocilia with a well-defined gradient in height across the rows, and very few microvilli-like stereocilia. Cross-links are only consistently observed in more differentiated bundles, where the rows of stereocilia are regular and have significant height increments across them. The links show a wide variety of forms and orientations not previously observed in other preparations. Spoke-like arrays of links project from the upper regions of many stereocilia and other stereocilia appear to bear distinct tip-to-side links, although with a variety of orientations. A similar variety of cross-links is observed in early postnatal cochleae in vivo, but not in the cochleae of adult mice, indicating that this variety may be a transient feature of sterociliary bundle development. In vitro, inner hair cell stereociliary bundles are often covered by overlying material from the developing tectorial membrane. The variations in morphology of inner hair cell bundles and their cross-links are similar to those of the outer hair cell bundles.     

A newly identified surface coat on cochlear hair cells

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.     

Pronounced infracuticular endocytosis in mammalian outer hair cells.

Endocytosis in cochlear hair cells was investigated by staining with the vital fluorescent dye FM 1-43, that partitions reversibly into membranes and is trapped in vesicles during endocytosis. The temporal development and spatial distribution of FM 1-43 induced fluorescence was investigated using confocal laser-scanning microscopy. FM 1-43 rapidly and intensely stained cochlear hair cells, leaving the supporting cells unstained. For short application (0.2-30 s), only the infracuticular region of outer hair cells (OHCs) was labeled, whereas for long application (30-60 s), the OHCs were also labeled in the infranuclear zone and along a central strand extending from the infracuticular zone down to the nucleus, as well as along the entire cell membrane. Except for the cell membrane, the infracuticular zone, directly below the cuticular plate, showed the most rapid and intense staining, and in most cases staining was spherically shaped with a diameter of 3-7 microm. Localization and size of this infracuticular staining coincided with Hensen's body, a specialized variant of the endoplasmic reticulum. In contrast to the OHCs, apical fluorescence of inner hair cells presented a homogeneous distribution. When OHCs were incubated in FM 1-43 for longer than 1 min, many points of contact between the central strand, the infracuticular zone and the lateral cell membrane were observed. Since Hensen's bodies are a specialty of OHCs and the fluorescent staining pattern of these cells was unique, it is proposed that Hensen's body is involved in the turnover of OHC-specific proteins, such as those involved in the molecular machinery of the motor action of the plasma membrane.     

The inner ear as an electrosensory sense organ]

A model for cochlear perception is introduced based on the consideration that the organ of Corti is an electrosensory organ like that found in fish physiology during phylogenetic development. The inner hair cells work as electroreceptors, the outer hair cells as electrocytes. A layer of potassium ions on the lower surface of the tectorial membrane causes the excitation of the inner hair cells as soon as contact with a stereocilium occurs. A model for the basilar membrane motion, based on mathematical considerations and in accordance with the typical tuning curves of single auditory nerve fibres can serve as basis to explain the results of frequency and intensity analyses if we assume an electric excitation of the inner hair cells.     

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.     

What have lizard ears taught us about auditory physiology?

The structure of the basilar papilla of the inner ear of lizards is the most diverse among all vertebrates. Research on a variety of lizard ears, animals that are remarkably robust under laboratory conditions, has provided the field of auditory research with valuable information, particularly on the minimum structural requirements for sensitive, selective hearing and on the importance of the tectorial membrane and active processes in this regard. Despite the absence of a tuned basilar membrane, lizard ears produce highly frequency selective hearing through micromechanical tuning of small, resonant hair-cell-tectorial units or of free-standing hair bundles. These units are driven by an active process that also underlies spontaneous and other otoacoustic emissions. Lizard ears provided the first in vivo evidence that the active process is calcium-sensitive and lies within the stereovillar bundles of the hair cells.     

Structure of the hairs on cochlear sensory cells

A study of cochlear sensory hairs has been made using transmission and scanning electron microscopy. The structure of the hairs and the relation to the hair cells and to the tectorial membrane is described. A number of micrographs demonstrate both the inner structure of the hairs and their relation to the surrounding structures.     

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.     

Localization of type II, IX and V collagen in the inner ear.

Types II and IX collagen are traditionally considered cartilage collagens; however, within the inner ear, types II and IX collagen have a more diverse distribution. In the adult gerbil, type II collagen is the major fibrillar component. In the otic capsule it is present surrounding the osteocytes embedded and branching in the periosteal layer, in the cartilaginous rests of the enchondral layer, and in the endosteal layer bordering the membranous labyrinth. In the regions of the sensory cells, type II collagen is found in the osseous spiral lamina, the connective tissue of the spiral limbus, the subepithelial tissue of the maculae in the vestibule and the cristae in the ampullae, and in the spiral ligament. It is present in the non-cartilaginous and acellular structures of the tectorial membrane over the cochlear hair cells and the vestibular membrane lining the semicircular canals. Type IX collagen, when present, in all cases co-localizes with type II collagen but is found in more limited regions. It is found only in the cartilaginous rests of the enchondral bone, the tectorial membrane and the vestibular membrane. Type V-like collagen, a connective tissue collagen, is found to have a complementary localization to types II and IX collagen within the interstitial bone of the otic capsule, the osseous spiral lamina and the tectorial membrane, but it is absent from the vestibular membrane. This report is the first documenting the co-localization of types II and IX collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Place code for pitch: a necessary revision.

It is widely believed that the location of the cochlear excitation maximum, which has been shown by Békésy to depend on sound frequency and move from the cochlear apex to its base as the frequency increases, is a code for subjective pitch. The pitch of a tone is known to be practically independent of sound intensity. If the location does determine the pitch, it too must remain invariant. At the 1990 meeting of the Collegium held in Basel, however, the first author reported compelling indirect evidence indicating that this may not be true. It suggested that, at least in the mid-portion of the cochlea, the most important for speech frequencies, the maximum moves toward the cochlear base as sound intensity is increased. We now have a direct verification of this inference. Recording alternating Hensen's cell potentials at two or three second-turn locations of each of several Mongolian gerbil cochleas, we observed that the maximum response produced by a single tone moved substantially toward the cochlear base as sound intensity increased. For example, an intensity increment of only 10 dB caused the maximum to move by about 0.225 mm. Since Hensen's cells are known to reflect closely the excitation pattern of the outer hair cells, similar to that of the inner hair cells, the discovery makes it impossible for the cochlear excitation maximum to be an adequate code for pitch. We observed, on the other hand, that the apical excitation cut-off did not depend on sound intensity. Every cochlear location investigated had its invariant characteristic cut-off frequency. It is possible, therefore, that the cut-off location provides the place code for pitch. These findings may have profound consequences for our understanding of auditory mechanisms as well as for the technology of cochlear implants.     

Potentiation of inner ear damage following electron beam irradiation with CDDP administration

The study was designed to examine the combined ototoxic effect of CDDP (Cisplatin, Cis-diammine dichloroplatinum) and electron beam irradiation, using guinea pigs. One group received physiological saline solution of 4 ml/kg/day, and another group received CDDP of 2 mg/kg/day for five days. And following the injection of saline or CDDP, the electron beam of 14Gy/day was applied to the both groups to the right ear for five days. Animals were sacrificed after 21 days, and temporal bones of these animals were removed for the inner ear histopathology. Temporal bones were classified into four groups (control, electron beam irradiation, CDDP administration, and combined administration group), and the inner ears were observed by the surface preparation technique with a phase contrast microscope, and by scanning and transmission electron microscopy, and by temporal bone study of serial sectioned slides. The main pathologic findings of the inner ear are as follows: Electron beam irradiation group showed no hair cell damage. CDDP group induced slight damage to the outer hair cells. Combined administration group (CDDP + electron beam irradiation) showed severe outer hair cell damage. Stria vascularis was degenerated moderately in the combined administration group and slightly in some animals of electron beam irradiation group. And Reissner's membrane and Hensen's cells, were damaged in the basal turn of cochlea of the combined administration group. All other cochlear structures (spiral ganglion, spiral lamina, basilar membrane, spiral prominence, tectorial membrane, blood vessels of the cochlea) and vestibular organs were lack of significant changes in all groups by a light microscopic observation. This study was clarified that combined administration of CDDP and electron beam irradiation showed severe ototoxic potentiation. Therefore, it is important that we must pay attention to the inner ear damage caused by combined therapy of CDDP and electron beam irradiation involving inner ear for the head and neck tumor.