Outer Hair Cell Stereocilia Research Articles

A characteristic protein highly expressed in guinea pig inner ear, defined by monoclonal antibody WH-1

A new monoclonal antibody (termed WH-1; isotype IgG 2b) was established using a homogenate of dissected guinea pig cochleas ( N = 60) as immunogen. Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) identified the WH-1 antigen as a protein or glycoprotein with M r ≈ 40 kDa. Immunoperoxidase treatment of histologie cryosections of guinea pig cochlea, followed by light microscopic examination, revealed strong positive staining at three sites: (i) parts of Hensen's stripe, marginal band, covering net, and Kimura's membrane (within the tectorial membrane [TM]); (ii) Deiters' cells, pillar cells, Hensen's cells, and stereocilia of outer hair cells (within the organ of Corti); (iii) interdental cells, inner and outer sulcus cells, Reissner's membrane, and surface membrane of stria vascularis epithelium. Similar staining patterns were observed for cryosections of rat and mouse cochleas. Only a trace quantity of cross-reacting protein was detectable in brainstem. The protein was not detectable in tongue extract by Western blotting. However, sections of brainstem and tongue did show positive immunohistological staining with WH-1. Localization of WH-1 antigen was further examined by electron microscopy. WH-1 positivity on outer hair cell stereocilia, certain sites on the TM, interdental cell surface, Reissner's membrane epithelia, and inner and outer sulcus cells was confirmed. WH-1 antigen was not detected on inner hair cell stereocilia by light or electron microscopy. The localization of WH-1 antigen on outer hair cell stereocilia and TM suggests that it may play some role in adhesion between these structures.

3-D analysis of F-actin in stereocilia of cochlear hair cells after loud noise exposure

Fluorescence microscopy can be a useful tool in the early detection of pathological changes in the stereocilia of outer hair cells which have undergone acoustic overstimulation. Fluorescent phalloidin, a highly specific F-actin stain, can be used to label F-actin in stereocilia. In this study, phalloidin label is used to determine quantitative changes of F-actin in the stereocilia of guinea pigs exposed to loud noise (117 dB; octave band noise, centered at 1 kHz; 4 h). Reliably determining three-dimensional (3-D) structural changes in stereocilia is a challenging problem in optical microscopy since stereocilia diameter is close to the optical resolution limit. In order to alleviate the problem, a computational 3-D microscopy technique is used (Avinash et al., 1992). Whole-mounts of the cochlear second and third turns were examined in a Leitz Orthoplan microscope through a Leitz Plan Apo objective lens (100 ×; 1.32 N. A.; 170 0.17 ). Images were acquired with a charge-coupled device camera where the focus was shifted in 0.2 μm steps using a piezoelectric translator. Images were processed with the appropriate point spread function of the optical system. Analysis of control cochleas indicate that our technique can resolve single stereocilia and distinguish between various intensities of label along each stereocilia. In noise-exposed cochleas, our data show length and intensity changes in the phalloidin label. These results suggest that both depolymerization and polymerization of F-actin can occur in stereocilia of outer hair cells after acoustic overstimulation. Our findings demonstrate the applicability of computational 3-D microscopy to quantitative and qualitative analysis of stereocilia.

Normal structure of stereocilia and recovery from ciliary damage in the organ of Corti after acoustic overstimulation

The normal structure of outer hair cell (OHC) stereocilia in the organ of Corti, as well as damage and the recovery of OHC stereocilia after acoustic overstimulation, were demonstrated using scanning electron microscopy and tannic acid-osmium staining techniques. The highest row of OHC stereocilia is known to show an orderly gradation in height along the length of the cochlea. The present study demonstrated that the middle and lower rows of stereocilia possess a similar height gradation pattern. These findings suggest that the orderly gradation of stereocilia may play an important role in the tuning capability of the organ of Corti sensory cell. To investigate the mechanisms of recovery from ciliary acoustic damage, guinea pigs were exposed to a 4.00 kHz pure tone at an intensity of 120 dB for 120 minutes. All animals showed temporarily elevated hearing thresholds, which had returned to normal one week later. The first detectable change after acoustic overstimulation was a derangement of the cilia with a loss of ciliary interconnections. The tip links connecting the tips of the stereocilia to their taller neighbours were also affected showing elongation or disappearance. In comparing ciliary damage immediately after and one week after acoustic overstimulation, no signs of recovery in hair cell cilia which had been already lost, could be detected, while stereocilia with slight damage seemed to recover in the early post-sound exposure stage. Some lost tip links also seem to reappear in surviving cilia. In addition side links have the possibility of recovery.

Atrophy of outer hair cell stereocilia and hearing loss in hydropic cochleae

We have earlier described selective atrophy of short and middle stereocilia on outer hair cells of the three upper cochlear turns in hydropic cochleae of guinea pigs. The present study describes sequential early stages of stereocilia degeneration leading to this specific atrophy. Comparison of the morpho-pathology with the ultimate CAP audiograms taken before sacrifice indicated a close association between the low frequency hearing loss and this atrophy of stereocilia. The atrophy appeared to be associated first with the short and then the middle stereocilia of the 2nd and 3rd rows of outer hair cells between 0.5 kHz and 2 kHz and with time included the 1st row of all outer hair cells of the upper cochlear turns down to the 8 kHz region.

Noise-induced Hearing Loss in Iron-deficient Rats

The role of iron deficiency in noise-induced hearing loss (NIHL) was evaluated in 64 rats of four different experimental groups. Iron-deficient rats (ID-rats) and normal rats (N-rats) were simultaneously exposed to a steady state white noise (20-10,000 Hz) at 110 dB SPL for 30 min. Unexposed ID- and N-rats served as controls. In N-rats the temporary threshold shifts (TTS) would have completely disappeared if the animals were allowed to survive for 72 h. No permanent threshold shift (PTS) was seen in any of the N-rats. The ultrastructural correlates in N-rats are stereocilia disarray and mitochondria swelling in outer hair cells (OHCs). The TTS in ID-rats were larger than those in the N-rats, and most ID-rats with larger threshold shifts showed varying degrees of PTSs at 11 days post-exposure. The ultrastructural correlates of NIHL in ID-rats are obvious pathology of the stereocilia, such as segmental coalescence of stereocilia of many continuous OHCs and fusion of the tips of stereocilia of OHCs, and a significant reduction of mitochondria as well as slight degeneration of nucleus in the OHCs. It is concluded that iron deficiency can provide a pathological basis for NIHL.

Does electrical stimulation of the crossed olivo-cochlear bundle produce movement of the organ of Corti?

Low-frequency microphonic waveforms have been recorded in the basal turn of the guinea pig cochlea with and without electrical stimulation of the crossed olivocochlear bundle (COCB) at the floor of the fourth ventricle. Stimulation of the COCB increased the amplitude of the microphonic waveforms as described previously, but did not alter the shape of the waveforms markedly. The changes observed with COCB stimulation are consistent with a reduction in the impedance of the basolateral wall of the outer hair cells by about 50%, and possibly a 20% increase in the vibration of the organ of Corti at low frequencies, but suggest little or no change in the operating point on the transfer curve relating deflection of the hair bundles to the receptor current through the hair cells. It therefore seems that if slow contraction of the outer hair cells occurs during acute efferent stimulation in vivo, then it produces only a small deflection of the outer hair cell stereocilia, equivalent to a transverse displacement of the organ of Corti of less than 1.5 nm.

Observation of the Glycocalyx of the Organ of Corti: An investigation by electron microscopy in the normal and gentamicin-treated guinea pig

The ultrastructure of the glycocalyx of the organ of Corti in the normal and gentamicin-treated guinea pig was investigated at the electron microscopic level by using the ruthenium red staining technique. The glycocalyx was evident over the whole apical surface of both sensory and supporting cells. The entire length of the stereocilia of both inner and outer hair cells was seen to be covered by a glycocalyx, which interconnected the neighbouring stereocilia. In the gentamicin-treated animals, the fusion process between the stereocilia was revealed and a decrease of the glycocalyx was noticed at the point where fusion started. These findings suggest that the glycocalyx may play an important role in holding the stereocilia together in a bundle, yet prevent their close contact by means of its strong negative charge.

音響刺激後のコルチ器聴毛間連結機構の変化

Changes in the glycocalyx structure and ciliary interconnections of the organ of Corti were demonstrated after acoustic overstimulation. A special high resolution scanning electron microscope and a tannic acid-osmium staining technique giving an almost three-dimensional view were used for this demonstration. Guinea pigs were exposed to a 3.85kHz pure tone at an intensity of 120dB for 22.5min. It was possible to observe the early changes of the ciliary interconnections of the hair cell stereocilia. The first detectable change was a disarrangement of the cilia with a loosening of the interconnections. The ciliary membrane had an abnormally smooth appearance. The tip links connecting the tips of the stereocilia to their neighbours were also affected showing elongation or even disappearance. The fine granules which cover the tips of the tallest stereocilia of outer hair cells were also decreased. These findings suggest that acoustic overstimulation probably affects carbohydrate metabolism which induces a regeneration of ciliary interconnections resulting in a disarrangement and detachment of cilia. The tip links, which appeared to be involved in sensory cell transduction, seem also to be affected by acoustic overstimulation.

Characteristics of the membrane of the stereocilia and cell apex in cochlear hair cells.

Freeze-fracture has been used to examine the membrane of the cell apex and of the stereocilia in cochlear hair cells. The apical (non-stereociliary) membrane of inner hair cells (IHCs) exhibited a lower density of intramembrane particles (IMP) than that of the outer hair cells (OHCs) but in both cell types the apical membrane responded to the effects of filipin. The distribution of IMP and of filipin-induced membrane deformations was uniform over the apical membranes in both IHC and OHC, thus, providing no evidence for local membrane differentiation on the non-stereociliary part of the hair cell apex. The stereociliary membranes of IHC and of OHC differed not only in the density of IMP, but also in their responses to filipin and to tomatin. IHC stereocilia responded intensely to both agents. OHC stereocilia showed a significantly lower density of filipin-induced lesions and appeared almost unaffected by tomatin. This suggests that the OHC stereocilial membrane may be structurally specialized. The membrane at the apical end of stereocilia appeared to be differentiated from the membrane of the stereociliary shaft. The tip region was free of the usual IMP and showed no filipin-induced lesions. The differentiation at the apical end was also apparent in samples which have been rapidly frozen without prior chemical fixation or cryoprotection, showing that the particle-free area was not an artefact induced by glutaraldehyde fixation. Close examination of the membrane at the apical-most tip of the stereocilium revealed the presence of a small number of large particles of 10.5-11.0 nm diameter. The occurrence of membrane differentiation localized to the tip of the stereocilium may be consistent with the suggestion that transduction channels in hair cells are situated at this point.

Sharp mechanical tuning in a cochlear model without negative damping.

It is possible for a cochlear model without active negative damping to exhibit a mechanical response peak that is arbitrarily high and arbitrarily wide. Conventional active models rely on external energy inputs to the cochlea to produce the required peak shape. The new model proposed here produces very similar response profiles by assuming that outer hair cell stereocilia stiffness suppresses the mechanical motion in all regions basal of the response peak. Therefore, the model is not active in the usual sense of adding energy to the cochlea. The model suggests a reason for the differing basilar membrane structure in the arcuate and pectinate zones and simulates in vivo and postmortem responses similar to those measured in the real cochlea.

Pure tone overstimulation changes the micromechanical properties of the inner hair cell stereocilia

The effect of permanent noise-induced hearing loss on the auditory brainstem response (ABR) and the micromechanical properties of cochlear hair cell stereocilia in guinea pigs was investigated. The threshold of movement of the stereocilia was measured by applying force from a fluid filled pipette. After exposure to a 1.0 kHz pure tone signal at 105 dB(A) for 72 h the threshold of the ABR was broadly elevated by approximately 50 dB. Inner hair cell stereocilia showed a decrease in threshold while the outer hair cell stereocilia bundles remained unaltered. This effect was localized to the 13–15 mm distance from the stapes corresponding to the region of maximal stimulation. The effect was recorded within 1 h of exposure and remained constant with exposures up to 7 days. Following a one month recovery period from sound exposure, normal threshold values of stereocilia movement were observed, indicating recovery. At this time, swelling of the afferent dendrites beneath the inner hair cells was observed throughout the cochlea together with approximately 30% scattered loss of outer hair cells in the 13 to 15 mm region. The ABR showed some recovery (approximately 20 dB), yet a threshold shift remained.

Intercellular cross-linkages between the stereociliary bundles of adjacent hair cells in the guinea pig cochlea.

Hair cells of the guinea pig organ of Corti have been examined using high resolution scanning electron microscopy. In addition to the extensive array of cross-links between the stereocilia of individual hair cells which have been reported previously, we have seen examples of attachments between the stereocilia of both adjacent inner and adjacent outer hair cells. The implications of these observations are discussed.

A model for active elements in cochlear biomechanics.

A linear, mathematical model of cochlear biomechanics is presented in this paper. In this model, active elements are essential for simulating the high sensitivity and sharp tuning characteristic of the mammalian cochlea. The active elements are intended to represent the motile action of outer hair cells; they are postulated to be mechanical force generators that are powered by electrochemical energy of the cochlear endolymph, controlled by the bending of outer hair cell stereocilia, and bidirectionally coupled to cochlear partition mechanics. The active elements are spatially distributed and function collectively as a cochlear amplifier. Excessive gain in the cochlear amplifier causes spontaneous oscillations and thereby generates spontaneous otoacoustic emissions.

Morphological changes to the stereociliary bundles in the guinea pig cochlea after kanamycin treatment.

Pigmented guinea pigs were treated with 400 mg/kg of kanamycin for 8 days and sacrificed 13 days later. The upper surface structures of the organ of Corti were studied using high resolution scanning electron microscopy to reveal fine details of stereociliary fusion and damage to the reticular lamina. Outer hair cell stereocilia showed from partial to complete fusion and loss whilst the inner hair cells showed very little evidence of any damage. In particular, the possible effects of kanamycin on the cross-links between the stereocilia have been investigated. The cross-links are found to be present on all outer and inner hair cell bundles which show no fusion of stereocilia. Partially-fused stereocilia which have free tips still possess the upward-pointing links, and the remaining undamaged stereocilia in these bundles possess the normal cross-links. Where fusion occurs along the sides of the stereocilia, the side-to-side links are missing although whether their absence either results in, or else is caused by, the fusion is not known.

Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells

The distribution of actin, alpha-actinin, fimbrin, tropomyosin and tubulin in the apical region of inner and outer hair cells was studied by immunofluorescent localization of antibodies to these proteins. The macromolecular distribution of actin and alpha-actinin was studied using post-embedding immunoelectron microscopic techniques. Actin is present in the stereocilia and cuticular plate of both inner and outer hair cells. Antibodies to actin were localized with fluorescence and colloidal gold. Colloidal gold particles were distributed uniformly over the stereocilia, stereocilia rootlets and cuticular plate. Fimbrin is present in the stereocilia and the cuticular plate. Immunofluorescent label was more intense over the cuticular plate of outer hair cells than over the cuticular plate of inner hair cells. Alpha-actinin is present in the cuticular plate only. At the ultrastructural level, antibodies to alpha-actinin were labeled throughout the cuticular plate, with larger accumulations of colloidal gold over the electron dense bodies in the cuticular plate, as well as over the electron dense region at the junctional complex. There was no label over the electron dense portion of the stereocilia rootlets. Tropomyosin is observed in the area of the stereocilia rootlets by immunofluorescent techniques, but like fimbrin, the antigenic sites of tropomyosin did not withstand processing for ultrastructural localization. Tubulin is not present in the apical region of inner or outer hair cells, although its presence could be documented in the hair cell body and in the supporting cells.

Scanning electron microscopy of the cochlea in rats with Streptococcus pneumoniae otitis media.

This investigation was performed to determine the morphologic effects of bacterial otitis media on the organ of Corti. The middle ear cavities of rats were inoculated with Streptococcus pneumoniae or saline and the animals were killed on days 1, 4, 7, 10, 14, and 21 after inoculation. Middle ear cultures were obtained and the cochleas were examined using scanning electron microscopy. All animals killed on day 1 had positive cultures, but by day 21, all cultures were negative. Cochlear changes observed were (1) damage to supporting cells (Deiters' cells), (2) morphologic changes of hair cell stereocilia, and (3) loss of inner and outer hair cell stereocilia and cell bodies (to a lesser extent), especially in the lower middle and basal turns. These changes appeared to occur in a definite sequence; ie, damage to the supporting cells, changes in stereocilia, and, finally, hair cell loss. These data show that cochlear damage and hair loss can be associated with bacterial otitis media.

Ultrastructural damage in cochleas used for studies of basilar membrane mechanics.

Cat cochleas used for interferometric studies of basilar membrane mechanics were examined with the electron microscope. The structures most severely damaged in the experimental cochleas are the outer hair cells and the radial afferent fibers to the inner hair cells. Since the basilar membrane and other supporting structures appear to be normal, mechanical changes observed in the experimental cochleas are most probably due to outer hair cell damage. Individual animals with varying degrees of damage showed large differences in the frequency of basilar membrane resonance at the same place in the cochlea. Shifts in tuning of this magnitude could occur as a consequence of hair cell damage only if the stiffness of the stereocilia and associated structures was greater initially than the stiffness of the basilar membrane and gradually decreased with damage. The present series of observations, therefore, suggest that the stiffness of the outer hair cell stereocilia determines basilar membrane tuning.

High-Voltage Electron Microscopic Study of the Inner Ear: Technique and Preliminary Results

The technique and some preliminary results of the application of high-voltage electron microscopy (HVEM) to the study of inner ear morphology in the guinea pig are reported in this paper. The main advantage of HVEM is that sharp images of thicker specimens can be obtained because of the greater penetrating power of high energy electrons. The optimum thickness of the sections examined with an accelerating voltage of 1,000 kV was found to be between 500 to 800 nm. The sections below 500 nm in thickness often had insufficient contrast, while those above 800 nm were rather difficult to interpret due to overlap of images of the organelles. The whole structure of the sensory hairs from the tip to the rootlet was more frequently observed in the 800-nm thick sections. Thus the fine details of the hair attachment to the tectorial membrane as well as the hair rootlet extension into the cuticular plate could be thoroughly studied in the HVEM. In specimens fixed in aldehyde containing 2% tannic acid, the attachment of the tips of the outer hair cell stereocilia to the tectorial membrane was observed. For the inner hair cells, however, the tips of the hairs were separated from the undersurface of the tectorial membrane. The majority of the rootlets of the outer hair cells terminated at the midportion of the cuticular plate, while most of the inner hair cell rootlets traversed the entire width of the cuticular plate and extended into the apical cytoplasm. These differences in ultrastructural appearance may indicate that the two kinds of hair cells play different roles in the acoustic transduction process. The three-dimensional arrangement of the nerve endings on the hair cells was also studied by the serial thick-sectioning technique in the HVEM. In general, an entire arrangement of the nerve endings was almost completely cut in less than ten 800-nm thick sections instead of the 50- to 100-ultrathin (ie, less than 100 nm) conventional sections for transmission electron microscopy. The present study confirms an earlier report that the first row outer hair cells in the third cochlear turn are innervated by nearly equal numbers of efferent and afferent endings, the average number being nine.

A nonlinear feedback model for the mechanical response of outer‐hair‐cell sterocilia

There is some experimental evidence that the mechanical cochlear function is strongly influenced by the efferent innervation of outer‐hair‐cell stereocilia (OHCS). [See, e.g., D. C. Mountain, Science 210, 71–72 (1980); J. H. Siegel and D. O. Kim, Hear. Res. 6, 171–182 (1982).] This suggests that the various nonlinear and active mechanical features of the cochlear response may be mainly rooted in the OHCS. We propose a specific nonlinear feedback model for the mechanics of the OHCS, which is a generalization of a linear model proposed by Mountain. It gives formally the combination of negative resistance and nonlinear damping used in previous calculations [S. Koshigoe and A. Tubis, J. Acoust. Soc. Am. Suppl. 1 71, S17 (1982)]. The parameters of the model are determined so as to account for experimental measurements of the basilar membrane response as well as stimulated and spontaneous emissions in the ear canal. [This work was supported by NSF.]

Correlation of audiometric data with changes in cochlear hair cell stereocilia resulting from impulse noise trauma.

In a previous experiment, after chinchillas had been exposed to impulse noise trauma, plastic-embedded surface preparations of the organ of Corti were examined with the light microscope. A consistent relationship between cochlear hair cell loss and hearing loss was not found (Hamernik et al., 1980). In the present study, four cochleas from that experiment were sectioned and examined with the transmission electron microscope to determine if their were consistent patterns of damage to the sensory cells at the ultrastructural level that would more closely correlate with the audiometric data. Alterations of the outer hair cell stereocilia were found when threshold was elevated 15 to 30 dB. The membranes of the stereocilia appeared loose and wrinkled and the stereocilia were no longer erect. In some cases, predominantly in the first row of outer hair cells, stereocilia were missing and in other cases, stereocilia were fused. Within these giant stereocilia, the rootlets of the individual stereocilia had disintegrated. Other alterations in sensory cell ultrastructure, though present, had no consistent pattern and could not be related to changes in hearing thresholds. Only the changes in the outer hair cell stereocilia appeared to correlate with hearing loss and the degree of damage was reflected in the amount of threshold elevation.