Stereocilium injury mediates hair bundle stiffness loss and recovery following intense water-jet stimulation.

Inner ear hair cells exhibit many pathologies following exposure to intense sound, and the hair bundle is a major site of damage. This paper measures in vitro hair bundle motion on chick cochlear hair cells after intense in vitro and in vivo stimulation to explore the nature of hair bundle injury. Hair bundle stiffness, as well as relative and asymmetric motion of individual stereocilia, is controlled largely by the extracellular tip links, and a change in hair bundle motion was used to assess tip-link destruction following overstimulation. Intense in vitro stimulation caused a loss in stiffness that fully recovered within 10 min post-exposure. Relative and asymmetric stereocilia motion, however, were unchanged following the exposure, implying that tip links remained intact while the core or rootlet of the stereocilia were damaged and subsequently repaired. Intense and prolonged in vivo sound exposures produced stereocilia movements, measured in vitro, that were indicative of damage to stereocilia and tip links. Finally, the relative susceptibility of hair bundles to overstimulation was addressed by comparing stiffness loss with morphological features in the hair bundles. The loss of stiffness significantly increased as the amount of curvature in the hair bundle contour increased.

Targeted mutagenesis of the POU-domain gene Brn4/Pou3f4 causes developmental defects in the inner ear.

Targeted mutagenesis in mice demonstrates that the POU-domain gene Brn4/Pou3f4 plays a crucial role in the patterning of the mesenchymal compartment of the inner ear. Brn4 is expressed extensively throughout the condensing mesenchyme of the developing inner ear. Mutant animals displayed behavioral anomalies that resulted from functional deficits in both the auditory and vestibular systems, including vertical head bobbing, changes in gait, and hearing loss. Anatomical analyses of the temporal bone, which is derived in part from the otic mesenchyme, demonstrated several dysplastic features in the mutant animals, including enlargement of the internal auditory meatus. Many phenotypic features of the mutant animals resulted from the reduction or thinning of the bony compartment of the inner ear. Histological analyses demonstrated a hypoplasia of those regions of the cochlea derived from otic mesenchyme, including the spiral limbus, the scala tympani, and strial fibrocytes. Interestingly, we observed a reduction in the coiling of the cochlea, which suggests that Brn-4 plays a role in the epithelial-mesenchymal communication necessary for the cochlear anlage to develop correctly. Finally, the stapes demonstrated several malformations, including changes in the size and morphology of its footplate. Because the stapes anlage does not express the Brn4 gene, stapes malformations suggest that the Brn4 gene also plays a role in mesenchymal-mesenchymal signaling. On the basis of these data, we suggest that Brn-4 enhances the survival of mesodermal cells during the mesenchymal remodeling that forms the mature bony labyrinth and regulates inductive signaling mechanisms in the otic mesenchyme.

Low calcium abolishes tip links and alters relative stereocilia motion in chick cochlear hair cells.

The role of stereocilia tip links in controlling hair bundle motion on chick hair cells was examined in this study. Hair cells from the apical end of the basilar papilla were maintained in culture medium and oriented so that the sensory hair bundles were viewed in profile. A water-jet was used to stimulate the hair bundle and stroboscopic illumination allowed slow motion viewing of a sensory hair motion at the bundle edges. Motion of the tallest stereocilium in the bundle was set to a criterion angular deflection and the excursion of the shortest stereocilium was measured. These measurements were made in a sample of hair cells maintained in culture medium containing either near normal levels of calcium or very low calcium levels supplemented with EGTA. In low calcium the angular deflection of the shortest hair was significantly reduced from that observed in normal media. The resting inward tilt of the hairs in the bundle, however, did not change. Scanning electron microscopy verified an almost complete destruction of tip links after exposure to low calcium. These results suggest that tip links contribute significantly to the relative motion of stereocilia and exhibit the mechanical properties of a relatively stiff linkage.

Morphometric changes in the chick nucleus magnocellularis following acoustic overstimulation.

The present investigation considered the effects of cochlear damage caused by exposure to intense sound on the nucleus magnocellularis of the chick. Neonatal chicks exposed to intense sound were separated into four groups with post-exposure recovery durations of 0, 15, 27, and 43 days. Four age-matched, non-exposed control groups were also formed. At each recovery interval, the control and exposed birds were sacrificed and their brains prepared for paraffin embedding. The brain stem region containing the nucleus magnocellularis (NM) was serially sectioned in the coronal plane. All sections containing NM cells were identified and then coded in terms of their percentile distance from the most caudolateral section. Sections along the nucleus at the 15th, 30th, 50th, 65th, 80th, and 95th percentile positions were selected for evaluation, and the cross-sectional areas of individual NM cells in these sections were then measured. Cell areas were corrected for the bias introduced by eccentricity of the nucleus. The number of NM cells per 1,000 microm2 was also calculated at the 50th and 65th percentile positions. These procedures were repeated for the age-matched, non-exposed control animals. The cross-sectional cell area in exposed animals, immediately after the exposure, was reduced significantly at all positions, but returned to near normal by 43 days of recovery. However, the coronal area of NM in the sections at the 50th and 65th percentile position, as well as NM cell density, were unaffected by the exposure at all recovery intervals. The observation of structural recovery in NM cells at 43 days post-exposure was remarkable because it occurred at least 4 weeks after complete functional restoration of single-cell activity in the NM. The shrinkage in NM cell size throughout the nucleus may be due to a general reduction in spontaneous activity in the cochlear nerve fibers caused by the acoustic injury to the chick basilar papilla.

The effect of acoustic overexposure on the tonotopic organization of the nucleus magnocellularis.

We assessed the effect a sound-induced cochlear lesion had on the tonotopic organization of the nucleus magnocellularis (NM) immediately after acoustic overexposure and following a twelve day recovery period. The acoustic overexposure was a 0.9 kHz tone at 120 dB sound pressure level (SPL) for 48 h. Initially after the acoustic overexposure, the tonotopic organization of the NM was statistically different from that of age-matched controls. Specifically, it appeared that the center frequencies of units in the frequency region of the NM associated with the acoustic overexposure had higher center frequencies than their control counterparts. Following a twelve day recovery period, when threshold sensitivity and frequency selectivity were operating normally, the tonotopic organization of the NM was not statistically different from age-matched controls. We suggest that the sound-induced changes in the tonotopic organization of the NM reflect peripheral damage in the basilar papilla. It has been well documented that similar exposure paradigms produce a loss of short hair cells and a degeneration of the tectorial membrane in the region of the basilar membrane associated with the overexposure. We postulate that the loss of these structures alters the micromechanics and tuning of the basilar membrane which is reflected in the observed changes in NM tonotopy. Following the recovery period, when those structures destroyed by the overexposure had regenerated and basilar membrane micromechanics were operating normally, the tonotopic organization of the NM returned to normal.

Recovery of auditory function and structure in the chick after two intense pure tone exposures.

Groups of neonatal chicks were examined in three experimental conditions that differed in the age and number of times they were exposed to a pure tone of 0.9 kHz at 120 dB SPL for 48 h. Several animals were exposed once at 2 or 16 days of age, while others were subjected twice to the above stimulus, first at 2 days and then at 16 days. Evoked potential measures of threshold shift, obtained at 0, 12 or 26 days post-exposure, were used to determine the degree of hearing loss and recovery. The average threshold loss in the mid-range frequencies was about 57 dB at 0 days for all three conditions. This level was reduced to about 15 dB in all three groups at 12 days of recovery, while in birds exposed once at 2 days, but allowed 26 days to recover, the post-exposure thresholds returned to pre-exposure levels. Scanning electron microscopic analysis of cochlear structure was conducted in groups of similarly exposed and recovered animals. Twelve days post-exposure, the structural analysis revealed regeneration of a single honeycomb-like tectorial membrane layer in both the once and twice-exposed cochleae. However, damage to, and repair of, the tectorial membrane after the second exposure revealed the production of a second honeycomb layer in about half the animals examined. The results indicated that chicks retain the capacity to repair receptor epithelium damage and recover considerably from hearing loss after multiple exposures to intense sound.

Recovery of auditory structure and function in neonatal chicks exposed to intense sound for 8 days.

This paper describes hearing loss and recovery as well as cochlear damage in chicks, after a 200-h exposure to an intense pure tone, and compares the results to similar data following a 48-h exposure to the same sound. The results revealed that the magnitude of initial hearing loss and the rate of recovery were nearly the same for both exposures. The initial cochlear damage produced by the 200-h exposure, however, was less severe than that seen after the 48-h exposure. In addition, new hair cells were observed in the lesion area immediately after the 200-h exposure. However, after the 48-h exposure, they were first identified after several days of recovery. These observations were consistent with the conclusion that cochlear repair began during the longer exposure itself. The fact that hearing loss and recovery was the same for the two exposure conditions, while the level of cochlear damage differed, suggests that functional recovery depended on processes other than the regeneration or repair of hair cells and supporting cells. These other processes are considered.

The structural and functional aspects of hair cell regeneration in the chick as a result of exposure to intense sound.

This paper summarizes the structural and functional damage caused by intense sound exposure in neonatal chicks. Scanning electron microscopy has been used to follow the structural changes to the papilla and their subsequent repair. Pure-tone exposures produced a localized lesion consisting of tectorial membrane destruction, changes in surface organization of the papilla, and hair cell loss. The papilla underwent significant repair following the exposure and new hair cells could be identified on the sensory surface after 4 days of recovery. In addition, various evoked-potential methods provided an objective assessment of auditory function and demonstrated that the peripheral ear was severely impaired by overstimulation. Auditory function returned to near normal levels within 3 days postexposure. The inescapable conclusion from these observations was that hair cell regeneration had little to do with the functional recovery observed during the first 3 days. Tectorial membrane regeneration and the restoration of cochlear micromechanics were combined to form a hypothesis to account for the restoration of auditory function.

Hydrofitness devices for strengthening upper extremity muscles.

Our evaluation of hydrofitness devices for strengthening arm muscles has revealed several important determinants of their performance. Most importantly, the device's frontal projection should be in the shape of a paddle whose surface area can be varied to meet the individual exerciser's needs. Its weight should be light to minimize the inertial forces encountered in charging the velocity of paddle movement. Its handle should have a relatively large circumference without digital profiling. These optimal performance parameters have been recently incorporated into a new hydrofitness device that will be commercially available for aquatic exercise programs for strengthening upper extremity muscles.

Immunocytochemical and quantitative studies of Na+,K+-ATPase distribution in the developing chick cochlea.

Immunocytochemical methods were used to examine cryosections of the embryonic and neonatal chicken cochlea in order to study the histological distribution of the Na+,K+-ATPase molecule during maturation. In complementary studies the Na+,K+-ATPase capacity of microdissected freeze-dried substructures of the cochlea was determined fluorometrically. The dark cell of the tegmentum vasculosum exhibited intense immunochemical staining of the convoluted basolateral infoldings. The adjacent light cells demonstrated very little staining. The plasma membrane of the hair cell was also stained as were the first order auditory neurons, including the cell soma. The homogene cells and the supporting cells were unstained. The dark cell was only lightly stained with the antibody at stage 45 but became more intense and selective by the seventh postnatal day. The other cells of the cochlear duct exhibited specific immunofluorescent staining of their plasma membranes from stage 45 onwards and the fluorescent intensity did not change. The tegmentum vasculosum exhibited very high activities of the Na+,K+-ATPase enzyme relative to the other structures of the cochlea. Furthermore, a pronounced gradient of enzymatic activity was detected longitudinally. The proximal tip (or high-frequency end) had a sixteen-fold greater capacity for Na+- and K+-dependent ATP hydrolysis relative to the distal tip (or low-frequency end). The appearance of this enzyme in the tegmentum vasculosum during the development of the cochlea paralleled the known rate of improvement in hearing thresholds.

Hair cell damage produced by acoustic trauma in the chick cochlea.

Examination of pure-tone acoustic damage in the chick basilar papilla revealed that the location and extent of hair cell damage was a function of both the stimulus intensity and the age at which the chicks were exposed. Scanning electron microscopic evaluation of noise-exposed cochleae at post-hatching days 1, 10 and 30 permitted the identification of discrete regions of damage, including hair cells with stereocilia injuries as well as those lost from the epithelium. The hair cell damage was tonotopically distributed along the cochlea according to frequency. However, for each exposure frequency two distinct sites of damage were often produced, and their locations were correlated with stimulus intensity. At low intensities, a longitudinal strip of hair cell damage ran along the superior edge of the basilar papilla. As exposure intensity increased, a second damage site developed along the inferior edge of the basilar papilla, distal to the longitudinal strip. This second type of damage initially took the form of a series of laterally-oriented wedges, but at higher intensities, the wedges coalesced to form a large crescent-shaped patch of damage. The location of the damage sites for each frequency did not shift with age. However, there were differences in the extent and position of the damage which could be correlated with stimulus intensity and with changes in middle ear admittance during development [(1983) Development of Auditory and Vestibular Systems, pp. 3-25. Editor: R. Romand. Academic Press, New York]. These results suggest that developmental changes in the location and extent of hair cell damage depend on the effective stimulus intensity reaching the cochlea, rather than on alterations in the frequency coding of the hair cells.

Frequency selectivity in the parakeet (Melopsittacus undulatus) studied with narrow-band noise masking.

Narrow-band noise masking was studied in the parakeet (Melopsittacus undulatus) using a modified method of limits and an instrumental avoidance-conditioning procedure. Masked thresholds were obtained from five subjects at 10 frequencies between 0.5 and 5.0 kHz for each of four sensation levels (26, 46, 66, and 86 db) of a 1/3-octave band noise masker centered at 1.6 kHz. The amount of masking was found to be linearly related to noise level, and the shape of the masking curve was symmetrical on both sides of the center frequency of the masker. In all cases, the greatest threshold shift occurred at the center frequency of the masker. The relative symmetry of the parakeet narrow-band masking curves contrasts with masking results reported in mammals.

Evoked responses from inferior colliculus as an index of hearing thresholds in guinea pigs.

Bipolar electrodes were implanted in the inferior colliculus of 10 healthy guinea pigs, and visual detection level thresholds of auditory evoked responses were obtained at 17 frequencies between 0.5 and 20.0 kHz. One microvolt isopotential curves from the round window for the same test frequencies were obtained in these animals. A comparison between the evoked response threshold curves and previous reports of the behavioral audiogram reveals a favorable correlation. The cochlear potential curve, however, appears to be a less efficient predictor of the audibility curve than the evoked response thresholds. The utility of evoked response audiometry from the inferior colliculus for quickly assessing auditory thresholds in the guinea pig is discussed.