Gary W. Harding

Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (Fgfr3) is a tyrosine kinase receptor expressed in developing bone, cochlea, brain and spinal cord. Achondroplasia, the most common genetic form of dwarfism, is caused by mutations in FGFR3. Here we show that mice homozygous for a targeted disruption of Fgfr3 exhibit skeletal and inner ear defects. Skeletal defects include kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae. Contrasts between the skeletal phenotype and achondroplasia suggest that activation of FGFR3 causes achondroplasia. Inner ear defects include failure of pillar cell differentiation and tunnel of Corti formation and result in profound deafness. Our results demonstrate that Fgfr3 is essential for normal endochondral ossification and inner ear development.

Histopathological differences between temporary and permanent threshold shift

The structural changes associated with noise-induced temporary threshold shift (TTS) were compared to the damage associated with permanent threshold shift (PTS). A within-animal paradigm involving survival-fixation was used to minimize problems with data interpretation from interanimal variability in response to noise. Auditory brainstem response thresholds for clicks and tone pips were determined pre- and 1-2 h post-exposure in 11 chinchillas. The animals were exposed for 24 h to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 86 dB. Three animals (0/0-day) had both cochleas terminal-fixed 2-3 h post-exposure. Two animals (27/27-day) had threshold shifts determined every other day for 1 week, every week thereafter, and underwent terminal-fixation of both cochleas 27 days after exposure. Six animals (0/n-day) had threshold shifts determined in both ears upon removal from the noise; their left cochlea was then survival-fixed 2-3 h post-exposure. Threshold shifts were determined in their right ear every 2-3 days until their hearing either returned to pre-exposure values or stabilized at a reduced level at which time their right cochlea was terminal-fixed (4-13 days post-exposure). All cochleas were prepared as plastic-embedded flat preparations. Missing hair cells were counted and supporting cells and nerve fibers were evaluated throughout the organ of Corti using phase-contrast microscopy. Post-exposure, all animals had moderate TTSs in their left and right ears which averaged 43 dB for 4-12 kHz. In the 0/0-day animals, the only abnormality which correlated with TTS was a buckling of the pillar bodies. In the 0/n-day animals, their left cochlea (survival-fixed 2-3 h post-exposure) had outer hair cell (OHC) stereocilia which were not embedded in the tectorial membrane in the region of the TTS whereas OHC stereocilia were embedded in the tectorial membrane throughout the cochleas of non-noise-exposed, survival-fixed controls. Three of six right cochleas (terminal-fixed 4-13 days post-exposure) from the 0/n-day animals developed a PTS and two of these cochleas had focal losses of inner and outer hair cells and afferent nerve fibers at the corresponding frequency location. The other cochlea with PTS had buckled pillars in the corresponding frequency region. These results suggest that with moderate levels of noise exposure, buckling of the supporting cells results in an uncoupling of the OHC stereocilia from the tectorial membrane which results in a TTS. The mechanisms resulting in TTS appear to be distinct from those that produce permanent hair cell damage and a PTS.

Morphological correlates of aging in the chinchilla cochlea

The inner ears from 80 chinchillas ranging in age from premature to 19.2 years were examined as plastic-embedded flat preparations to determine the morphological changes associated with aging. Three of the four forms of human presbycusis defined by Schuknecht were found in the chinchillas. All animals had losses of sensory cells or sensory presbycusis. Inner (IHCs) and outer hair cells (OHCs) degenerated at a rate of about 0.29% and 1.0% per year, respectively. Age-related degeneration of inner (IPs) and outer pillars (OPs) occurred at a much slower rate. In four animals (5%) the dendritic processes of some of the spiral ganglion cells had degenerated in areas where the loss of sensory cells was minimal. This pathological change is likely equivalent to neural presbycusis. Six animals (7.5%) had regions of degeneration of the stria vascularis or strial presbycusis. The other common finding in the aging cochleas was the presence of lipofuscin or age pigment. Lipofuscin deposits were found to accumulate in the subcuticular region of OHCs, IPs and OPs, near the endolymphatic surfaces of many of the supporting cells and in the epithelial cells of Reissners membrane. The IHCs accumulated much less lipofuscin. The morphological changes seen in the ears of aging chinchillas were qualitatively similar to those seen in the temporal bones of aging humans although the magnitude of the changes was considerably less. These results suggest that some of the damage found in aging human cochleas may be due to aging plus exposure to one or more ototraumatic agents.

Spiral CT image deblurring for cochlear implantation

Cochlear implantation is the standard treatment for profound hearing loss, Preimplantation and postimplantation spiral computed tomography (CT) is essential in several key clinical and research aspects. The maximum image resolution with commercial spiral CT scanners is insufficient to define clearly anatomical features and implant electrode positions in the inner ear, In this paper, the authors develop an expectation maximization (EM)-like iterative deblurring algorithm to achieve spiral CT image super-resolution for cochlear implantation, assuming a spatially invariant linear spiral CT system with a three-dimensional (3-D) separable Gaussian point spread function (PSF). The authors experimentally validate the 3-D Gaussian blurring model via phantom measurement and profile fitting. The imaging process is further expressed as convolution of an isotropic 3-D Gaussian PSF and a blurred underlying volumetric image. Under practical conditions, an oblique reconstructed section is approximated as convolution of an isotropic two dimensional (2-D) Gaussian PSF and the corresponding actual cross section. The spiral CT image deblurring algorithm is formulated with sieve and resolution kernels for suppressing noise and edge artifacts. A typical cochlear cross section is used for evaluation, demonstrating a resolution gain up to 30%-40% according to the correlation criterion. Physical phantoms, preimplantation and postimplantation patients are reconstructed into volumes of 0.1-mm cubic voxels. The patient images are digitally unwrapped along the central axis of the cochlea and the implanted electrode array respectively, then oblique sections orthogonal to the central axis formed. After deblurring, representation of structural features is substantially improved in all the cases.

Otoconial agenesis in tilted mutant mice

The sense of balance is one of the phylogenetically oldest sensory systems. The vestibular organs, consisting of sensory hair cells and an overlying extracellular membrane, have been conserved throughout vertebrate evolution. To better understand mechanisms regulating vestibular development and mechanisms of vestibular pathophysiology, we have analyzed the mouse mutant, tilted (tlt), which has dysfunction of the gravity receptors. The tilted mouse arose spontaneously and has not been previously analyzed for a developmental or physiological deficit. Here we demonstrate that the tilted mouse, like the head tilt (het) mouse, specifically lacks otoconia and consequently does not sense spatial orientation relative to the force of gravity. Unlike other mouse mutations affecting the vestibular system (such as pallid, mocha and tilted head), the defect in the tilted mouse is highly penetrant, results in the nearly complete absence of otoconia, exhibits no degeneration of the sensory epithelium and has no apparent abnormal phenotype in other organ systems. We further demonstrate that protein expression in the macular sensory epithelium is qualitatively unaltered in tilted mutant mice.

DPOAE level shifts and ABR threshold shifts compared to detailed analysis of histopathological damage from noise.

A detailed comparison of 2f(1)-f(2) distortion product otoacoustic emission (DPOAE) level shifts (LS) and auditory brainstem response (ABR) threshold shifts with noise-induced histopathology was conducted in chinchillas. DPOAE levels (i.e., L(1) and L(2)) at f(1) and f(2), respectively, ranged from 55-75 dB sound pressure level (SPL), with f(2)/f(1)=1.23, 6 points/octave, f(2)=0.41-20 kHz, and ABR thresholds at 0.5-20 kHz, 2 points/octave, were determined pre-exposure. The exposure was a 108 dB SPL octave band of noise centered at 4 kHz (1-1.75 h, n=6) or 80-86 dB SPL (24 h, n=5). DPOAE LSs (magnitude pre- minus post-exposure) and ABR threshold shifts (TS) were determined at 0 days and up to 28 days post-exposure. The cochleae were fixed, embedded in plastic and dissected into flat preparations. The length of the organ of Corti (OC) was measured; missing inner (IHC) and outer (OHC) hair cells counted; stereocilia damage rated; and regions of OC and nerve-fiber loss determined. Cytocochleograms were made showing functional loss and structural damage with the LS and TS overlaid. Some unexpected results were obtained. First, the best correlation of LS with histopathology required plotting the DPOAE data at f(1) with respect to the chinchilla-place map. The best correlation of TS was with IHC and nerve-fiber loss. Second, wide regions of up to 10% scattered OHC loss in the apical half of the OC showed little or no LS. Third, with the 108 dB SPL noise, there was 20-40 dB of recovery for DPOAEs at mid-high frequencies (3-10 kHz) in eight of 12 cochleae where there was 70-100% OHC loss in the basal half of the OC. The largest recovery at mid-high frequencies occurred in regions where the OC was entirely missing. Fourth, with the 80-86 dB SPL noise, there was no LS at small focal lesions (100% loss of OHCs over 0.4 mm) when the frequency place of either f(1) or f(2) was within the lesion but not both. There was no correlation of LS with OHC stereocilia loss, fusion or disarray. These results suggest that, after noise exposure, DPOAEs at mid-high frequencies can originate from or be augmented by generators located at someplace other than the frequency place of f(2), possibly the basal 20% of the OC when this region is intact. Also, noise-induced DPOAE LSs seemed to reflect differing mechanisms for temporary and permanent hearing loss.

Noise damage in the C57BL/CBA mouse cochlea

The present study was designed to determine the response to noise of the auditory system of a genetically well-defined laboratory mouse in preparation for examining the effect of noise on mice with specific genetic mutations. The mice were C57BL/CBA F1 hybrids. Eight mice served as non-noise-exposed controls and 39 mice were exposed for 1-24 h to an octave band of noise with a center frequency of 2, 4 or 8 kHz and a sound pressure level of 100-120 dB. Auditory brainstem response thresholds were measured pre-exposure and several times post-exposure (i.e., 0-27 days) to determine the magnitude of the temporary threshold shift (TTS) and permanent threshold shift (PTS). After fixation by cardiac perfusion, the cochleas from each mouse were embedded in plastic, dissected into quarter turns of the cochlear duct and analyzed quantitatively. Immediately post-exposure, all mice had sizable TTSs at the tested frequencies (i.e., 3-50 kHz). At this time, two mice were killed. Thresholds of the other 37 mice recovered somewhat in the first 4 days post-exposure. One mouse fully recovered from its TTS; 10 mice were left with PTSs at all frequencies; 26 mice recovered at some frequencies but not others. Most mice with PTSs for 30-50 kHz had focal losses of inner and outer hair cells in the basal 20% of the organ of Corti, often with degeneration of adjacent myelinated nerve fibers in the osseous spiral lamina. On the other hand, mice with PTSs for the lower frequencies (i.e., 3-20 kHz) had stereocilia disarray without significant hair cell losses in the second and first turns. Considerable variability was found in the magnitude of hair cell losses in those mice that received identical noise exposures, despite their genetic homogeneity.

Death pathways in noise-damaged outer hair cells

Using morphological criteria, death pathways in outer hair cells (OHCs) were determined in chinchilla organs of Corti that had been exposed to a high- or moderate-level octave band of noise (OBN) centered at either 0.5 or 4-kHz. The specimens were part of our large collection of plastic-embedded flat preparations of chinchilla cochleae. Three death pathways were identified: (1) oncotic - swollen, pale-staining cell with a swollen nucleus, (2) apoptotic - shrunken, dark-staining cell with a pyknotic nucleus and (3) a newly defined third pathway - no basolateral plasma membrane but cellular debris arranged in the shape of an intact OHC with a nucleus deficient in nucleoplasm. To minimize the secondary loss of OHCs from the entrance of endolymph into the organ of Corti, the specimens used for quantitative analysis of death pathways had the following characteristics: (1) the level to which they were exposed was less than or equal to 95dB SPL, (2) the exposure duration was 6-216h, (3) fixation for microscopic examination took place in vivo 1-2h post-exposure and (4) there were no focal OHC lesions in the organs of Corti. Fifty-eight noise-exposed cochleae met these criteria. In these specimens, degenerating and missing OHCs were classified as to which death pathway the cells had followed or were following. Nine non-noise-exposed cochleae were also evaluated for OHC death pathways. The number of OHCs following the third death pathway was significantly greater in the noise-exposed cochleae than the non-noise-exposed cochleae for total exposure energies greater than those produced by 75dB SPL for 216h to a 0.5-kHz OBN and 57dB SPL for 48h to a 4-kHz OBN. In cochleae exposed to either octave band, OHCs dying by oncosis or apoptosis were uncommon.

Time course of nerve-fiber regeneration in the noise-damaged mammalian cochlea.

The time course of events which are essential for nerve‐fiber regeneration in the mammalian cochlea was determined using a group of chinchillas that had been exposed for 3.5 hr to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. The animals recovered from 40 min (0 days) to 100 days at which times their inner ears were fixed and the organs of Corti prepared for phase‐contrast and bright‐field microscopy as plastic‐embedded flat preparations. Selected areas identified in the flat preparations were semi‐thick and thin sectioned at radial or tangential angles for examination by bright‐field and transmission electron microscopy. The following time‐ordered events appeared critical for nerve‐fiber regeneration: (1) The area of the basilar membrane in which regeneration had a possibility of occurring showed signs of severe injury. Outer hair cells degenerated first followed by outer pillars, inner pillars, inner hair cells and other supporting cells; (2) Myelinated nerve fibers in the osseous spiral lamina became fragmented, starting at the distal ends of the fibers. This degeneration gradually extended back to Rosenthals canal; (3) Fibrous processes, originating from Schwann‐like cells in the osseous spiral lamina, extended laterally on the basilar membrane; (4) Schwann cells lined up medial to the habenulae perforata in the areas of severest damage, apparently ready to migrate through the habenulae onto the basilar membrane; (5) Schwann‐cell nuclei appeared on the basilar membrane beneath the developing layer of squamous epithelium which was in the process of replacing the degenerated portion of the organ of Corti; (6) Regenerated nerve fibers with thin myelin sheaths or a simple investment of Schwann cell cytoplasm appeared in areas of total loss of the organ of Corti; and (7) The myelin sheaths on the regenerated nerve fibers gradually became thicker.

The effect of an age-related hearing loss gene (Ahl) on noise-induced hearing loss and cochlear damage from low-frequency noise.

Inbred C57BL/6J mice carry two copies of an age-related hearing loss gene (Ahl). It has been shown that these mice begin losing high-frequency hearing at two months. Several functional studies have reported that the Ahl gene renders mice more susceptible to noise-induced hearing loss (NIHL) than strains which do not carry this gene [e.g., Hear. Res. 93 (1996) 181; Hear. Res. 155 (2001) 82; J. Assoc. Res. Otolaryngol. 2 (2001) 233]. Johnson et al. [Hear. Res. 114 (1997) 83] developed a congenic B6.CAST-+Ahl mouse which carries the wild-type allele from Mus musculus castaneus at the Ahl locus. Five each of young C57BL/6J males and females, and B6.CAST-+Ahl males were exposed to a 4-kHz octave band of noise at 108 dB SPL for 4 h. Non-noise-exposed mice of the same strains and age served as controls. The noise-exposed mice were functionally tested for ABR thresholds and DPOAE levels pre-exposure and three times post-exposure: 0 days to determine the magnitude of temporary threshold shift (TTS); 6 days to determine rate of recovery; and 20 days to determine the magnitude of permanent threshold shift (PTS). At 20 days post-exposure, the animals underwent cardiac perfusion to fix their cochleae. The isolated cochleae were embedded in plastic and dissected into flat preparations. By phase-contrast microscopy, each cochlea was evaluated from apex to base to quantify the losses of hair cells, nerve fibers and stria vascularis and to localize stereocilia damage. Functional data from each mouse were aligned with the cytocochleogram using the frequency-place map of Ou et al. [Hear. Res. 145 (2000) 111; Hear. Res. 145 (2000) 123]. Sizable variation in the magnitude of TTS, PTS and hair-cell loss was found among mice of the same genetic strain. The congenic B6.CAST-+Ahl male mice had significantly less TTS immediately post-exposure than C57BL/6J males or females but not less PTS or hair-cell losses at 20 days post-exposure. These results indicate that, at one month of age, mice carrying two copies of the Ahl gene have an increased susceptibility to TTS from a low-frequency noise before they have any indication of age-related hearing or hair-cell loss. However, this appeared not to be the case for PTS. The Ahl gene appears to play a role in susceptibility to NIHL but, other genes as well as systemic and local factors must also be involved.