Barbara A. Bohne

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.

A frequency‐position map for the chinchilla cochlea

Frequencies of tones are mapped on to distances along the organ of Corti by associating behaviorally measured threshold shifts with regions of hair-cell loss. The central tendency found for 95 frequency-position matches by four observers on 21 ears is approximated by a straight-line, log-linear relation between frequency and position. Only a small portion of the considerable variation of individual matches around this function could be explained by length of the organ of Corti. Other unidentified factors appear to be responsible for most of these variations.

Neuronal and transneuronal degeneration of auditory axons in the brainstem after cochlear lesions in the chinchilla: Cochleotopic and non-cochleotopic patterns

Terminal axonal degeneration in the brain following cochlear lesions was studied with the Nauta-Rasmussen method. Losses of hair cells and myelinated cochlear fibers were assessed. The cochleotopic map projected, from apex to base, on the ventral-to-dorsal axes of the cochlear nuclei. The cochleotopic correspondence was better for loss of cochlear nerve fibers and inner hair cells, than for outer hair cells. Cochlear fibers were traced to all parts of the cochlear nucleus, including the small-cell shell, also to cell-group Y and the flocculus. Terminal axonal degeneration in nuclei of the superior olivary complex, lateral lemniscus, and inferior colliculus was interpreted as transynaptic, since degenerated axons could not be traced to these locations from the cochlear nerve or trapezoid body. Moreover, biotinylated dextran amine injection in the basal turn of scala media of a normal cochlea labeled cochlear nerve fibers projecting to the high-frequency regions of the cochlear nuclei and to the flocculus, but not to more central auditory nuclei. This is the first detailed account of transynaptic degeneration in the ascending auditory pathway resulting from cochlear damage in an adult mammal. These findings are consistent with a dystrophic process depending on hair-cell loss and/or direct damage to cochlear nerve fibers.

Degeneration of axons in the brainstem of the chinchilla after auditory overstimulation

The patterns of axonal degeneration following acoustic overstimulation of the cochlea were traced in the brainstem of adult chinchillas. The Nauta-Rasmussen method for axonal degeneration was used following survivals of 1-32 days after a 105 min exposure to an octave-band noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. Hair-cell and myelinated nerve-fiber loss were assessed in the cochlea. The cochleotopic pattern of terminal degeneration in the ventral cochlear nucleus correlated with the sites of myelinated fiber and inner-hair-cell loss: this correlation was less rigorous with outer-hair-cell loss, especially in the dorsal cochlear nucleus. These results are consistent with a dystrophic process with a slow time course depending on hair-cell loss and/or direct cochlear nerve-fiber damage. However, in a number of cases with no damage in the apical cochlea, fine fiber degeneration occurred with a faster course in low-frequency regions in the dorsal cochlear nucleus and, transynaptically, in a non-cochleotopic pattern in the superior olive and inferior colliculus. These findings suggest that neuronal hyperactivity plays a role in the central degeneration following acoustic overstimulation, possibly by an excitotoxic process.

Delayed effects of ionizing radiation on the ear

The question of damage to the ear from exposure to ionizing radiation was addressed by exposing groups of chinchillas to fractioned doses of radiation (2 Gy per day) for total doses ranging from 40 to 90 Gy. In order to allow any delayed effects of radiation to become manifest, the animals were sacrificed two years after completion of treatment and their temporal bones were prepared for microscopic examination. The most pronounced effect of treatment was degeneration of sensory and supporting cells and loss of eighth nerve fibers in the organ of Corti. Damage increased with increasing dose of radiation. The degree of damage found in many of these ears was of sufficient magnitude to produce a permanent sensorineural hearing loss.

Holes in the reticular lamina after noise exposure: Implication for continuing damage in the organ of Corti

Three different histological techniques (scanning electron microscopy, phase contrast microscopy and light microscopy) were used to examine the organ of Corti after a damaging noise exposure. In the region of maximal injury, all outer hair cells were missing 1-2 h after the exposure had ended. Degenerated hair cells are eventually replaced by phalangeal scars. However, in these short-term recovery ears, a series of holes which were the exact size and shape of the missing outer hair cell apices were found in the reticular lamina. These holes may provide a communication route between the endolymphatic space and the fluid spaces of the organ of Corti for a period of time following a damaging noise exposure. The noise-related degeneration of supporting cells, nerve fibers and even some sensory cells may be secondary to contamination of the fluid spaces with potassium-rich endolymph.

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.

Spontaneous otoacoustic emissions in chinchilla ear canals correlation with histopathology and suppression by external tones

Two cases of spontaneous otoacoustic emissions (SOAEs) have been found among a sample of 28 chinchilla ears after noise exposure, and no cases of SOAEs have been found among 28 unexposed ears. Further observations of the characteristics of SOAEs recorded in the ear canals of two chinchillas after noise exposure are described. These signals were tonal, robust, and could be suppressed by presenting external tones to the ear. Histopathological evaluation of the cochlea of emitting ears revealed discrete basal-turn lesions near the positions corresponding to the frequencies of the emissions. Behavioral threshold shifts measured in one animal after noise exposure and acoustic intermodulation distortion product behavior in the other both suggest a region of increased vibratory response of the cochlear partition near the location of the SOAE. Results from these emitting ears support a hypothesis that a punctate loss of the organ of Corti (OC) may facilitate the occurrence of an SOAE. We further hypothesize that the following conditions are both necessary and sufficient for an SOAE to occur: (1) functional disruption of a normally present biomechanical control mechanism in a region of the OC; (2) presence of functionally active OC (especially outer hair cells) adjacent to the region. The observations from ears possessing SOAEs provide strong, though indirect support for active and nonlinear models in interpreting cochlear biomechanical phenomena.

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.