Apoptotic hair cell death after transient cochlear ischemia in gerbils

The mechanisms of cochlear hair cell death following exposure to transient inner ear ischemia were investigated in gerbils histologically. The animals were subjected to ischemic insult by occluding both vertebral arteries for 15 min. Hoechst 33342 nuclear staining showed that inner hair cells (IHCs) underwent sporadic degeneration via nuclear condensation, which peaked 12 hours after the ischemia. Furthermore, nuclear DNA fragmentation was noted by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling method. Transmission electron microscopy revealed morphological changes in the IHCs characteristic of apoptosis, including karyopyknosis, chromatin condensation. These findings suggest that apoptotic cell death is the major process in hair cell degeneration in this animal model.     

Free radical scavenger protects against inner hair cell loss after cochlear ischemia

We investigated the protective effects of edaravone, a free radical scavenger, against ischemic damage of inner hair cells (IHCs) in gerbils. Cochlear ischemia was induced in the animals by occluding the vertebral arteries bilaterally for 15 min. Edaravone (1 mg/kg, i.v.) or saline was administered 1 h after ischemia. Hearing was assessed by auditory brain response (ABR). In animals treated with saline, the ABR threshold shift was 24.1 dB and there was a 26.5% decrease in the number of IHCs. By contrast, in animals treated with edaravone, the threshold shift was 7.5 dB and only 8.8% of IHCs was lost. These results suggest that edaravone protects against damage to the inner ear following transient ischemia.     

Ultrastructure of the horseshoe bat's organ of Corti. I. Scanning electron microscopy.

The organ of Corti of the echolocating horseshoe bat (Rhinolophus rouxi) was investigated with scanning electron microscopy in order to provide a comparison with non-echolocating mammals. Throughout the cochlea of horseshoe bats, each outer hair cell (OHC) possesses three rows of stereocilia and there are no morphological distinctions among the different rows of OHCs. However, there are morphological differences between different regions along the cochlea. In the lower and upper basal turn, the receptor surfaces of OHCs are characterized by extremely wide W-shaped stereocilia bundles and wingshaped cuticular plates. The cuticular plates of OHCs of the middle and outermost rows are arranged parallel to each other. Stereocilia length is only 0.8 microns and there is an exaggerated angle of inclination of the shortest row of stereocilia towards the next taller one. Stereocilia arrangements in the apex of the horseshoe bat's cochlea closely resembles those observed in the midbasal region of the rat cochlea. Inner hair cells (IHC) in the lower basal turn appear specialized. They possess only two rows of stereocilia and only 7-8 stereocilia per row. Their cuticular plates are small and oval and widely separated from one another in the longitudinal direction. IHCs at all other locations possess three and up to four rows of stereocilia and 17-20 stereocilia per row. Their cuticular plates are elongated and closely spaced. The transition from specialized to typical mammalian morphology occurs abruptly (over a distance of about 100-150 microns) at the border between the lower and the upper basal turn. This transition is not accompanied by a change in OHC morphology. In the subsurface of the tectorial membrane, throughout the cochlea, there are distinct imprints of the tallest row of stereocilia of all three rows of OHCs and of the IHCs. Data are discussed in relation to specialized aspects of the cochlear frequency map in horseshoe bats and as possible micromechanical adaptations to ultra-high frequency hearing.     

Changes in MAP2 and tyrosinated alpha-tubulin expression in cochlear inner hair cells after amikacin treatment in the rat

The expression of MAP2 (microtubule-associated protein 2) and of tyrosinated alpha-tubulin was investigated immunocytochemically in the cochleas of normal and amikacin-treated rats. For MAP2, two different antibodies were used: anti-MAP2ab, against the high molecular weight forms, and anti-MAP2abc, additionally against the embryonic form c. In the cochlea of the normal rat, the outer (OHCs) and inner (IHCs) hair cells were labeled for MAP2abc. The labeling was weaker in IHCs than in OHCs. The hair cells were rarely labeled for MAPab. Both OHCs and IHCs were labeled for tyrosinated alpha-tubulin. In the cochlea of the amikacin-treated rat, aggregates of anti-MAP2abc and anti-tyrosinated alpha-tubulin antibodies were seen in the apical region of the IHCs as early as the end of the antibiotic treatment. In rats investigated during the following week, the cell body of most of the surviving IHCs were not labeled for MAP2abc and tyrosinated alpha-tubulin. Then, labeling for these two antibodies reappeared in the surviving IHCs, including their giant stereocilia. Fewer surviving IHCs were labeled for tyrosinated alpha-tubulin than for MAP2abc. The amikacin-poisoned IHCs were rarely labeled for MAP2ab. These results suggest that cochlear hair cells essentially express form c of MAP2. In the amikacin-damaged cochlea, the apical aggregation of MAP2c and tyrosinated alpha-tubulin within the poisoned IHCs could be implicated in a cell degenerative process. By contrast, the extinction and recovery of MAP2c and tyrosinated alpha-tubulin labeling in the remaining IHCs suggest the occurrence of a limited repair process. A possible role of MAP2 and tubulin in hair cell survival is discussed.     

Histochemical and scanning electron microscopic studies of supernumerary hair cells in embryonic rat cochlea in vitro

In the embryonic organ of Corti supernumerary hair cells were observed when developed in organotypic cultures. Hair cells ranging in up to two rows of inner hair cells (IHCs) and up to nine rows of outer hair cells (OHCs), were observed by phalloidin histochemistry. The total number of hair cells may double in some explanted cochlea compared to control ones. Cuticular plates of hair cells displayed an actin-free zone corresponding to the kinocilium location, differently located and indicating different degrees of differentiation and maturation. Moreover, some hair cells had a small apical surface area and a centrally located kinocilium, revealing immaturity. Under scanning electron microscopy, stereocilia appeared to differentiate normally, as compared to the in vivo development. The staircase pattern of the stereociliary bundles was reached on most of the hair cells with a ‘V’ shape on the OHCs and hemispherical one on the IHCs. Hair cell polarity was not homogeneous along the length of the tissue. Organs of Corti explanted at birth developed a weaker number of supernumerary hair cells showing a decrease of supernumerary hair cells with the developmental stage of the explant. These results provide evidence for supernumerary hair cells in the mammalian cochlea in culture, without loss or injury to preexisting hair cells.     

Time course of efferent fiber and spiral ganglion cell degeneration following complete hair cell loss in the chinchilla

Ethacrynic acid (EA) is known to interact with aminoglycoside antibiotics such as gentamicin (GM). In the chinchilla, co-administration of GM and EA can produce hair cell lesions ranging from a small loss of outer hair cells (OHCs) in the base of the cochlea to complete destruction of all hair cells, depending on dosing parameters. Although hair cell loss has been characterized, little is known about the fate of efferent fibers or spiral ganglion neurons (SGNs) in this model. To study the time course of efferent fiber and SGN loss, chinchillas were injected with GM (125 mg/kg IM) followed immediately by EA (40 mg/kg IV). Estimates of efferent fiber loss and density changes were made after 3 days or 1, 2, 3, or 4 weeks of survival. Estimates of SGN loss and density changes were made after 15 days or 1, 2, 4, or 6 months of survival. Cochlear function was rapidly abolished and all cochlear hair cells were missing within 24 h after treatment. Inner hair cells (IHCs) in the middle turn of the cochlea died earlier than cells in the apex or base, and OHCs in Rows 1 and 2 died earlier than OHCs in Row 3. Degeneration of efferent nerve fibers began 3-7 days post-injection, versus 15-30 days for SGNs, and the loss of efferent fibers was essentially complete within 1 month, versus 2-4 months for SGNs. The rapid time course of efferent fiber and SGN loss in the chinchilla may make it a practical model for studying mechanisms of neural loss and survival in the mammalian inner ear.     

Hearing loss and glutamate efflux in the perilymph following transient hindbrain ischemia in gerbils

The mechanism underlying ischemia-induced hearing loss was studied in gerbils with transient hindbrain ischemia. Occlusion of the vertebral arteries caused an increase in the concentration of glutamate in the perilymph and elevated the compound action potential (CAP) threshold to 24.6 dB at 5 minutes. the CAP threshold subsequently recovered on reperfusion, gradually reaching 8.3 dB 120 minutes after reperfusion. Under electron microscopy, afferent dendrites of the cochlear nerve in contact with inner hair cells exhibited abnormal swelling 5 minutes after ischemia/reperfusion. These morphological changes were not observed in cochleas treated with an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist, 6-7-dinitroquinoxaline-2,3-dione (DNQX), before hindbrain ischemia; an N-methyl-D-aspartate (NMDA)-type receptor antagonist, D-2-amino-5-phosphonopentanoate (D-AP5), was ineffective. Moreover, the histopathological alterations noted 5 minutes after reperfusion were spontaneously ameliorated 120 minutes after ischemia/reperfusion. These findings suggest that the ischemia-induced increase in extracellular glutamate concentration with subsequent activation of AMPA/kainate receptors is responsible for neurite degeneration and hearing loss in the early stages following transient hindbrain ischemia.     

Effects of delayed treatment with combined GDNF and continuous electrical stimulation on spiral ganglion cell survival in deafened guinea pigs

Electrical stimulation (ES) of spiral ganglion cells (SGC) via a cochlear implant is the standard treatment for profound sensor neural hearing loss. However, loss of hair cells as the morphological correlate of sensor neural hearing loss leads to deafferentation and death of SGC. Although immediate treatment with ES or glial cell line–derived neurotrophic factor (GDNF) can prevent degeneration of SGC, only few studies address the effectiveness of delayed treatment. We hypothesize that both interventions have a synergistic effect and that even delayed treatment would protect SGC. Therefore, an electrode connected to a pump was implanted into the left cochlea of guinea pigs 3 weeks after deafening. The contralateral untreated cochleae served as deafened intraindividual controls. Four groups were set up. Control animals received intracochlear infusion of artificial perilymph (AP/?). The experimental groups consisted of animals treated with AP in addition to continuous ES (AP/ES) or treated with GDNF alone (GDNF/?) or GDNF combined with continuous ES (GDNF/ES). Acoustically and electrically evoked auditory brain stem responses were recorded. All animals were killed 48 days after deafening; their cochleae were histologically evaluated. Survival of SGC increased significantly in the GDNF/? and AP/ES group compared with the AP/? group. A highly significant increase in SGC density was observed in the GDNF/ES group compared with the control group. Additionally, animals in the GDNF/ES group showed reduced EABR thresholds. Thus, delayed treatment with GDNF and ES can protect SGC from degeneration and may improve the benefits of cochlear implants. © 2008 Wiley‐Liss, Inc.     

Development of the organ of corti in horseshoe bats: Scanning and transmission electron microscopy

The late prenatal and early postnatal development of the organ of Corti were studied in the horseshoe bat (Rhinolophus rouxi) by using scanning and transmission electron microscopy. Arrangements and dimensions of stereocilia bundles, together with their contacts with the tectorial membrane, were found to be adult-like shortly before birth, and thus before the biological onset of hearing (3–5 days after birth). During the first postnatal week, there were baso-apical gradients in disappearing kinocilia on inner hair cells (IHC), microvillis of supporting cells, and marginal pillars. The lower basal cochlear turn was mature with respect to these regressing structures at 3 days after birth, the apical turn at 10 days after birth. At birth, cytodifferentiation was found to be completed, and the tunnel of Corti and innermost spaces of Nuel had opened. The ultrastructure of IHCs was not markedly different from that at later ages. In outer hair cells (OHC), the adult-like regular arrangement of a single layer of subsurface cisternae and pillars was seen as soon as protrusions of supporting cells had withdrawn from the lateral wall of OHCs (basal turn at birth and throughout the cochlea 2 days after birth). Numerous efferent endings contacted the somata of IHCs up to the second postnatal week. Since the medial olivocochlear system is absent in horseshoe bats, the adult-like innervation pattern of OHCs was established at the biological onset of hearing. During the first 2 postnatal weeks, the cytoskeleton of pillar and Deiters cells, and the specialized Deiters cups developed. The organ of Corti appeared adult-like at 14 days, apart from the persistence of a reduced tympanic cover layer attached to the basilar membrane. Morphological data support physiological findings that the first broadly tuned auditory responses arise from the basal turn. The distinct low to high frequency gradient in development of sensitivity during the first 2 postnatal weeks of the horseshoe bat was not, however, matched by morphological gradients, and it would appear that the development of the cytoskeleton of supporting cells contributed to the establishment of tuning in the auditory fovea. Adult-like morphology of the organ of Corti coincided with the emergence of sharply tuned responses from the auditory fovea, but there was no clear-cut correlate for the shift in tuned foveal frequency representation that occurred during the following 3 weeks. J. Comp. Neurol. 377:520–534, 1997. © 1997 Wiley-Liss, Inc.     

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line-derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad-GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad-GDNF or ES or both. Ad-GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES-treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad-GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES-treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF- and ES-mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. .     

The resting transducer current drives spontaneous activity in prehearing Mammalian cochlear inner hair cells

Spontaneous Ca(2+)-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately -75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca(2+) concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately -55 mV). We propose that, in vivo, spontaneous Ca(2+) action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.     

Longitudinal gradients of KCNQ4 expression in spiral ganglion and cochlear hair cells correlate with progressive hearing loss in DFNA2

Mutations in the human KCNQ4 gene were recently found by Kubisch et al. [Cell 96 (1999) 437-446] to cause a non-syndromic, autosomal dominant, progressive hearing loss, DFNA2. The mouse Kcnq4 orthologue was previously localized to the outer hair cells (OHCs) of the inner ear, suggesting the pathophysiological effects were due to dysfunctional OHCs. Yet, OHC dysfunction does not provide a plausible explanation for the progressive nature of the frequency specific hearing loss. We have re-examined and extended the expression analyses of KCNQ4 in the murine inner ear using RT-PCR and whole mount in situ hybridization. Our results confirmed that the rat KCNQ4 orthologue is expressed in both inner and outer hair cells. Reciprocal longitudinal gradients were found in inner hair cells (IHCs) and OHCs. The strongest expression of KCNQ4 in IHCc was in the base of the cochlea and in the apex for OHCs. Similar to the IHCs, a basal to apical gradient was present in the spiral sensory neurons. IHCs mediate hearing via their afferent sensory neurons, whereas OHCs function as active cochlear amplifiers. The complete absence of OHCs leads only to severe sensitivity reduction, but not complete hearing loss. Our data suggest that the primary defect leading to initial high frequency loss and subsequent progressive hearing loss for all frequencies may be due to spiral ganglion and/or IHC dysfunction, rather than an OHC aberration.     

Spatiotemporal patterns of neuronal programmed cell death during postnatal development of the gerbil cochlea

During early postnatal development, afferent neurons of the cochlear (spiral) ganglion progressively refine their projections to auditory hair cells so that, by hearing onset, most cochlear nerve fibers innervate a single hearing receptor. One mechanism that might contribute to these changes in cochlear innervation is the programmed cell death (apoptosis) of developing neurons within the spiral ganglion. In the present study, we used the TUNEL method and morphological criteria to identify apoptotic cells within the spiral ganglion of the Mongolian gerbil during the first week of postnatal life when afferent projections to the cochlea are actively refined in this species. The locations of individual apoptotic spiral ganglion cells were mapped onto three-dimensional reconstructions of the entire ganglion for an age-graded series of gerbils to produce the first high-resolution, spatiotemporal maps of apoptotic ganglion cell death for the postnatal cochlea. We observed a significant increase in apoptosis in the spiral ganglion from postnatal day (P) 4 through P6. During this time, the most intense apoptotic activity occurred in regions of the spiral ganglion providing innervation to the lower middle and apical turns of the cochlea. The time course and regional variation of programmed cell death within the developing gerbil spiral ganglion are discussed in terms of the postnatal refinement of cochlear innervation and its possible functional significance for hearing in gerbils.     

Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.     

Expression of Myh9 in the mammalian cochlea: localization within the stereocilia

Mutations of non-muscle myosin Type IIA or MYH9 are linked to syndromic or nonsyndromic hearing loss. The biologic function of MYH9 in the auditory organ and the pathophysiology of its dysfunction remain to be determined. The mouse represents an excellent model for investigating the biologic role of MYH9 in the cells and tissues affected by its dysfunction. A primary step toward the understanding of the role of MYH9 in hearing and its dysfunction is the documentation of its cellular and sub-cellular localization within the cochlea, the auditory organ. We describe the localization of Myh9 within the mouse cochlea using a polyclonal anti-Myh9-antibody, generated against an 18 amino acid long peptide corresponding to the sequence at the C-terminus of mouse Myh9. The anti-Myh9 antibody identified a single, specific, immunoreactive band of 220 kDa in immunoblot analysis of homogenate from a variety of different mouse tissues. The Myh9 antibody cross-reacts with the rat but not the human orthologue. Myh9 is expressed predominantly within the spiral ligament as well as in the sensory hair cells of the organ of Corti. Confocal microscopy of cochlear surface preparations, identified Myh9 within the inner and outer hair cells and their stereocilia. Localization of Myh9 within the stereocilia raises the possibility that mutations of MYH9 may effect hearing loss though disruption of the stereocilia structure.     

Postnatal development of the hamster coclea. I. Growth of hair cells and the organ of Corti

A morphometric analysis of the developing organ of Corti and its component hair cells was carried out in an age-graded series of Syrian golden hamsters with the aid of scanning electron microscopy. The purpose was to establish a quantitative framework that would provide insight into the rules and principles by which the mammalian cochlea attains its adult proportions. This study examined postnatal development at two day intervals from birth to 22 days after birth. Our analysis included measures of cochlear length and hair cell numbers as well as measures of hair cell sizes in each of five sectors along the cochlear spiral. Our results demonstrate several principles of cochlear development: (1) The full two and one-fourths turns seen in the adult cochlea are already present at birth, but the cochlea continues to elongate for the next 10–12 days. (2) Development of hair cells in the apex generally lags behind that in the base. Whereas the stereocilia and apical margins of hair cells are clearly defined in the basal turn, they become well defined in the apex only postnatally. (3) Growth in cochlear length occurs mainly by increases in cell size rather than in cell numbers; although hair cells do increase in numbers during the first 4 days of cochlear growth, this increase involves addition of hair cells only to preexisting regions of the cochlear apex. Moreover, the full complement of hair cells is established 6 days before the full size of the cochlea is attained; in contrast, hair cell growth occurs at all positions along the cochlear spiral and spans the entire period of cochlear elongation. (4) The period of hair cell growth exceeds the period of organ of Corti growth and appears to be possible by decreases in intercellular spacing, primarily in the apical region of the cochlea; inner and outer hair cell growth was complete between 16 and 18 days after birth. (5) Inner and outer hair cell neighbors remain virtually constant at different ages indicating that the spatial relationships between the two hair cell populations is preserved as the cochlea grows. (6) Comparison with previous developmental studies of auditory function in the hamster reveals that the age of 16 days after birth, when hair cells attain their mature sizes, coincides with the onset of brainstem auditory evoked responses. Growth of hair cell somas alone, however, cannot explain either the subsequent maturation of evoked potential thresholds or changes in frequency representation in the developing cochlea. © Wiley-Liss, Inc.     

An efficient strategy for establishing a model of sensorineural deafness in rats

Ototoxic drugs can be used to produce a loss of cochlear hair cells to create animal models of deafness. However, to the best of our knowledge, there is no report on the establishment of a rat deafness model through the combined application of aminoglycosides and loop diuretics. The aim of this study was to use single or combined administration of furosemide and kanamycin sulfate to establish rat models of deafness. The rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. The auditory brainstem response was measured to determine the hearing threshold after drug application. Immunocytochemistry and confocal microscopy were performed to evaluate inner ear morphology. In the group receiving combined administration of furosemide and kanamycin, the auditory brainstem response threshold showed significant elevation 3 days after administration, higher than that produced by furosemide or kanamycin alone. The hair cells showed varying degrees of injury, from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. The spiral ganglion cells maintained a normal morphology during the first week after the hair cells completely disappeared, and then gradually degenerated. After 2 months, the majority of spiral ganglion cells disappeared, but a few remained. These findings demonstrate that the combined administration of furosemide and kanamycin has a synergistic ototoxic effect, and that these drugs can produce hair cell loss and hearing loss in rats. These findings suggest that even in patients with severe deafness, electronic cochlear implants may partially restore hearing.     

Loxhd1 Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells

Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase α-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the mechanotransduction phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphologic defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multirepeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.     

Caspase-3-deficiency induces hyperplasia of supporting cells and degeneration of sensory cells resulting in the hearing loss

Caspase-3 is one of the cystein proteases that play essential roles in programmed cell death. As such, brain development is profoundly affected by caspase-3-deficiency, resulting in hyperplasia and abnormal cell organization (Kuida et al., Nature 1996;384:368-372). In the present study, we used caspase-3 (-/-) mice to show that caspase-3 deficiency results in severe hearing loss, hyperplasia of supporting cells and degeneration of sensory hair cells. The greater epithelial ridge, a remnant of the primordial organ of Corti, persists throughout all of the turns of cochlea in 2-week-old caspase-3 (-/-) mice, which indicates that the morphology of the cochlea is immature. The number of border cells, that develop from the greater epithelial ridge and are one of the supporting cells of the inner hair cell, increase significantly in both 2- and 5-week-old caspase-3 (-/-) mice. On the other hand, abnormal fused stereocilia can be seen in both 2- and 5-week-old caspase-3 (-/-) mice, and disarrangement and loss of sensory hair cells are observed in 5-week-old caspase-3 (-/-) mice. Taken together, both hyperplasia and degeneration occur simultaneously in the inner ear of the caspase-3 (-/-) mice, suggesting that caspase-3-dependent apoptosis is necessary for the development and formation of a properly functioning auditory system in mammals.     

Conchlear inner hair cells exist transiently in the fetal Bronx waltzer (bv/bv) mouse

In the cochlea of the adult Bronx waltzer (bv/bv) mouse, the majority of inner hair cells are missing or deformed. As a result, Bronx waltzer mice are severely hearing impaired or deaf. Previous studies determined that most inner hair cells in these mice are missing by the time of birth, but no studies have resolved whether the missing inner hair cells ever exist in the mutant cochlea. The present study used light and electron microscopy to locate inner hair cells in the mutant mouse before birth. Most, and possibly all, inner hair cells exist in the embryonic day (E) 17 mouse. The shapes of the cells vary from normal and elongated in the youngest animals, to round and protruding through the reticular lamina a few days later. The density of sensory cells in the inner hair cell region (inner hair cells/millimeter) decreases in the basal turn between E17 and birth, and in the apical turn between birth and the third postnatal day. The initial presence of the full complement of inner hair cells, taken together with the temporospatial pattern of degeneration, suggests that the cause of inner hair cell death in the Bronx waltzer mouse is related to a differentiation event subsequent to cell birth. © 1996 Wiley-Liss, Inc.