Influence of CX3CR1 Deletion on Cochlear Hair Cell Survival and Macrophage Expression in Chronic Suppurative Otitis Media.

OBJECTIVE: Our objective was to determine whether the receptor CX3CR1 is necessary for the recruitment of macrophages to the cochlea in chronic suppurative otitis media (CSOM) and if its deletion can prevent hair cell loss in CSOM. BACKGROUND: CSOM is a neglected disease that afflicts 330 million people worldwide and is the most common cause of permanent hearing loss among children in the developing world. It is characterized by a chronically discharging infected middle ear. We have previously demonstrated that CSOM causes macrophage associated sensory hearing loss. The receptor CX3CR1 is expressed on macrophages, which have been shown to be increased at the time point of outer hair cell (OHC) loss in CSOM. METHODS: In this report, we examine the influence of CX3CR1 deletion (CX3CR1-/-) in a validated model of Pseudomonas aeruginosa (PA) CSOM. RESULTS: The data show no difference in OHC loss between the CX3CR1-/- CSOM group and CX3CR1+/+ CSOM group (p = 0.28). We observed partial OHC loss in the cochlear basal turn, no OHC loss in the middle and apical turns in both CX3CR1-/- and CX3CR1+/+ CSOM mice at 14 days after bacterial inoculation. No inner hair cell (IHC) loss was found in all cochlear turns in all groups. We also counted F4/80 labeled macrophages in the spiral ganglion, spiral ligament, stria vascularis and spiral limbus of the basal, middle, and apical turn in cryosections. We did not find a significant difference in the total number of cochlear macrophages between CX3CR1-/- mice and CX3CR1+/+ mice (p = 0.97). CONCLUSION: The data did not support a role for CX3CR1 macrophage associated HC loss in CSOM.

Povidone-Iodine Fails to Eradicate Chronic Suppurative Otitis Media and Demonstrates Ototoxic Risk in Mice.

HYPOTHESIS: Commercially available povidone-iodine solution can eliminate biofilms and persister cells rapidly in in vivo achievable concentrations without inducing ototoxicity. BACKGROUND: Chronic suppurative otitis media (CSOM) is a substantial global problem. Current treatment options often induce a temporary remission without leading to a permanent cessation of symptoms secondary to the treatments' inability to eliminate persister cells. Povidone-iodine has been shown to be able to clear biofilm and planktonic cells in in vitro assays, but there are reports of ototoxic effects limiting its clinical utility. METHODS: Bacterial and biofilm growth with quantification by spectrophotomer, murine auditory brainstem response (ABR), and distortion product otoacoustic emissions, immunohistochemistry, in vivo povidone-iodine treatment of murine CSOM, persister cell assay. RESULTS: Commercially available 10% povidone-iodine solution is able to completely eradicate multiple clinical strains of Pseudomonas aeruginosa and Staphylococcus aureus in vitro with 10 minutes of exposure. Mice that have received a transtympanic injection of 1% povidone-iodine solution did not have significantly different auditory brainstem response or distortion product otoacoustic emission results compared with the control. Mice that received a povidone-iodine scrub or 10% povidone-iodine solution had significantly worsened hearing (25- and 13-dB increase in threshold, respectively; p < 0.05). In vivo CSOM infection recurred in all mice after the completion of treatment with 10% povidone-iodine solution, and there was no improvement in the bacterial load after treatment, indicating in vivo failure of therapy. CONCLUSION: Povidone-iodine solution is effective at eliminating biofilm and persister cells in vitro at in vivo achievable concentrations but fails in vivo most likely because of kinetics of distribution in vivo. Even if drug distribution could be improved, the therapeutic window is likely to be too small given that the diluted solution does not have ototoxic potential, whereas while the scrub variant, which contains detergents, and the undiluted solution are ototoxic after a single treatment.

The impact of targeted ablation of one row of outer hair cells and Deiters' cells on cochlear amplification.

The mammalian cochlea contains three rows of outer hair cells (OHCs) that amplify the basilar membrane traveling wave with high gain and exquisite tuning. The pattern of OHC loss caused by typical methods of producing hearing loss in animal models (noise, ototoxic exposure, or aging) is variable and not consistent along the length of the cochlea. Thus, it is difficult to use these approaches to understand how forces from multiple OHCs summate to create normal cochlear amplification. Here, we selectively removed the third row of OHCs and Deiters' cells in adult mice and measured cochlear amplification. In the mature cochlear epithelia, expression of the Wnt target gene Lgr5 is restricted to the third row of Deiters' cells, the supporting cells directly underneath the OHCs. Diphtheria toxin administration to Lgr5DTR-EGFP/+ mice selectively ablated the third row of Deiters' cells and the third row of OHCs. Basilar membrane vibration in vivo demonstrated disproportionately lower reduction in cochlear amplification by about 13.5 dB. On a linear scale, this means that the 33% reduction in OHC number led to a 79% reduction in gain. Thus, these experimental data describe the impact of reducing the force of cochlear amplification by a specific amount. Furthermore, these data argue that because OHC forces progressively and sequentially amplify the traveling wave as it travels to its peak, the loss of even a relatively small number of OHCs, when evenly distributed longitudinally, will cause a substantial reduction in cochlear amplification.NEW & NOTEWORTHY Normal cochlear physiology involves force production from three rows of outer hair cells to amplify and tune the traveling wave. Here, we used a genetic approach to target and ablate the third row of outer hair cells in the mouse cochlea and found it reduced cochlear amplification by 79%. This means that the loss of even a relatively small number of OHCs, when evenly distributed, causes a substantial reduction in cochlear amplification.

Topical Delivery of Elastic Liposomal Vesicles for Treatment of Middle and Inner Ear Diseases.

We present a topical drug delivery mechanism through the ear canal to the middle and inner ear using liposomal nanoparticles without disrupting the integrity of the tympanic membrane. The current delivery method provides a noninvasive and safer alternative to transtympanic membrane injections, ear tubes followed by ear drops administration, and systemic drug formulations. We investigate the capability of liposomal NPs, particularly transfersomes (TLipo), used as drug delivery vesicles to penetrate the tympanic membrane (TM) and round window membrane (RWM) with high affinity, specificity, and retention time. The TLipo is applied to the ear canal and found to pass through the tympanic membrane quickly in 3 h post drug administration. They are identified in the middle ear cavity 6 h and in the inner ear 24 h after drug administration. We performed cytotoxicity in vitro and ototoxicity in vivo studies. Cell viability shows no significant difference between the applied TLipo concentration and control. Furthermore, auditory brainstem response (ABR) reveals no hearing loss in 1 week and 1 month post-administration. Immunohistochemistry results demonstrate no evidence of hair cell loss in the cochlea at 1 month following TLipo administration. Together, the data suggested that TLipo can be used as a vehicle for topical drug delivery to the middle ear and inner ear.

Chronic suppurative otitis media causes macrophage-associated sensorineural hearing loss.

BACKGROUND: Chronic suppurative otitis media (CSOM) is the most common cause of permanent hearing loss in children in the developing world. A large component of the permanent hearing loss is sensory in nature and our understanding of the mechanism of this has so far been limited to post-mortem human specimens or acute infection models that are not representative of human CSOM. In this report, we assess cochlear injury in a validated Pseudomonas aeruginosa (PA) CSOM mouse model. METHODS: We generated persisters (PCs) and inoculated them into the mouse middle ear cavity. We tracked infection with IVIS and detected PA using RT-PCR. We assessed cochlear damage and innate immunity by Immunohistochemistry. Finally, we evaluated cytokines with multiplex assay and quantitative real-time PCR. RESULTS: We observed outer hair cell (OHC) loss predominantly in the basal turn of the cochlear at 14 days after bacterial inoculation. Macrophages, not neutrophils are the major immune cells in the cochlea in CSOM displaying increased numbers and a distribution correlated with the observed cochlear injury. The progression of the morphological changes suggests a transition from monocytes into tissue macrophages following infection. We also show that PA do not enter the cochlea and live bacteria are required for cochlear injury. We characterized cytokine activity in the CSOM cochlea. CONCLUSIONS: Taken together, this data shows a critical role for macrophages in CSOM-mediated sensorineural hearing loss (SNHL).

Gold nanocluster adjuvant enables the eradication of persister cells by antibiotics and abolishes the emergence of resistance.

Persister cells are responsible for relapses of infections common in cystic fibrosis and chronic suppurative otitis media (CSOM). Yet, there are no Food and Drug Administration (FDA) approved antibiotics to eradicate persister cells. Frustratingly, the global preclinical bacterial pipeline does not contain antibacterial agents targeting persister cells. Therefore, we report a nontraditional antimicrobial chemotherapy strategy based on gold nanoclusters adjuvant to eradicate persister cells by existing antibiotics belonging to that different class. Compared to killing with antibiotics alone, combining antibiotics and AuNC@CPP sterilizes persister cells and biofilms. Enhanced killing of up to 4 orders of magnitude in a validated mouse model of CSOM with Pseudomonas aeruginosa infection was observed when combining antibiotics and AuNC@CPP, informing a potential approach to improve the treatment of CSOM. We established that the mechanism of action of AuNC@CPP is due to disruption of the proton gradient and membrane hyperpolarization. The method presented here could compensate for the lack of new antibiotics to combat persister cells. This method could also benefit the current effort to slow resistance development because AuNC@CPP abolished the emergence of drug-resistant strains induced by antibiotics.

Minimally invasive trans-tympanic eustachian tube occlusion animal model.

OBJECTIVE: Eustachian tube dysfunction is believed to be involved in the pathogenesis of many middle ear diseases including chronic suppurative otitis media. We aimed to describe a simple and reliable animal model of Eustachian Tube obstruction to further research into middle ear disorders. STUDY DESIGN: Prospective cohort study in animals. SETTING: University laboratory. SUBJECTS AND METHODS: 30 mice C57Bl/6J (n = 15) and CBA/CaJ (n = 15) aged 6-8 weeks received transtympanic Eustachian tube occlusion on left ear trough an acute tympanic membrane perforation using thermoplastic latex used in dental procedures (gutta percha). Control mice (n = 6) received tympanic membrane perforation only. At two and four weeks, the mice were observed for signs of Eustachian tube dysfunction and compared to control ears. ET dysfunction was defined as presence of effusion in the middle ear. RESULTS: 100% (n = 30) of the treated ears had otoscopic signs of Eustachian tube dysfunction at two weeks and the endpoint time of four weeks, compared to 0% in control mice (0/6). Temporary head tilt lasting up to 2 days were observed in 3 mice (10%). No other potential adverse events were recorded. No bacterial growth was determined in the middle ear fluid. CONCLUSION: We describe a technically easy and reliable method for Eustachian tube occlusion in mice with an excellent success rate and minimal morbidity.

Topical Therapy Failure in Chronic Suppurative Otitis Media is Due to Persister Cells in Biofilms.

OBJECTIVE: Chronic suppurative otitis media (CSOM) is characterized by a chronically draining middle ear. CSOM is typically treated with multiple courses of antibiotics or antiseptics which are successful in achieving quiescence; however, the disease is prone to relapse. Understanding why these treatment failures occur is essential. STUDY DESIGN: The minimum inhibitory concentration (MIC), minimal biofilm eradication concentration, and the inhibitory zone were determined for ototopicals and ofloxacin for the laboratory strains and CSOM-derived isolates. The percentage of persister cells and bacterial biofilm formation were measured. Disease eradication was tested in a validated in-vivo model of CSOM after treatment with ofloxacin. SETTING: Microbiology Laboratory. METHODS: Basic science experiments were performed to measure the effectiveness of a number of compounds against CSOM bacteria in a number of distinct settings. RESULTS: The minimal biofilm eradication concentration is higher than is physiologically achievable with commercial preparations, except for povo-iodine. Clincial isolates of CSOM have equivalent biofilm-forming ability but increased proportions of persister cells. Ofloxacin can convert to inactive disease temporarily but fails to eradicate disease in an in-vivo model. CONCLUSIONS: Higher percentages of persister cells in clinical CSOM isolates are associated with resistance to ototopicals. Current ototopicals, except povo-iodine, have limited clinical effectiveness; however, it is unknown what the maximum achievable concentration is and there are ototoxicity concerns. Fluoroquinolones, while successful in producing inactive disease in the short term, have the potential to encourage antimicrobial resistance and disease recalcitrance and do not achieve a permanent remission. Given these limitations, clinicians should consider surgery earlier or use of clinically safe concentrations of povo-iodine earlier into the treatment algorithm.

Short-Wave Infrared Fluorescence Chemical Sensor for Detection of Otitis Media.

Otitis media (OM) or middle ear infection is one of the most common diseases in young children around the world. The diagnosis of OM is currently performed using an otoscope to detect middle ear fluid and inflammatory changes manifested in the tympanic membrane. However, conventional otoscopy cannot visualize across the tympanic membrane or sample middle ear fluid. This can lead to low diagnostic certainty and overdiagnoses of OM. To improve the diagnosis of OM, we have developed a short-wave infrared (SWIR) otoscope in combination with a protease-cleavable biosensor, 6QC-ICG, which can facilitate the detection of inflammatory proteases in the middle ear with an increase in contrast. 6QC-ICG is a fluorescently quenched probe, which is activated in the presence of cysteine cathepsin proteases that are up-regulated in inflammatory immune cells. Using a preclinical model and custom-built SWIR otomicroscope in this proof-of-concept study, we successfully demonstrated the feasibility of robustly distinguishing inflamed ears from controls (p = 0.0006). The inflamed ears showed an overall signal-to-background ratio of 2.0 with a mean fluorescence of 81 ± 17 AU, while the control ear exhibited a mean fluorescence of 41 ± 11 AU. We envision that these fluorescently quenched probes in conjunction with SWIR imaging tools have the potential to be used as an alternate/adjunct tool for objective diagnosis of OM.

Locally administered heparin-binding epidermal growth factor-like growth factor reduces radiation-induced oral mucositis in mice.

Oral mucositis refers to lesions of the oral mucosa observed in patients with cancer being treated with radiation with or without chemotherapy, and can significantly affect quality of life. There is a large unmet medical need to prevent oral mucositis that can occur with radiation either alone or in combination with chemotherapy. We investigated the efficacy of locally administered heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent epithelial proliferation and migration stimulator of the oral mucosa as a potential therapy to prevent radiation induced oral mucositis. Using a single dose (20 Gy) of radiation to the oral cavity of female C57BL/6 J mice, we evaluated the efficacy of HB-EGF treatment (5 µl of 10 µg/ml) solution. The results show that HB-EGF delivered post radiation, significantly increased the area of epithelial thickness on the tongue (dorsal tongue (42,106 vs 53,493 µm2, p < 0.01), ventral tongue (30,793 vs 39,095 µm2, *p < 0.05)) compared to vehicle control, enhanced new epithelial cell division, and increased the quality and quantity of desmosomes in the oral mucosa measured in the tongue and buccal mucosa. This data provides the proof of concept that local administration of HB-EGF has the potential to be developed as a topical treatment to mitigate oral mucositis following radiation.

A novel mouse model of chronic suppurative otitis media and its use in preclinical antibiotic evaluation.

Chronic suppurative otitis media (CSOM) is a neglected pediatric disease affecting 330 million worldwide for which no new drugs have been introduced for over a decade. We developed a mouse model with utility in preclinical drug evaluation and antimicrobial discovery. Our model used immune-competent mice, tympanic membrane perforation and inoculation with luminescent Pseudomonas aeruginosa that enabled bacterial abundance tracking in real-time for 100 days. The resulting chronic infection exhibited hallmark features of clinical CSOM, including inhibition of tympanic membrane healing and purulent ear discharge. We evaluated the standard care fluoroquinolone ofloxacin and demonstrated that this therapy resulted in a temporary reduction of bacterial burden. These data are consistent with the clinical problem of persistent infection in CSOM and the need for therapeutic outcome measures that assess eradication post-therapeutic endpoint. We conclude that this novel mouse model of CSOM has value in investigating new potential therapies.

Nr2f1 heterozygous knockout mice recapitulate neurological phenotypes of Bosch-Boonstra-Schaaf optic atrophy syndrome and show impaired hippocampal synaptic plasticity.

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) has been identified as an autosomal-dominant disorder characterized by a complex neurological phenotype, with high prevalence of intellectual disability and optic nerve atrophy/hypoplasia. The syndrome is caused by loss-of-function mutations in NR2F1, which encodes a highly conserved nuclear receptor that serves as a transcriptional regulator. Previous investigations to understand the protein's role in neurodevelopment have mostly used mouse models with constitutive and tissue-specific homozygous knockout of Nr2f1. In order to represent the human disease more accurately, which is caused by heterozygous NR2F1 mutations, we investigated a heterozygous knockout mouse model and found that this model recapitulates some of the neurological phenotypes of BBSOAS, including altered learning/memory, hearing defects, neonatal hypotonia and decreased hippocampal volume. The mice showed altered fear memory, and further electrophysiological investigation in hippocampal slices revealed significantly reduced long-term potentiation and long-term depression. These results suggest that a deficit or alteration in hippocampal synaptic plasticity may contribute to the intellectual disability frequently seen in BBSOAS. RNA-sequencing (RNA-Seq) analysis revealed significant differential gene expression in the adult Nr2f1+/- hippocampus, including the up-regulation of multiple matrix metalloproteases, which are known to be critical for the development and the plasticity of the nervous system. Taken together, our studies highlight the important role of Nr2f1 in neurodevelopment. The discovery of impaired hippocampal synaptic plasticity in the heterozygous mouse model sheds light on the pathophysiology of altered memory and cognitive function in BBSOAS.

Identification of early inflammatory changes in the tympanic membrane with Raman spectroscopy.

The tympanic membrane (TM) is a dynamic structure that separates the middle ear from the external auditory canal. It is also integral for the transmission of sound waves. In this study, we demonstrate the feasibility of using Raman spectroscopy to identify early chemical changes resulting from inflammation in the TM that can serve as an indicator of acute otitis media. Bacterial lipopolysaccharide (LPS) was injected trans-tympanicaly in a murine model. Presence of inflammatory response was assessed with binocular microscopy, confirmed with histopathology and immunofluorescence staining. Successful discrimination suggesting spectral differences among the control and LPS treated groups was achieved using principal component analysis. Raman imaging revealed major differences in collagen distribution and nucleic acid content. Image segmentation analysis on the trichrome stained tissue sections was performed to corroborate the Raman spectra. The spectral co-localization study suggests changes in the expression of collagen IV specific signals in LPS treated samples. The overall findings of the study support prospective application of RS in the diagnosis and therapeutic monitoring of otitis media.

Elimination of peripheral auditory pathway activation does not affect motor responses from ultrasound neuromodulation.

BACKGROUND: Recent studies in a variety of animal models including rodents, monkeys, and humans suggest that transcranial focused ultrasound (tFUS) has considerable promise for non-invasively modulating neural activity with the ability to target deep brain structures. However, concerns have been raised that motor responses evoked by tFUS may be due to indirect activation of the auditory pathway rather than direct activation of motor circuits. OBJECTIVE: In this study, we sought to examine the involvement of peripheral auditory system activation from tFUS stimulation applied to elicit motor responses. The purpose was to determine to what extent ultrasound induced auditory artifact could be a factor in ultrasound motor neuromodulation. METHODS: In this study, tFUS-induced electromyography (EMG) signals were recorded and analyzed in wild-type (WT) normal hearing mice and two strains of genetically deaf mice to examine the involvement of the peripheral auditory system in tFUS-stimulated motor responses. In addition, auditory brainstem responses (ABRs) were measured to elucidate the effect of the tFUS stimulus envelope on auditory and motor responses. We also varied the tFUS stimulation duration to measure its effect on motor response duration. RESULTS: We show, first, that the sharp edges in a tFUS rectangular envelope stimulus activate the peripheral afferent auditory pathway and, second, that smoothing these edges eliminates the auditory responses without affecting the motor responses in normal hearing WT mice. We further show that by eliminating peripheral auditory activity using two different strains of deaf knockout mice, motor responses are the same as in normal hearing WT mice. Finally, we demonstrate a high correlation between tFUS pulse duration and EMG response duration. CONCLUSION: These results support the concept that tFUS-evoked motor responses are not a result of stimulation of the peripheral auditory system.

Organ of Corti vibration within the intact gerbil cochlea measured by volumetric optical coherence tomography and vibrometry.

There is indirect evidence that the mammalian cochlea in the low-frequency apical and the more commonly studied high-frequency basal regions function in fundamentally different ways. Here, we directly tested this hypothesis by measuring sound-induced vibrations of the organ of Corti (OoC) at three turns of the gerbil cochlea using volumetric optical coherence tomography vibrometry (VOCTV), an approach that permits noninvasive imaging through the bone. In the apical turn, there was little frequency selectivity, and the displacement-vs.-frequency curves had low-pass filter characteristics with a corner frequency of ~0.5-0.9 kHz. The vibratory magnitudes increased compressively with increasing stimulus intensity at all frequencies. In the middle turn, responses were similar except for a slight peak in the response at ~2.5 kHz. The gain was ~50 dB at the peak and 30-40 dB at lower frequencies. In the basal turn, responses were sharply tuned and compressively nonlinear, consistent with observations in the literature. These data demonstrated that there is a transition of the mechanical response of the OoC along the length of the cochlea such that frequency tuning is sharper in the base than in the apex. Because the responses are fundamentally different, it is not appropriate to simply frequency shift vibratory data measured at one cochlear location to predict the cochlear responses at other locations. Furthermore, this means that the number of hair cells stimulated by sound is larger for low-frequency stimuli and smaller for high-frequency stimuli for the same intensity level. Thus the mechanisms of central processing of sounds must vary with frequency. NEW & NOTEWORTHY A volumetric optical coherence tomography and vibrometry system was used to probe cochlear mechanics within the intact gerbil cochlea. We found a gradual transition of the mechanical response of the organ of Corti along the length of the cochlea such that tuning at the base is dramatically sharper than that at the apex. These data help to explain discrepancies in the literature regarding how the cochlea processes low-frequency sounds.

Mammalian Auditory Hair Cell Bundle Stiffness Affects Frequency Tuning by Increasing Coupling along the Length of the Cochlea.

The stereociliary bundles of cochlear hair cells convert mechanical vibrations into the electrical signals required for auditory sensation. While the stiffness of the bundles strongly influences mechanotransduction, its influence on the vibratory response of the cochlear partition is unclear. To assess this, we measured cochlear vibrations in mutant mice with reduced bundle stiffness or with a tectorial membrane (TM) that is detached from the sensory epithelium. We found that reducing bundle stiffness decreased the high-frequency extent and sharpened the tuning of vibratory responses obtained postmortem. Detaching the TM further reduced the high-frequency extent of the vibrations but also lowered the partition's resonant frequency. Together, these results demonstrate that the bundle's stiffness and attachment to the TM contribute to passive longitudinal coupling in the cochlea. We conclude that the stereociliary bundles and TM interact to facilitate passive-wave propagation to more apical locations, possibly enhancing active-wave amplification in vivo.

Osmotic stabilization prevents cochlear synaptopathy after blast trauma.

Traumatic noise causes hearing loss by damaging sensory hair cells and their auditory synapses. There are no treatments. Here, we investigated mice exposed to a blast wave approximating a roadside bomb. In vivo cochlear imaging revealed an increase in the volume of endolymph, the fluid within scala media, termed endolymphatic hydrops. Endolymphatic hydrops, hair cell loss, and cochlear synaptopathy were initiated by trauma to the mechanosensitive hair cell stereocilia and were K+-dependent. Increasing the osmolality of the adjacent perilymph treated endolymphatic hydrops and prevented synaptopathy, but did not prevent hair cell loss. Conversely, inducing endolymphatic hydrops in control mice by lowering perilymph osmolality caused cochlear synaptopathy that was glutamate-dependent, but did not cause hair cell loss. Thus, endolymphatic hydrops is a surrogate marker for synaptic bouton swelling after hair cells release excitotoxic levels of glutamate. Because osmotic stabilization prevents neural damage, it is a potential treatment to reduce hearing loss after noise exposure.

Hair cell force generation does not amplify or tune vibrations within the chicken basilar papilla.

Frequency tuning within the auditory papilla of most non-mammalian species is electrical, deriving from ion-channel resonance within their sensory hair cells. In contrast, tuning within the mammalian cochlea is mechanical, stemming from active mechanisms within outer hair cells that amplify the basilar membrane travelling wave. Interestingly, hair cells in the avian basilar papilla demonstrate both electrical resonance and force-generation, making it unclear which mechanism creates sharp frequency tuning. Here, we measured sound-induced vibrations within the apical half of the chicken basilar papilla in vivo and found broadly-tuned travelling waves that were not amplified. However, distortion products were found in live but not dead chickens. These findings support the idea that avian hair cells do produce force, but that their effects on vibration are small and do not sharpen tuning. Therefore, frequency tuning within the apical avian basilar papilla is not mechanical, and likely derives from hair cell electrical resonance.

Two-Dimensional Cochlear Micromechanics Measured In Vivo Demonstrate Radial Tuning within the Mouse Organ of Corti.

UNLABELLED: The exquisite sensitivity and frequency discrimination of mammalian hearing underlie the ability to understand complex speech in noise. This requires force generation by cochlear outer hair cells (OHCs) to amplify the basilar membrane traveling wave; however, it is unclear how amplification is achieved with sharp frequency tuning. Here we investigated the origin of tuning by measuring sound-induced 2-D vibrations within the mouse organ of Corti in vivo Our goal was to determine the transfer function relating the radial shear between the structures that deflect the OHC bundle, the tectorial membrane and reticular lamina, to the transverse motion of the basilar membrane. We found that, after normalizing their responses to the vibration of the basilar membrane, the radial vibrations of the tectorial membrane and reticular lamina were tuned. The radial tuning peaked at a higher frequency than transverse basilar membrane tuning in the passive, postmortem condition. The radial tuning was similar in dead mice, indicating that this reflected passive, not active, mechanics. These findings were exaggerated in Tecta(C1509G/C1509G) mice, where the tectorial membrane is detached from OHC stereocilia, arguing that the tuning of radial vibrations within the hair cell epithelium is distinct from tectorial membrane tuning. Together, these results reveal a passive, frequency-dependent contribution to cochlear filtering that is independent of basilar membrane filtering. These data argue that passive mechanics within the organ of Corti sharpen frequency selectivity by defining which OHCs enhance the vibration of the basilar membrane, thereby tuning the gain of cochlear amplification. SIGNIFICANCE STATEMENT: Outer hair cells amplify the traveling wave within the mammalian cochlea. The resultant gain and frequency sharpening are necessary for speech discrimination, particularly in the presence of background noise. Here we measured the 2-D motion of the organ of Corti in mice and found that the structures that stimulate the outer hair cell stereocilia, the tectorial membrane and reticular lamina, were sharply tuned in the radial direction. Radial tuning was similar in dead mice and in mice lacking a tectorial membrane. This suggests that radial tuning comes from passive mechanics within the hair cell epithelium, and that these mechanics, at least in part, may tune the gain of cochlear amplification.

Changes in the regulation of the Notch signaling pathway are temporally correlated with regenerative failure in the mouse cochlea.

Sensorineural hearing loss is most commonly caused by the death of hair cells in the organ of Corti, and once lost, mammalian hair cells do not regenerate. In contrast, other vertebrates such as birds can regenerate hair cells by stimulating division and differentiation of neighboring supporting cells. We currently know little of the genetic networks which become active in supporting cells when hair cells die and that are activated in experimental models of hair cell regeneration. Several studies have shown that neonatal mammalian cochlear supporting cells are able to trans-differentiate into hair cells when cultured in conditions in which the Notch signaling pathway is blocked. We now show that the ability of cochlear supporting cells to trans-differentiate declines precipitously after birth, such that supporting cells from six-day-old mouse cochlea are entirely unresponsive to a blockade of the Notch pathway. We show that this trend is seen regardless of whether the Notch pathway is blocked with gamma secretase inhibitors, or by antibodies against the Notch1 receptor, suggesting that the action of gamma secretase inhibitors on neonatal supporting cells is likely to be by inhibiting Notch receptor cleavage. The loss of responsiveness to inhibition of the Notch pathway in the first postnatal week is due in part to a down-regulation of Notch receptors and ligands, and we show that this down-regulation persists in the adult animal, even under conditions of noise damage. Our data suggest that the Notch pathway is used to establish the repeating pattern of hair cells and supporting cells in the organ of Corti, but is not required to maintain this cellular mosaic once the production of hair cells and supporting cells is completed. Our results have implications for the proposed used of Notch pathway inhibitors in hearing restoration therapies.