Electrocochleographic frequency-following responses as a potential marker of age-related cochlear neural degeneration.

Auditory nerve (AN) fibers that innervate inner hair cells in the cochlea degenerate with advancing age. It has been proposed that age-related reductions in brainstem frequency-following responses (FFR) to the carrier of low-frequency, high-intensity pure tones may partially reflect this neural loss in the cochlea (Märcher-Rørsted et al., 2022). If the loss of AN fibers is the primary factor contributing to age-related changes in the brainstem FFR, then the FFR could serve as an indicator of cochlear neural degeneration. In this study, we employed electrocochleography (ECochG) to investigate the effects of age on frequency-following neurophonic potentials, i.e., neural responses phase-locked to the carrier frequency of the tone stimulus. We compared these findings to the brainstem-generated FFRs obtained simultaneously using the same stimulation. We conducted recordings in young and older individuals with normal hearing. Responses to pure tones (250 ms, 516 and 1086 Hz, 85 dB SPL) and clicks were recorded using both ECochG at the tympanic membrane and traditional scalp electroencephalographic (EEG) recordings of the FFR. Distortion product otoacoustic emissions (DPOAE) were also collected. In the ECochG recordings, sustained AN neurophonic (ANN) responses to tonal stimulation, as well as the click-evoked compound action potential (CAP) of the AN, were significantly reduced in the older listeners compared to young controls, despite normal audiometric thresholds. In the EEG recordings, brainstem FFRs to the same tone stimulation were also diminished in the older participants. Unlike the reduced AN CAP response, the transient-evoked wave-V remained unaffected. These findings could indicate that a decreased number of AN fibers contributes to the response in the older participants. The results suggest that the scalp-recorded FFR, as opposed to the clinical standard wave-V of the auditory brainstem response, may serve as a more reliable indicator of age-related cochlear neural degeneration.

Surface electrical stimulation of the auditory cortex preserves efferent medial olivocochlear neurons and reduces cochlear traits of age-related hearing loss.

The auditory cortex is the source of descending connections providing contextual feedback for auditory signal processing at almost all levels of the lemniscal auditory pathway. Such feedback is essential for cognitive processing. It is likely that corticofugal pathways are degraded with aging, becoming important players in age-related hearing loss and, by extension, in cognitive decline. We are testing the hypothesis that surface, epidural stimulation of the auditory cortex during aging may regulate the activity of corticofugal pathways, resulting in modulation of central and peripheral traits of auditory aging. Increased auditory thresholds during ongoing age-related hearing loss in the rat are attenuated after two weeks of epidural stimulation with direct current applied to the surface of the auditory cortex for two weeks in alternate days (Fernández del Campo et al., 2024). Here we report that the same cortical electrical stimulation protocol induces structural and cytochemical changes in the aging cochlea and auditory brainstem, which may underlie recovery of age-degraded auditory sensitivity. Specifically, we found that in 18 month-old rats after two weeks of cortical electrical stimulation there is, relative to age-matched non-stimulated rats: a) a larger number of choline acetyltransferase immunoreactive neuronal cell body profiles in the ventral nucleus of the trapezoid body, originating the medial olivocochlear system.; b) a reduction of age-related dystrophic changes in the stria vascularis; c) diminished immunoreactivity for the pro-inflammatory cytokine TNFα in the stria vascularis and spiral ligament. d) diminished immunoreactivity for Iba1 and changes in the morphology of Iba1 immunoreactive cells in the lateral wall, suggesting reduced activation of macrophage/microglia; d) Increased immunoreactivity levels for calretinin in spiral ganglion neurons, suggesting excitability modulation by corticofugal stimulation. Altogether, these findings support that non-invasive neuromodulation of the auditory cortex during aging preserves the cochlear efferent system and ameliorates cochlear aging traits, including stria vascularis dystrophy, dysregulated inflammation and altered excitability in primary auditory neurons.

Evidence of cochlear neural degeneration in normal-hearing subjects with tinnitus.

Tinnitus, reduced sound-level tolerance, and difficulties hearing in noisy environments are the most common complaints associated with sensorineural hearing loss in adult populations. This study aims to clarify if cochlear neural degeneration estimated in a large pool of participants with normal audiograms is associated with self-report of tinnitus using a test battery probing the different stages of the auditory processing from hair cell responses to the auditory reflexes of the brainstem. Self-report of chronic tinnitus was significantly associated with (1) reduced cochlear nerve responses, (2) weaker middle-ear muscle reflexes, (3) stronger medial olivocochlear efferent reflexes and (4) hyperactivity in the central auditory pathways. These results support the model of tinnitus generation whereby decreased neural activity from a damaged cochlea can elicit hyperactivity from decreased inhibition in the central nervous system.

Morphological and morphometric study of the superior vestibular nerve trunk in guinea pigs.

The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve dissection. Little data have been reported on the normality of these nerves. The vestibular nerve is composed of the superior vestibular, inferior vestibular, and branch nerves. This study aimed to study the microscopic anatomy of the superior vestibular nerve (SVN) of guinea pigs using light microscopy and to search for normality patterns for use in experimental models in basic otologic research. We used eight male albino guinea pigs (Cavia porcellus, English strain), weighing between 400 and 500 g. After anesthetizing, the animals were perfused with a fixative solution of 2.5% glutaraldehyde. Dissection was performed by the access method to the temporal bone, coming to the rock and exposing the cochlea and vestibular nerve. The NVS fragments were removed, postfixed in osmium tetroxide, and embedded in the epoxy plastic resin Poly/Bed 812® (Polysciences Inc., Warrington, PA). Semi-thin transverse serial sections (0.5 µm) were made using a microtome MT6000-XL, RMC, Inc. and stained with toluidine blue. Morphology and morphometry were described and evaluated using the KS 400 application (Kontron 2.0, EchingBei, Munich, Germany) by macro, a computer program specially designed and developed for the study of the VIII nerve. The SVN was found to be devoid of epineurium, with only a thin conjunctive tissue layer. The myelin sheath of guinea pigs is relatively thin compared to the sensory and motor nerves found in mammals. The average fascicular area SVN was 0.19 ± 0.05 mm2 , with the largest area found to be 0.24 mm2 and the lowest was 0.12 mm2 . The average number of fibers was 5,753.00 ± 538 fibers. The density of myelinated fibers reached 32,316.08 ± 11,375.29 fibers/mm2 . Its diameter ranged from 1.0 to 9 µm and its peak was 3 µm. The measured results confirm the results of another study, indicating that the methodology is appropriate and reproducible. These findings are important for the evaluation of injured nerves in experimental models of peripheral neuropathy and basic ear disease.

Loss of Pex1 in Inner Ear Hair Cells Contributes to Cochlear Synaptopathy and Hearing Loss.

Peroxisome Biogenesis Disorders (PBD) and Zellweger syndrome spectrum disorders (ZSD) are rare genetic multisystem disorders that include hearing impairment and are associated with defects in peroxisome assembly, function, or both. Mutations in 13 peroxin (PEX) genes have been found to cause PBD-ZSD with ~70% of patients harboring mutations in PEX1. Limited research has focused on the impact of peroxisomal disorders on auditory function. As sensory hair cells are particularly vulnerable to metabolic changes, we hypothesize that mutations in PEX1 lead to oxidative stress affecting hair cells of the inner ear, subsequently resulting in hair cell degeneration and hearing loss. Global deletion of the Pex1 gene is neonatal lethal in mice, impairing any postnatal studies. To overcome this limitation, we created conditional knockout mice (cKO) using Gfi1Creor VGlut3Cre expressing mice crossed to floxed Pex1 mice to allow for selective deletion of Pex1 in the hair cells of the inner ear. We find that Pex1 excision in inner hair cells (IHCs) leads to progressive hearing loss associated with significant decrease in auditory brainstem responses (ABR), specifically ABR wave I amplitude, indicative of synaptic defects. Analysis of IHC synapses in cKO mice reveals a decrease in ribbon synapse volume and functional alterations in exocytosis. Concomitantly, we observe a decrease in peroxisomal number, indicative of oxidative stress imbalance. Taken together, these results suggest a critical function of Pex1 in development and maturation of IHC-spiral ganglion synapses and auditory function.

ISL1 is necessary for auditory neuron development and contributes toward tonotopic organization.

A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.

Chromatin remodeler CHD7 is critical for cochlear morphogenesis and neurosensory patterning.

Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.

The possibility of cochlear synaptopathy in young people using a personal listening device.

OBJECTIVE: To evaluate the association of listening to music loudly through personal listening devices with cochlear synaptopathy in young adults. METHODS: Fifty healthy young adults selected among 109 volunteers were included in the study. Participants of high risk (n=25) and low risk (n=25) groups estimated according to ETDNL (estimated total daily noise level) were evaluated using pure tone audiometry, tympanometry, matrix test, electrocochleography (EcochG) and auditory brainstem response (ABR) to evaluate the occurrence of cochlear synaptopathy. RESULTS: Audiometric thresholds between the groups were not significantly different (p>0.05). High risk group participants showed poorer performance than the low-risk group on the TurMatrix test, in non-adaptive noise with -5 SNR and -7.5 SNR, and at the 50% understanding SNR level with headphones (p<0.01). There was no difference in the adaptive free field in noise test at which 50% understanding was achieved (p>0.05). The AP amplitudes on EcochG and wave V amplitudes on ABR were significantly smaller in the high-risk group (p<0.05). There was no association between ETDNL and I/V ratio on ABR. CONCLUSION: Poorer performance in TurMatrix and other electrophysiologic tests revealed the negative effect of personal listening devices on the auditory system. Our findings support the hypothesis that personal listening devices could cause cochlear synaptopathy. Long-term studies are needed to determine the effects of binaural hearing and duration of noise exposure on the auditory system.

Cytoarchitecture and innervation of the mouse cochlear amplifier revealed by large-scale volume electron microscopy.

In mammalian cochlea, sound-induced vibration is amplified by a three-row lattice of Y-shaped microstructures consisting of electromotile outer hair cell and supporting Deiters cell. This highly organized structure is thought to be essential for hearing of low-level sounds. Prior studies reported differences in geometry and synaptic innervation of the outer hair cells between rows, but how these fine features are achieved at subcellular level still remains unclear. Using serial block-face electron microscopy, we acquired few-hundred-micron-sized cytoarchitecture of mouse organ of Corti at nanometer resolution. Structural quantifications were performed on the Y-shapes as well as afferent and efferent projections to outer hair cells (OHCs). Several new features, which support the previously observed inter-row heterogeneity, are described. Our result provides structural bases for the gradient of mechanical properties and diverse centrifugal regulation of OHC rows.

Profound bilateral deafness complicating aortic arch surgery.

Hearing loss is a rare complication of cardiac surgery; bilateral profound deafness has never been reported in this setting. A 45-year-old male presented with profound bilateral sudden deafness following arch surgery and frozen elephant trunk. Patient's presentation, surgery details and aetiological mechanisms are discussed.

Neuroprotective Effect of Huperzine A on d-Galactose-Induced Hearing Dysfunction.

BACKGROUND: Administration of d-galactose (d-gal) has been used to create animal models of neurodegenerative diseases, and huperzine A has been used to treat the neurodegenerative diseases such as Alzheimer disease. METHODS: An animal model of hearing dysfunction was established by administration of d-gal in the rats, and the effect of huperzine A on d-gal-induced abnormal hearing function and cochlear damage was investigated. Senescence of the cochlear tissues was examined by ß-galactase staining, and messenger RNA expression of inflammatory cytokines was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: It was found that d-gal significantly increased auditory brainstem response (ABR) threshold and cellular senescence and decreased neurofilament in the cochlear tissues. Huperzine A could significantly attenuate d-gal-induced increase of ABR threshold and cellular senescence as well as reduction of neurofilament. Moreover, huperzine A could inhibit d-gal-induced activation of nuclear factor kappa-B (NF-κB) in Schwann cells and significantly blocked d-gal-stimulated gene expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α. CONCLUSION: These findings suggested that d-gal causes hearing dysfunction by inflammatory injury of cochlear neurons and that huperzine A could prevent hearing loss by protecting d-gal-induced physical damage of cochlear tissues.

Mathematical model of the auditory nerve response to stimulation by a micro-machined cochlea.

We report a novel mathematical model of an artificial auditory system consisting of a micro-machined cochlea and the auditory nerve response it evokes. The modeled micro-machined cochlea is one previously realized experimentally by mimicking functions of the cochlea [Shintaku et al, Sens. Actuat. 158 (2010) 183-192; Inaoka et al, Proc. Natl. Acad. Sci. USA 108 (2011) 18390-18395]. First, from the viewpoint of mechanical engineering, the frequency characteristics of a model device were experimentally investigated to develop an artificial basilar membrane based on a spring-mass-damper system. In addition, a nonlinear feedback controller mimicking the function of the outer hair cells was incorporated in this experimental system. That is, the developed device reproduces the proportional relationship between the oscillation amplitude of the basilar membrane and the cube root of the sound pressure observed in the mammalian auditory system, which is what enables it to have a wide dynamic range, and the characteristics of the control performance were evaluated numerically and experimentally. Furthermore, the stimulation of the auditory nerve by the micro-machined cochlea was investigated using the present mathematical model, and the simulation results were compared with our previous experimental results from animal testing [Shintaku et al, J. Biomech. Sci. Eng. 8 (2013) 198-208]. The simulation results were found to be in reasonably good agreement with those from the previous animal test; namely, there exists a threshold at which the excitation of the nerve starts and a saturation value for the firing rate under a large input. The proposed numerical model was able to qualitatively reproduce the results of the animal test with the micro-machined cochlea and is thus expected to guide the evaluation of micro-machined cochleae for future animal experiments.

Hidden Hearing Loss Impacts the Neural Representation of Speech in Background Noise.

Many individuals with seemingly normal hearing abilities struggle to understand speech in noisy backgrounds. To understand why this might be the case, we investigated the neural representation of speech in the auditory midbrain of gerbils with "hidden hearing loss" through noise exposure that increased hearing thresholds only temporarily. In noise-exposed animals, we observed significantly increased neural responses to speech stimuli, with a more pronounced increase at moderate than at high sound intensities. Noise exposure reduced discriminability of neural responses to speech in background noise at high sound intensities, with impairment most severe for tokens with relatively greater spectral energy in the noise-exposure frequency range (2-4 kHz). At moderate sound intensities, discriminability was surprisingly improved, which was unrelated to spectral content. A model combining damage to high-threshold auditory nerve fibers with increased response gain of central auditory neurons reproduced these effects, demonstrating that a specific combination of peripheral damage and central compensation could explain listening difficulties despite normal hearing thresholds.

Inner-ear malformations as a cause of single-sided deafness.

OBJECTIVE: To determine the prevalence and distribution of inner-ear malformations in congenital single-sided deafness cases, as details of malformation type are crucial for disease prognosis and management. METHODS: A retrospective study was conducted of 90 patients aged under 16 years with congenital single-sided deafness. Radiological findings were evaluated using computed tomography and magnetic resonance imaging. Inner-ear malformations were identified and cochlear nerve status was determined in affected ears. RESULTS: Out of 90 ears, 42 (46.7 per cent) were found to have inner-ear malformation. Isolated cochlear aperture stenosis was the most common anomaly (n = 18, 20 per cent), followed by isolated cochlear aperture atresia (n = 11, 12.2 per cent) and cochlear hypoplasia (n = 7, 7.8 per cent). Cochlear nerve deficiency was encountered in 41 ears (45.6 per cent). The internal auditory canal was also stenotic in 49 ears (54.4 per cent). CONCLUSION: Inner-ear malformations, especially cochlear aperture anomalies, are involved in the aetiology of single-sided deafness more than expected. The cause of single-sided deafness differs greatly between congenital and adult-onset cases. All children with single-sided deafness should undergo radiological evaluation, as the prognosis and management, as well as the aetiology, may be significantly influenced by inner-ear malformation type.

Macromolecular and electrical coupling between inner hair cells in the rodent cochlea.

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.

Long-term moderate noise exposure enhances the medial olivocochlear reflex.

OBJECTIVE: To investigate the effects of long-term moderate noise on hearing functions, MOCR, and MEMR. METHODS: Mice were exposed to the moderate noise (11.2 - 22.4 kHz, 80 dB SPL, 6 h/day, 4 weeks). Subsequently, the hearing functions, including threshold and input-output roles of ABR (auditory brainstem response) and cubic (2f1-f2) DPOAEs (distortion product otoacoustic emissions) were evaluated. Also, MEMR and MOCR were assessed shortly after or at four weeks following the termination of exposure to the noise. RESULTS: The mice's acoustic suppression reflex was strengthened, hearing functions and MEMR were unaffected four weeks after the moderate noise. For primary tones of 16, 20 and 24 kHz, the strengths of contralateral and ipsilateral suppression in the noise group were about double those recorded in the control group. In order to further determine whether the functional changes of the afferent or efferent nerves increased the strengths of acoustic suppression, the mouse's left ear was inserted the earplug, and then exposed the moderate noise for four weeks. The strengths of contralateral suppression at 16, 20 and 24 kHz were increased for the noise + earplug than for the control group and were indistinguishable between the noise + earplug and the noise group. While no significant changes were found in the strengths of ipsilateral suppression at all frequencies for the noise + earplug group compared with the control group. Under ketamine/xylazine anesthesia, the broadband suppressor noise did not stimulate the MEMR by 20 min post-induction at all frequencies in three groups. CONCLUSION: Our data demonstrated that the long-term moderate noise-exposure strengthened mice's MOCR by changing its afferent nerves, and unaffected cochlear hair cells and type I spiral ganglion neurons.

Circumvention of common labelling artefacts using secondary nanobodies.

A standard procedure to study cellular elements is via immunostaining followed by optical imaging. This methodology typically requires target-specific primary antibodies (1.Abs), which are revealed by secondary antibodies (2.Abs). Unfortunately, the antibody bivalency, polyclonality, and large size can result in a series of artifacts. Alternatively, small, monovalent probes, such as single-domain antibodies (nanobodies) have been suggested to minimize these limitations. The discovery and validation of nanobodies against specific targets are challenging, thus only a minimal amount of them are currently available. Here, we used STED, DNA-PAINT, and light-sheet microscopy, to demonstrate that secondary nanobodies (1) increase localization accuracy compared to 2.Abs; (2) allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; (3) penetrate thick tissues more efficiently; and (4) avoid probe-induced clustering of target molecules observed with conventional 2.Abs in living or poorly fixed samples. Altogether, we show how secondary nanobodies are a valuable alternative to 2.Abs.

FGF22 promotes generation of ribbon synapses through downregulating MEF2D.

Cochlear ribbon synapses play a pivotal role in the prompt and precise acoustic signal transmission from inner hair cells (IHCs) to the spiral ganglion neurons, while noise and aging can damage ribbon synapses, resulting in sensorineural hearing loss. Recently, we described reduced fibroblast growth factor 22 (FGF22) and augmented myocyte enhancer factor 2D (MEF2D) in an ototoxicity mouse model with impaired ribbon synapses. Here, we investigated the mechanisms that underlie the FGF22/MEF2D- regulated impairment of ribbon synapses. We generated adeno-associated virus (AAV) carrying FGF22, shFGF22, MEF2D, shMEF2D, calcineurin (CalN), shCalN or corresponding scramble controls for transduction of cultured mouse hair cells. We found that FGF22 was a suppressor for MEF2D, but not vice versa. Moreover, FGF22 likely induced increases in the calcium influx into IHCs to activate CalN, which subsequently inhibited MEF2D. Cochlear infusion of AAV-shFGF22 activated MEF2D, reduced ribbon synapse number and impaired hearing function, which were all abolished by co-infusion of AAV-shMEF2D. Hence, our data suggest that the ribbon synapses may be regulated by FGF22/calcium/CalN/MEF2D signaling, which implied novel therapeutic targets for hearing loss.

Knockdown of Foxg1 in supporting cells increases the trans-differentiation of supporting cells into hair cells in the neonatal mouse cochlea.

Foxg1 is one of the forkhead box genes that are involved in morphogenesis, cell fate determination, and proliferation, and Foxg1 was previously reported to be required for morphogenesis of the mammalian inner ear. However, Foxg1 knock-out mice die at birth, and thus the role of Foxg1 in regulating hair cell (HC) regeneration after birth remains unclear. Here we used Sox2CreER/+ Foxg1loxp/loxp mice and Lgr5-EGFPCreER/+ Foxg1loxp/loxp mice to conditionally knock down Foxg1 specifically in Sox2+ SCs and Lgr5+ progenitors, respectively, in neonatal mice. We found that Foxg1 conditional knockdown (cKD) in Sox2+ SCs and Lgr5+ progenitors at postnatal day (P)1 both led to large numbers of extra HCs, especially extra inner HCs (IHCs) at P7, and these extra IHCs with normal hair bundles and synapses could survive at least to P30. The EdU assay failed to detect any EdU+ SCs, while the SC number was significantly decreased in Foxg1 cKD mice, and lineage tracing data showed that much more tdTomato+ HCs originated from Sox2+ SCs in Foxg1 cKD mice compared to the control mice. Moreover, the sphere-forming assay showed that Foxg1 cKD in Lgr5+ progenitors did not significantly change their sphere-forming ability. All these results suggest that Foxg1 cKD promotes HC regeneration and leads to large numbers of extra HCs probably by inducing direct trans-differentiation of SCs and progenitors to HCs. Real-time qPCR showed that cell cycle and Notch signaling pathways were significantly down-regulated in Foxg1 cKD mice cochlear SCs. Together, this study provides new evidence for the role of Foxg1 in regulating HC regeneration from SCs and progenitors in the neonatal mouse cochlea.

Cisplatin Toxicology: The Role of Pro-inflammatory Cytokines and GABA Transporters in Cochlear Spiral Ganglion.

BACKGROUND: The current study was conducted to examine the specific activation of pro-inflammatory cytokines (PICs), namely IL-1ß, IL-6 and TNF-α in the cochlear spiral ganglion of rats after ototoxicity induced by cisplatin. Since γ-aminobutyric acid (GABA) and its receptors are involved in pathophysiological processes of ototoxicity, we further examined the role played by PICs in regulating expression of GABA transporter type 1 and 3 (GAT-1 and GAT-3), as two essential subtypes of GATs responsible for the regulation of extracellular GABA levels in the neuronal tissues. METHODS: ELISA and western blot analysis were employed to examine the levels of PICs and GATs; and auditory brainstem response was used to assess ototoxicity induced by cisplatin. RESULTS: IL-1ß, IL-6 and TNF-α as well as their receptors were significantly increased in the spiral ganglion of ototoxic rats as compared with sham control animals (P<0.05, ototoxic rats vs. control rats). Cisplatin-ototoxicity also induced upregulation of the protein levels of GAT-1 and GAT-3 in the spiral ganglion (P<0.05 vs. controls). In addition, administration of inhibitors to IL-1ß, IL-6 and TNF-α attenuated amplification of GAT-1 and GAT-3 and improved hearing impairment induced by cisplatin. CONCLUSION: Our data indicate that PIC signals are activated in the spiral ganglion during cisplatin-ototoxicity which thereby leads to upregulation of GABA transporters. As a result, it is likely that de-inhibition of GABA system is enhanced in the cochlear spiral ganglion. This supports a role for PICs in engagement of the signal mechanisms associated with cisplatin-ototoxicity, and has pharmacological implications to target specific PICs for GABAergic dysfunction and vulnerability related to cisplatin-ototoxicity.