Inner Hair Cell Loss Research Articles

Multifunctional redox modulator prevents blast-induced loss of cochlear and vestibular hair cells and auditory spiral ganglion neurons

Blast wave exposure, a leading cause of hearing loss and balance dysfunction among military personnel, arises primarily from direct mechanical damage to the mechanosensory hair cells and supporting structures or indirectly through excessive oxidative stress. We previously reported that HK-2, an orally active, multifunctional redox modulator (MFRM), was highly effective in reducing both hearing loss and hair cells loss in rats exposed to a moderate intensity workday noise that likely damages the cochlea primarily from oxidative stress versus direct mechanical trauma. To determine if HK-2 could also protect cochlear and vestibular cells from damage caused primarily from direct blast-induced mechanical trauma versus oxidative stress, we exposed rats to six blasts of 186 dB peak SPL. The rats were divided into four groups: (B) blast alone, (BEP) blast plus earplugs, (BHK-2) blast plus HK-2 and (BEPHK-2) blast plus earplugs plus HK-2. HK-2 was orally administered at 50 mg/kg/d from 7-days before to 30-day after the blast exposure. Cochlear and vestibular tissues were harvested 60-d post-exposure and evaluated for loss of outer hair cells (OHC), inner hair cells (IHC), auditory nerve fibers (ANF), spiral ganglion neurons (SGN) and vestibular hair cells in the saccule, utricle and semicircular canals. In the untreated blast-exposed group (B), massive losses occurred to OHC, IHC, ANF, SGN and only the vestibular hair cells in the striola region of the saccule. In contrast, rats treated with HK-2 (BHK-2) sustained significantly less OHC (67%) and IHC (57%) loss compared to the B group. OHC and IHC losses were smallest in the BEPHK-2 group, but not significantly different from the BEP group indicating lack of protective synergy between EP and HK-2. There was no loss of ANF, SGN or saccular hair cells in the BHK-2, BEP and BEPHK-2 groups. Thus, HK-2 not only significantly reduced OHC and IHC damage, but completely prevented loss of ANF, SGN and saccule hair cells. The powerful protective effects of this oral MFRM make HK-2 an extremely promising candidate for human clinical trials.

Delayed hearing loss after cochlear implantation: Re-evaluating the role of hair cell degeneration

Delayed loss of residual acoustic hearing after cochlear implantation is a common but poorly understood phenomenon due to the scarcity of relevant temporal bone tissues. Prior histopathological analysis of one case of post-implantation hearing loss suggested there were no interaural differences in hair cell or neural degeneration to explain the profound loss of low-frequency hearing on the implanted side (Quesnel et al., 2016) and attributed the threshold elevation to neo-ossification and fibrosis around the implant. Here we re-evaluated the histopathology in this case, applying immunostaining and improved microscopic techniques for differentiating surviving hair cells from supporting cells. The new analysis revealed dramatic interaural differences, with a > 80 % loss of inner hair cells in the cochlear apex on the implanted side, which can account for the post-implantation loss of residual hearing. Apical degeneration of the stria further contributed to threshold elevation on the implanted side. In contrast, spiral ganglion cell survival was reduced in the region of the electrode on the implanted side, but apical counts in the two ears were similar to that seen in age-matched unimplanted control ears. Almost none of the surviving auditory neurons retained peripheral axons throughout the basal half of the cochlea. Relevance to cochlear implant performance is discussed.

The human OPA1delTTAG mutation induces adult onset and progressive auditory neuropathy in mice

Dominant optic atrophy (DOA) is one of the most prevalent forms of hereditary optic neuropathies and is mainly caused by heterozygous variants in OPA1, encoding a mitochondrial dynamin-related large GTPase. The clinical spectrum of DOA has been extended to a wide variety of syndromic presentations, called DOAplus, including deafness as the main secondary symptom associated to vision impairment. To date, the pathophysiological mechanisms underlying the deafness in DOA remain unknown. To gain insights into the process leading to hearing impairment, we have analyzed the Opa1delTTAG mouse model that recapitulates the DOAplus syndrome through complementary approaches combining morpho-physiology, biochemistry, and cellular and molecular biology. We found that Opa1delTTAG mutation leads an adult-onset progressive auditory neuropathy in mice, as attested by the auditory brainstem response threshold shift over time. However, the mutant mice harbored larger otoacoustic emissions in comparison to wild-type littermates, whereas the endocochlear potential, which is a proxy for the functional state of the stria vascularis, was comparable between both genotypes. Ultrastructural examination of the mutant mice revealed a selective loss of sensory inner hair cells, together with a progressive degeneration of the axons and myelin sheaths of the afferent terminals of the spiral ganglion neurons, supporting an auditory neuropathy spectrum disorder (ANSD). Molecular assessment of cochlea demonstrated a reduction of Opa1 mRNA level by greater than 40%, supporting haploinsufficiency as the disease mechanism. In addition, we evidenced an early increase in Sirtuin 3 level and in Beclin1 activity, and subsequently an age-related mtDNA depletion, increased oxidative stress, mitophagy as well as an impaired autophagic flux. Together, these results support a novel role for OPA1 in the maintenance of inner hair cells and auditory neural structures, addressing new challenges for the exploration and treatment of OPA1-linked ANSD in patients.

Dexamethasone treatment of murine auditory hair cells and cochlear explants attenuates tumor necrosis factor-α-initiated apoptotic damage.

The most common cause of sensorineural hearing loss is damage of auditory hair cells. Tumor necrosis factor-alpha (TNF-α) is closely associated with sensorineural hearing loss. The present study examined the preconditioning effect of dexamethasone (DEX) on TNF-α-induced ototoxicity in mouse auditory hair cells (HEI-OC1) and cochlear explants. Treatment of HEI-OC1 with 10 ng/ml TNF-α for 24 h decreased cell viability, increased the accumulation of reactive oxygen species (ROS), and induced caspase-mediated apoptotic signaling pathways. Pretreatment with 10 nM DEX for 6 h before TNF-α exposure restored cell viability, decreased ROS accumulation, and attenuated apoptotic signaling activation induced by TNF-α. Incubation of cochlear explants with 20 ng/ml TNF-α for 24 h resulted in significant loss of both inner hair cells (IHCs) and outer hair cells (OHCs) and an increase in apoptotic activation accessed by annexin V staining. The cochlear explants pre-incubated with 10 nM DEX attenuated TNF-α ototoxicity in both IHCs and OHCs and apoptotic cell death. These results indicated that DEX plays a protective role in ototoxicity induced by TNF-α through attenuation of caspase-dependent apoptosis signaling pathway and ROS accumulation.

Testing phenomenological auditory-nerve model predictions for selective inner- and outer-hair-cell dysfunction

Sensorineural hearing loss (SNHL) effects on neural coding and perception have been largely associated with outer-hair-cell (OHC) dysfunction (e.g., reduced cochlear gain, reduced compression, broadened tuning). However, both inner-hair-cell (IHC) and OHC dysfunction occur in common hearing-loss etiologies, e.g., noise-induced and age-related (metabolic). Although IHC effects are largely ignored based on the insensitivity of audiograms to IHC loss up to 80%, the same selective IHC damage affects suprathreshold hearing (e.g., tone-in-noise detection, EFRs to periodic stimuli). Phenomenological auditory-nerve (AN) models are useful for studying neural-coding effects of SNHL, but their parametric control of OHC and IHC function (e.g., cohc and cihc parameters, Bruce et al., 2018) are designed based on data from animals with noise-induced hearing loss (OHC/IHC dysfunction combined). Although this approach has proven successful for OHC effects, testing of IHC-based effects has been largely indirect. Here, we compare model predictions to our previously collected AN-fiber responses to amplitude-modulated stimuli in animals with either carboplatin-induced selective IHC dysfunction or gentamicin-induced selective OHC damage to provide more direct testing of IHC and OHC effects. Model parameters were estimated from histology, ABR thresholds, AN-fiber rates, and spike-train statistical metrics (e.g., vector strength, discriminability, mutual information).

The SNARE protein SNAP-25 is required for normal exocytosis at auditory hair cell ribbon synapses.

Hearing depends on fast and sustained calcium-dependent synaptic vesicle fusion at the ribbon synapses of cochlear inner hair cells (IHCs). The implication of the canonical neuronal SNARE complex in this exocytotic process has so far remained controversial. We investigated the role of SNAP-25, a key component of this complex, in hearing, by generating and analyzing a conditional knockout mouse model allowing a targeted postnatal deletion of Snap-25 in IHCs. Mice subjected to IHC Snap-25 inactivation after hearing onset developed severe to profound deafness because of defective IHC exocytosis followed by ribbon degeneration and IHC loss. Viral transfer of Snap-25 in these mutant mice rescued their hearing function by restoring IHC exocytosis and preventing synapses and hair cells from degeneration. These results demonstrate that SNAP-25 is essential for normal hearing function, most likely by ensuring IHC exocytosis and ribbon synapse maintenance.

AAV-ie-mediated UCP2 overexpression accelerates inner hair cell loss during aging in vivo

BackgroundUncoupling protein 2 (UCP2), activated by excessive reactive oxygen species (ROS) in vivo, has the dual effect of reducing ROS to protect against oxidative stress and reducing ATP production to regulate cellular metabolism. Both the UCP2 and ROS are increased in cochleae in age-related hearing loss (ARHL). However, the role of UCP2 in sensory hair cells in ARHL remains unclear.MethodsMale C57BL/6 J mice were randomly assigned to an 8-week-old group (Group 1), a 16-week-old group (Group 2), a 16-week-old + adeno-associated virus-inner ear (AAV-ie) group (Group 3), and a 16-week-old + AAV-ie-UCP2 group (Group 4). Mice aged 8 weeks were administrated with AAV-ie-GFP or AAV-ie-UCP2 via posterior semicircular canal injection. Eight weeks after this viral intervention, hearing thresholds and wave-I amplitudes were tested by auditory brainstem response (ABR). Subsequently, the cochlear basilar membrane was dissected for investigation. The number of hair cells and inner hair cell (IHC) synapses, the level of ROS, and the expression of AMP-activated protein kinase α (AMPKα), were assessed by immunofluorescence staining. In addition, mitochondrial function was determined, and the expression of AMPKα and UCP2 proteins was further evaluated using western blotting.ResultsMice with early-onset ARHL exhibited enhanced oxidative stress and loss of outer hair cells and IHC synapses, while UCP2 overexpression aggravated hearing loss and cochlear pathophysiological changes in mice. UCP2 overexpression resulted in a notable decrease in the number of IHCs and IHC synapses, caused ATP depletion and excessive ROS generation, increased AMPKα protein levels, and promoted IHC apoptosis, especially in the apical and middle turns of the cochlea.ConclusionCollectively, our data suggest that UCP2 overexpression may cause mitochondrial dysfunction via energy metabolism, which activates mitochondrion-dependent cellular apoptosis and leads to IHC loss, ultimately exacerbating ARHL.

No Reduction in the 226-Hz Probe Tone Acoustic Reflex Amplitude Following Severe Inner Hair Cell Loss in Chinchillas.

The relationship between the middle ear acoustic reflex (AR) and inner hair cell (IHC) loss is currently unknown. Given that IHC are believed to convey nearly all acoustic information to the central auditory nervous system, it has been assumed that loss of IHC would significantly impact the AR. To evaluate this relationship, we assessed the presence and amplitude of the AR in chinchillas before and after treatment with carboplatin, an anticancer drug that reliably and selectively destroys IHC in this species. Baseline measures of hearing sensitivity, including auditory brainstem response (ABR) thresholds and distortion product otoacoustic emissions (DPOAE), were assessed and then re-evaluated following carboplatin treatment. Post-carboplatin ABR thresholds and DPOAE were found to be unchanged or slightly elevated; results were consistent with published reports. Our main hypothesis was that loss of IHC would abolish the reflex or significantly reduce its amplitude. Contrary to our hypothesis, the ipsilateral 226-Hz AR continued to be reliably elicited following carboplatin treatment. Post-mortem histological analysis confirmed significant IHC loss (65-85 %), but no measurable loss of outer hair cells (OHCs). Given that loss of IHC alone does not significantly reduce the 226-Hz AR, our results suggest that few IHC are needed to maintain the 226-Hz AR response. These results suggest additional studies are needed to better understand the role of IHC in the reflex arc, present opportunities to further study the reflex pathway, and could change how we use the clinical AR as a potential diagnostic tool for IHC dysfunction, including those related to IHC synaptopathy.

A humanized murine model, demonstrating dominant progressive hearing loss caused by a novel KCNQ4 mutation (p.G228D) from a large Chinese family.

The pathogenic variants in KCNQ4 cause DFNA2 nonsyndromic hearing loss. However, the understanding of genotype-phenotype correlations between KCNQ4 and hearing is limited. Here, we identified a novel KCNQ4 mutation p.G228D from a Chinese family, including heterozygotes characterized by high-frequency hearing loss that is progressive across all frequencies and homozygotes with more severe hearing loss. We constructed a novel murine model with humanized homologous Kcnq4 mutation. The heterozygotes had mid-frequency and high-frequency hearing loss at 4 weeks, and moved toward all frequencies hearing loss at 12 weeks, while the homozygotes had severe-to-profound hearing loss at 8 weeks. The degeneration of outer hair cells (OHCs) was observed from basal to apical turn of cochlea. The reduced K+ currents and depolarized resting potentials were revealed in OHCs. Remarkably, we observed the loss of inner hair cells (IHCs) in the region corresponding to the frequency above 32 kHz at 8-12 weeks. The results suggest the degeneration of OHCs and IHCs may contribute to high-frequency hearing loss in DFNA2 over time. Our findings broaden the variants of KCNQ4 and provide a novel mouse model of progressive hearing loss, which contributes to an understanding of pathogenic mechanism and eventually treatment of DFNA2 progressive hearing loss.

Stiffening of Circumferential F-Actin Bands Correlates With Regenerative Failure and May Act as a Biomechanical Brake in the Mammalian Inner Ear.

The loss of inner ear hair cells causes permanent hearing and balance deficits in humans and other mammals, but non-mammals recover after supporting cells (SCs) divide and replace hair cells. The proliferative capacity of mammalian SCs declines as exceptionally thick circumferential F-actin bands develop at their adherens junctions. We hypothesized that the reinforced junctions were limiting regenerative responses of mammalian SCs by impeding changes in cell shape and epithelial tension. Using micropipette aspiration and atomic force microscopy, we measured mechanical properties of utricles from mice and chickens. Our data show that the epithelial surface of the mouse utricle stiffens significantly during postnatal maturation. This stiffening correlates with and is dependent on the postnatal accumulation of F-actin and the cross-linker Alpha-Actinin-4 at SC-SC junctions. In chicken utricles, where SCs lack junctional reinforcement, the epithelial surface remains compliant. There, SCs undergo oriented cell divisions and their apical surfaces progressively elongate throughout development, consistent with anisotropic intraepithelial tension. In chicken utricles, inhibition of actomyosin contractility led to drastic SC shape change and epithelial buckling, but neither occurred in mouse utricles. These findings suggest that species differences in the capacity for hair cell regeneration may be attributable in part to the differences in the stiffness and contractility of the actin cytoskeletal elements that reinforce adherens junctions and participate in regulation of the cell cycle.

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

Effect of Selective Carboplatin-Induced Inner Hair Cell Loss on Temporal Integration in Chinchillas.

Integration of acoustic information over time is essential for processing complex stimuli, such as speech, due to its continuous variability along the time domain. In both humans and animals, perception of acoustic stimuli is a function of both stimulus intensity and duration. For brief acoustic stimuli, as duration increases, thresholds decrease by approximately 3dB for every doubling in duration until stimulus duration reaches 500ms, a phenomenon known as temporal integration. Although hearing loss and damage to outer hair cells (OHC) have been shown to alter temporal integration in some studies, the role of cochlear inner hair cells (IHC) on temporal integration is unknown. Because IHC transmit nearly all acoustic information to the central auditory system and are believed to code both intensity and timing information, these sensory cells likely play a critical role in temporal integration. To test the hypothesis that selective IHC loss degrades the temporal integration function, behaviorally trained chinchillas were treated with carboplatin, a drug known to selectively destroy IHC with little to no effect on OHC in this species. Pure-tone thresholds were assessed across frequencies (1, 2, 4, 8, 12kHz) as a function of signal duration (500, 100, 50, 10, and 5ms). Baseline testing showed a significant effect of duration on thresholds. Threshold decreased as a function of increasing duration, as expected. Carboplatin treatment (75mg/kg) produced a moderate to severe loss of IHC (45-85%) with little-to-no loss of OHC. Contrary to our hypothesis, post-carboplatin temporal integration thresholds showed no significant differences from baseline regardless of stimulus duration or frequency. These data suggest that few IHC are necessary for temporal integration of simple stimuli. Temporal integration may be sensitive to loss of OHC and loss of cochlear non-linearities but does not appear to be sensitive to selective IHC loss.

Threshold Equalizing Noise Test Reveals Suprathreshold Loss of Hearing Function, Even in the "Normal" Audiogram Range.

Objectives:The threshold equalizing noise (TEN(HL)) is a clinically administered test to detect cochlear “dead regions” (i.e., regions of loss of inner hair cell [IHC] connectivity), using a “pass/fail” criterion based on the degree of elevation of a masked threshold in a tone-detection task. With sensorineural hearing loss, some elevation of the masked threshold is commonly observed but usually insufficient to create a “fail” diagnosis. The experiment reported here investigated whether the gray area between pass and fail contained information that correlated with factors such as age or cumulative high-level noise exposure (>100 dBA sound pressure levels), possibly indicative of damage to cochlear structures other than the more commonly implicated outer hair cells.Design:One hundred and twelve participants (71 female) who underwent audiometric screening for a sensorineural hearing loss, classified as either normal or mild, were recruited. Their age range was 32 to 74 years. They were administered the TEN test at four frequencies, 0.75, 1, 3, and 4 kHz, and at two sensation levels, 12 and 24 dB above their pure-tone absolute threshold at each frequency. The test frequencies were chosen to lie either distinctly away from, or within, the 2 to 6 kHz region where noise-induced hearing loss is first clinically observed as a notch in the audiogram. Cumulative noise exposure was assessed by the Noise Exposure Structured Interview (NESI). Elements of the NESI also permitted participant stratification by music experience.Results:Across all frequencies and testing levels, a strong positive correlation was observed between elevation of TEN threshold and absolute threshold. These correlations were little-changed even after noise exposure and music experience were factored out. The correlations were observed even within the range of “normal” hearing (absolute thresholds ≤15 dB HL).Conclusions:Using a clinical test, sensorineural hearing deficits were observable even within the range of clinically “normal” hearing. Results from the TEN test residing between “pass” and “fail” are dominated by processes not related to IHCs. The TEN test for IHC-related function should therefore only be considered for its originally designed function, to generate a binary decision, either pass or fail.

Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells

ABSTRACT The loss of inner ear hair cells leads to irreversible acoustic injury in mammals, and regeneration of inner ear hair cells to restore hearing loss is challenging. ATOH1 is a key gene in the development and regeneration of hair cells. Small activating RNAs (saRNAs) can target a gene to specifically upregulate its expression. This study aimed to explore whether small activating RNAs could induce the differentiation of human adipose-derived mesenchymal stem cells into hair cell-like cells with a combination of growth factors in vitro and thus provide a new strategy for hair cell regeneration and the treatment of sensorineural hearing loss. Fifteen small activating RNAs targeting the human ATOH1 gene were designed and screened in 293 T and human adipose-derived mesenchymal stem cells, and 3 of these candidates were found to be capable of effectively and stably activating ATOH1 gene expression. The selected small activating RNAs were then transfected into hair cell progenitor cells, and hair cell markers were examined 10 days after transfection. After transfection of the selected small activating RNAs, the expression of the characteristic markers of inner ear hair cells, POU class 4 homeobox 3 (POU4F3) and myosin VIIA (MYO7A), was detected. Human adipose-derived mesenchymal stem cells have the potential to differentiate into human hair cell progenitor cells. In vitro, small activating RNAs were able to induce the differentiation of hair cell progenitor cells into hair cell-like cells. Therefore, RNA activation technology has the potential to provide a new strategy for the regeneration of hair cells.

Human vestibular schwannoma reduces density of auditory nerve fibers in the osseous spiral lamina

Hearing loss in patients with vestibular schwannoma (VS) is commonly attributed to mechanical compression of the auditory nerve, though recent studies suggest that this retrocochlear pathology may be augmented by cochlear damage. Although VS-associated loss of inner hair cells, outer hair cells, and spiral ganglion cells has been reported, it is unclear to what extent auditory-nerve peripheral axons are damaged in VS patients. Understanding the degree of damage VSs cause to auditory nerve fibers (ANFs) is important for accurately modeling clinical outcomes of cochlear implantation, which is a therapeutic option to rehabilitate hearing in VS-affected ears. A retrospective analysis of human temporal-bone histopathology was performed on archival specimens from the Massachusetts Eye and Ear collection. Seven patients met our inclusion criteria based on the presence of sporadic, unilateral, untreated VS. Tangential sections of five cochlear regions were stained with hematoxylin and eosin, and adjacent sections were stained to visualize myelinated ANFs and efferent fibers. Following confocal microscopy, peripheral axons of ANFs within the osseous spiral lamina were quantified manually, where feasible, and with a “pixel counting” method, applicable to all sections. ANF density was substantially reduced on the VS side compared to the unaffected contralateral side. In the upper basal turn, a significant difference between the VS side and unaffected contralateral side was found using both counting methods, corresponding to the region tuned to 2000 Hz. Even spiral ganglion cells (SGCs) contralateral to VS were affected by the tumor as the majority of contralateral SGC counts were below average for age. This observation provides histological insight into the clinical observation that unilateral vestibular schwannomas pose a long-term risk of progression of hearing loss in the contralateral ear as well. Our pixel counting method for ANF quantification in the osseous spiral lamina is applicable to other pathologies involving sensorineural hearing loss. Future research is needed to classify ANFs into morphological categories, accurately predict their electrical properties, and use this knowledge to inform optimal cochlear implant programming strategies.

Selective Inner Hair Cell Loss in a Neonate Harbor Seal (Phoca vitulina).

Simple SummaryCongenital hearing loss (i.e., hearing impairment present at birth) is recognized in humans and other terrestrial species, but there is a lack of information on congenital malformations and associated hearing loss in pinnipeds (seals, sea lions, and walruses). Baseline knowledge on marine mammal inner ear malformations is essential to differentiate between congenital and acquired abnormalities, which may be caused by infectious agents, age, or anthropogenic interactions, such as noise exposure. Analysis of the cochlea of a neonate harbor seal (Phoca vitulina) revealed bilateral loss of inner hair cells (sensory cells responsible for transducing the auditory signal) while the outer hair cells (sensory cells responsible for sound amplification and frequency selectivity and sensitivity) were intact. The selective inner hair cell loss (up to 84.6% of loss) was more severe in the basal turn, where the high frequencies are encoded. Potential causes and consequences are discussed. This is the first report of a case of selective inner hair cell loss in a marine mammal neonate, likely congenital.Congenital hearing loss is recognized in humans and other terrestrial species. However, there is a lack of information on its prevalence or pathophysiology in pinnipeds. It is important to have baseline knowledge on marine mammal malformations in the inner ear, to differentiate between congenital and acquired abnormalities, which may be caused by infectious pathogens, age, or anthropogenic interactions, such as noise exposure. Ultrastructural evaluation of the cochlea of a neonate harbor seal (Phoca vitulina) by scanning electron microscopy revealed bilateral loss of inner hair cells with intact outer hair cells. The selective inner hair cell loss was more severe in the basal turn, where high-frequency sounds are encoded. The loss of inner hair cells started around 40% away from the apex or tip of the spiral, reaching a maximum loss of 84.6% of hair cells at 80–85% of the length from the apex. Potential etiologies and consequences are discussed. This is believed to be the first case report of selective inner hair cell loss in a marine mammal neonate, likely congenital.

Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage.

Noise exposure causes noise-induced hearing loss (NIHL). NIHL exhibits loss of inner ear sensory hair cells and is often irreparable. Although oxidative stress is involved in hearing loss, the complex mechanisms involved in NIHL are unclear. Hypoxia-inducible factor 1α (HIF-1α) has been suggested to be essential for protecting sensory hair cells. Additionally, it has been shown that ROS is involved in modulating the stability of HIF-1α. To investigate the NIHL pathogenesis, we established a tert-butyl hydroperoxide (t-BHP)-induced oxidative stress damage model in hair-like HEI-OC1 cells and an NIHL model in C57BL/6 mice. Protein and mRNA expression were determined, and biochemical parameters including reactive oxygen species (ROS) accumulation, glucose uptake, adenosine triphosphat (ATP) production, and mitochondrial content were evaluated. In HEI-OC1 cells, t-BHP induced ROS accumulation and reduced mitochondrial content and oxygen consumption, but the ATP level was unaffected. Additionally, there was increased glucose uptake and lactate release along with elevated expression of HIF-1α, glucose transporter 1, and several glycolytic enzymes. Consistently, noise trauma induced oxidative stress and the expression of HIF-1α and glycolytic enzymes in mice. Thus, we concluded that ROS induced HIF-1α expression, which promoted glycolysis, suggesting a metabolic shift maintained the ATP level to attenuate hair cell damage in NIHL.

Combined antioxidants and anti-inflammatory therapies fail to attenuate the early and late phases of cyclodextrin-induced cochlear damage and hearing loss

Niemann-Pick C1 (NPC1) is a fatal neurodegenerative disease caused by aberrant cholesterol metabolism. The progression of the disease can be slowed by removing excess cholesterol with high-doses of 2-hyroxypropyl-beta-cyclodextrin (HPβCD). Unfortunately, HPβCD causes hearing loss; the initial first phase involves a rapid destruction of outer hair cells (OHCs) while the second phase, occurring 4–6 weeks later, involves the destruction of inner hair cells (IHCs), pillar cells, collapse of the organ of Corti and spiral ganglion neuron degeneration. To determine whether the first and/or second phase of HPβCD-induced cochlear damage is linked, in part, to excess oxidative stress or neuroinflammation, rats were treated with a single-dose of 3000 mg/kg HPβCD alone or together with one of two combination therapies. Each combination therapy was administered from 2-days before to 6-weeks after the HPβCD treatment. Combination 1 consisted of minocycline, an antibiotic that suppresses neuroinflammation, and HK-2, a multifunctional redox modulator that suppresses oxidative stress. Combination 2 was comprised of minocycline plus N-acetyl cysteine (NAC), which upregulates glutathione, a potent antioxidant. To determine if either combination therapy could prevent HPβCD-induced hearing impairment and cochlear damage, distortion product otoacoustic emissions (DPOAE) were measured to assess OHC function and the cochlear compound action potential (CAP) was measured to assess the function of IHCs and auditory nerve fibers. Cochleograms were prepared to quantify the amount of OHC, IHC and pillar cell (PC) loss. HPβCD significantly reduced DPOAE and CAP amplitudes and caused significant OHC, IHC and OPC losses with losses greater in the high-frequency base of the cochlea than the apex. Neither minocycline + HK-2 (MIN+ HK-2) nor minocycline + NAC (MIN+NAC) prevented the loss of DPOAEs, CAPs, OHCs, IHCs or IPCs caused by HPβCD. These results suggest that oxidative stress and neuroinflammation are unlikely to play major roles in mediating the first or second phase of HPβCD-induced cochlear damage. Thus, HPβCD-induced ototoxicity must be mediated by some other unknown cell-death pathway possibly involving loss of trophic support from damaged support cells or disrupted cholesterol metabolism.

Age-related reduction in frequency-following responses as a potential marker of cochlear neural degeneration

Healthy aging may be associated with neural degeneration in the cochlea even before clinical hearing loss emerges. Reduction in frequency-following responses (FFRs) to tonal carriers in older clinically normal-hearing listeners has previously been reported, and has been argued to reflect an age-dependent decline in temporal processing in the central auditory system. Alternatively, age-dependent loss of auditory nerve fibers (ANFs) may have little effect on audiometric sensitivity and yet compromise the precision of neural phase-locking relying on joint activity across populations of fibers. This peripheral loss may, in turn, contribute to reduced neural synchrony in the brainstem as reflected in the FFR. Here, we combined human electrophysiology and auditory nerve (AN) modeling to investigate whether age-related changes in the FFR would be consistent with peripheral neural degeneration. FFRs elicited by pure tones and frequency sweeps at carrier frequencies between 200 and 1200 Hz were obtained in older (ages 48–76) and younger (ages 20–30) listeners, both groups having clinically normal audiometric thresholds up to 6 kHz. The same stimuli were presented to a computational model of the AN in which age-related loss of hair cells or ANFs was modelled using human histopathological data. In the older human listeners, the measured FFRs to both sweeps and pure tones were found to be reduced across the carrier frequencies examined. These FFR reductions were consistent with model simulations of age-related ANF loss. In model simulations, the phase-locked response produced by the population of remaining fibers decreased proportionally with increasing loss of the ANFs. Basal-turn loss of inner hair cells also reduced synchronous activity at lower frequencies, albeit to a lesser degree. Model simulations of age-related threshold elevation further indicated that outer hair cell dysfunction had no negative effect on phase-locked AN responses. These results are consistent with a peripheral source of the FFR reductions observed in older normal-hearing listeners, and indicate that FFRs at lower carrier frequencies may potentially be a sensitive marker of peripheral neural degeneration.

LGR5-Positive Supporting Cells Survive Ototoxic Trauma in the Adult Mouse Cochlea.

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters’ cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.