ABR/DPOAE publications

Over 27 million people worldwide currently receive daily antiretroviral therapy for the management of HIV/AIDS. In order to prevent the continued spread of HIV, the World Health Organization (WHO) recommends the use of highly active antiretroviral therapy by pregnant and nursing women. There is currently little research into the auditory effects of this therapy on children exposed during pregnancy and breastfeeding, and research to date on the direct effects of antiretroviral exposure on the auditory system is inconclusive. The current study examined the effects of WHO-recommended first-line antiretrovirals in a well-controlled animal model to evaluate the potential for auditory damage and dysfunction following these exposures. Female breeding mice were each exposed to one of four antiretroviral cocktails or a vehicle control once daily during pregnancy and breastfeeding. Offspring of these mice had their auditory status evaluated after weaning using auditory brainstem responses and distortion-product otoacoustic emissions (DPOAEs). Auditory brainstem response thresholds following antiretroviral exposure during gestation and breastfeeding showed elevated thresholds and increased wave latencies in offspring of exposed mice when compared to unexposed controls, but no corresponding decrease in DPOAE amplitude. These differences in threshold were small and so may explain the lack of identified hearing loss in antiretroviral-exposed children during hearing screenings at birth. Minimal degrees of hearing impairment in children have been correlated with decreased academic performance and impaired auditory processing, and so these findings, if also seen in human children, suggest significant implications for children exposed to antiretrovirals during development despite passing hearing screenings at birth.

The progressive motor neuropathy (PMN) mouse is a model of an inherited motor neuropathy disease with progressive neurodegeneration. Axon degeneration associates with homozygous mutations of the TBCE gene encoding the tubulin chaperone E protein. TBCE is responsible for the correct dimerization of alpha and beta-tubulin. Strikingly, the PMN mouse also develops a progressive hearing loss after normal hearing onset, characterized by degeneration of the auditory nerve and outer hair cell (OHC) loss. However, the development of this neuronal and cochlear pathology is not fully understood yet. Previous studies with pegylated insulin-like growth factor 1 (peg-IGF-1) treatment in this mouse model have been shown to expand lifespan, weight, muscle strength, and motor coordination. Accordingly, peg-IGF-1 was evaluated for an otoprotective effect. We investigated the effect of peg-IGF-1 on the auditory system by treatment starting at postnatal day 15 (p15). Histological analysis revealed positive effects on OHC synapses of medial olivocochlear (MOC) neuronal fibers and a short-term attenuation of OHC loss. Peg-IGF-1 was able to conditionally restore the disorganization of OHC synapses and maintain the provision of cholinergic acetyltransferase in presynapses. To assess auditory function, frequency-specific auditory brainstem responses and distortion product otoacoustic emissions were recorded in animals on p21 and p28. However, despite the positive effect on MOC fibers and OHC, no restoration of hearing could be achieved. The present work demonstrates that the synaptic pathology of efferent MOC fibers in PMN mice represents a particular form of “efferent auditory neuropathy.” Peg-IGF-1 showed an otoprotective effect by preventing the degeneration of OHCs and efferent synapses. However, enhanced efforts are needed to optimize the treatment to obtain detectable improvements in hearing performances.

Anxiety is characteristic comorbidity of noise-induced hearing loss (NIHL), which causes physiological changes within the dentate gyrus (DG), a subfield of the hippocampus that modulates anxiety. However, which DG circuit underlies hearing loss-induced anxiety remains unknown. We utilize an NIHL mouse model to investigate short- and long-term synaptic plasticity in DG networks. The recently discovered longitudinal DG-DG network is a collateral of DG neurons synaptically connected with neighboring DG neurons and displays robust synaptic efficacy and plasticity. Furthermore, animals with NIHL demonstrate increased anxiety-like behaviors similar to a response to chronic restraint stress. These behaviors are concurrent with enhanced synaptic responsiveness and suppressed short- and long-term synaptic plasticity in the longitudinal DG-DG network but not in the transverse DG-CA3 connection. These findings suggest that DG-related anxiety is typified by synaptic alteration in the longitudinal DG-DG network.

Objectives To systematically explore the differences in acoustic changes and healing outcomes of tympanic membranes (TMs) with pars flaccida perforation (PFP) and pars tensa perforation (PTP). Methods We created PFPs and PTPs of various sizes in Sprague–Dawley rats, and evaluated TM umbo velocity and hearing function using laser Doppler vibrometry and auditory brainstem response (ABR) measurement before and immediately after perforation. Two weeks later, hearing was reevaluated and TMs were investigated by immunohistochemical staining. Results Small PFPs and PTPs did not significantly affect umbo velocity and hearing function. Large PFPs increased umbo velocity loss at low frequency (1.5 kHz) and elevated ABR thresholds within 1–2 kHz. Large PTP caused significant velocity loss at low frequencies from 1.5 to 3.5 kHz and threshold elevations at full frequencies (1–2 kHz). Two weeks after the perforation, the hearing function of rats with healed PFPs recovered completely. However, high‐frequency hearing loss (16–32 kHz) persisted in rats with healed PTPs. Morphological staining revealed that no increase in the thickness and obvious increase in collagen I level of regenerated par flaccida; regenerated pars tensa exhibited obvious increase in thickness and increased collagen I, while the collagen II regeneration was limited with discontinuous and disordered structure in regenerated pars tensa. Conclusion The hearing loss caused by large PFP limits at low frequencies while large PTP can lead to hearing loss at wide range frequencies. PFP and PTP have different functional outcomes after spontaneous healing, which is determined by the discrepant structure reconstruction and collagen regeneration.

The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.

Age-related hearing loss (AHL) is the most common sensory disorder amongst the elderly population. Although the degeneration of spiral ganglion neurons (SGNs) and hair cells (HCs) is considered to play a critical role in AHL, the mechanism has not been fully outlined. The repressor element 1-silencing transcription factor (REST) has recently been associated with mediating cell death in neurodegenerative diseases. However, whether REST induces degeneration of cochlear HCs and SGNs to contribute to AHL remains unknown. Here, we report that REST expression was decreased in HCs and SGNs in AHL mice. Conditional deletion of Rest in HCs and SGNs of 2-month-old mice resulted in hearing loss accompanied by the upregulation of p53, TNFR1(tumor necrosis factor receptor-1), and cleaved caspase-3. The p53 inhibitor pifithrin-α significantly attenuated SGN and HC damage and rescued hearing impairment in Rest cKO mice. Furthermore, downregulation of REST by H 2 O 2 treatment induced apoptosis in the House Ear Institute Organ of Corti 1 cell, through the upregulation of p53. In contrast, overexpression of REST reversed the changes in p53 expression. In addition, REST was further shown to bind directly to the p53 promoter site, thereby inhibiting the effect of p53. Finally, in aged mice, the p53 inhibitor significantly reduced loss of HCs and SGNs, and subsequently improved hearing. In summary, our findings indicate that REST has a protective role in AHL, and that its deficiency upregulates p53 and induces cochlear cell apoptosis, which that leads to deafness.

Background Endolymphatic hydrops (EH) is considered as the pathological correlate of Menière’s disease (MD) and cause of hearing loss. The mechanism of EH, remaining unrevealed, poses challenges for formalized clinical trials. Objective This study aims to investigate the development of hearing loss, as well as the effect of dehydration treatment on EH animal models. Methods In this study, different severity EH animal models were created. The laser Doppler vibrometer (LDV) and auditory brainstem responses (ABR) were used to study the effects of EH and the dehydration effects of mannitol. The LDV was used to measure the vibration of the round window membrane (RWM) reflecting the changes in inner ear impedance. ABR was used to evaluate the hearing changes. Furthermore, tissue section and scanning electron microscopy (SEM) observations were used to analyze the anatomical change to the cochlea and outer hair cells. Results The RWM vibrations decreased with the severity of EH, indicating an increase in the cochlear impedance. The dehydration therapy lowered the impedance to restore acoustic transduction in EH 10- and 20-day animal models. Simultaneously, the ABR thresholds increased in EH models and were restored after dehydration. Moreover, a difference in the hearing was found between ABR and LDV results in severe EH animal models, and the dehydration therapy was less effective, indicating a sensorineural hearing loss (SNHL). Conclusion Endolymphatic hydrops causes hearing loss by increasing the cochlear impedance in all tested groups, and mannitol dehydration is an effective therapy to restore hearing. However, SNHL occurs for the EH 30-day animal models, limiting the effectiveness of dehydration. Our results suggest the use of dehydrating agents in the early stage of EH.

The laryngeal echolocation is regarded as one of the conspicuous traits that play major roles in flourishing bats. Whether the laryngeal echolocation in bats originated once, however, is still controversial. We here address this question by performing molecular convergence analyses between ancestral branches of bats and toothed whales. Compared with controls, the molecular convergences were enriched in hearing-related genes for the last common ancestor of bats (LCAB) and extant echolocating bats, but not for the LCA of Old World fruit bats (LCAP). And the convergent hearing gene prestin of the LCAB and the extant echolocating bats functionally converged. More importantly, the high-frequency hearing of the LCAP-prestin knock-in mice decreased with lower cochlear outer hair cell function compared with the LCAB-prestin knock-in mice. Together, our findings provide multiple lines of evidence suggesting a single origin of laryngeal echolocation in the LCAB and the subsequent loss in the LCAP.

Voltage-gated Ca 2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α 2 δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α 2 δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α 2 δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α 2 δ-4 knockout (KO) mice. α 2 δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α 2 δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor coordination in the rotarod assay. α 2 δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α 2 δ-4 in sensorimotor gating and motor function and identify α 2 δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Age-related hearing loss (ARHL) is a major hearing impairment characterized by pathological changes in both the peripheral and central auditory systems. Low-grade inflammation was observed in the cochlea of deceased human subjects with ARHL and animal models of early onset ARHL, which suggests that inflammation contributes to the development of ARHL. However, it remains elusive how chronic inflammation progresses during normal aging in the cochlea, and especially the accompanying changes of neuroinflammation in the central auditory system. To address this, we investigated chronic inflammation in both the cochlea and the cochlear nucleus (CN) of CBA/CaJ mice, an inbred mouse strain that undergoes normal aging and develops human, like-late-onset ARHL. Using immunohistochemistry, confocal microscopy, and quantitative image processing, we measured the accumulation and activation of macrophages in the cochlea and microglia in the CN using their shared markers: ionized calcium binding adaptor molecule 1 (Iba1) and CD68—a marker of phagocytic activity. We found progressive increases in the area covered by Iba1-labeled macrophages and enhanced CD68 staining in the osseous spiral lamina of the cochlea that correlated with elevated ABR threshold across the lifespan. During the process, we further identified significant increases in microglial activation and C1q deposition in the CN, indicating increased neuroinflammation and complement activation in the central auditory system. Our study suggests that during normal aging, chronic inflammation occurs in both the peripheral and the central auditory system, which may contribute in coordination to the development of ARHL.

CRISPR/RfxCas13d (CasRx) editing system can specifically and precisely cleave single-strand RNAs, which is a promising treatment for various disorders by downregulation of related gene expression. Here, we tested this RNA-editing approach on Beethoven ( Bth ) mice, an animal model for human DFNA36 due to a point mutation in Tmc1 . We first screened 30 sgRNAs in cell cultures and found that CasRx with sgRNA3 reduced the Tmc1 Bth transcript by 90.8%, and the Tmc1 wild type transcript ( Tmc1 + ) by 44.3%. We then injected a newly developed AAV vector (AAV-PHP.eB) based CasRx into the inner ears of neonatal Bth mice, and we found that Tmc1 Bth was reduced by 70.2% in 2 weeks with few off-target effects in the whole transcriptome. Consistently, we found improved hair cell survival, rescued hair bundle degeneration, and reduced mechanoelectrical transduction current. Importantly, the hearing performance, measured in both ABR and DPOAE thresholds, was improved significantly in all ages over 8 weeks. We, therefore, have validated the CRISPR/CasRx-based RNA editing strategy in treating autosomal-dominant hearing loss, paving way for its further application in many other hereditary diseases in hearing and beyond.

The sound-evoked electrical compound potential known as auditory brainstem response (ABR) represents the firing of a heterogenous population of auditory neurons in response to sound stimuli, and is often used for clinical diagnosis based on wave amplitude and latency. However, recent ABR applications to detect human cochlear synaptopathy have led to inconsistent results, mainly due to the high variability of ABR wave-1 amplitude. Here, rather than focusing on the amplitude of ABR wave 1, we evaluated the use of ABR wave curvature to detect cochlear synaptic loss. We first compared four curvature quantification methods using simulated ABR waves, and identified that the cubic spline method using five data points produced the most accurate quantification. We next evaluated this quantification method with ABR data from an established mouse model with cochlear synaptopathy. The data clearly demonstrated that curvature measurement is more sensitive and consistent in identifying cochlear synaptic loss in mice compared to the amplitude and latency measurements. We further tested this curvature method in a different mouse model presenting with otitis media. The change in curvature profile due to middle ear infection in otitis media is different from the profile of mice with cochlear synaptopathy. Thus, our study suggests that curvature quantification can be used to address the current ABR variability issue, and may lead to additional applications in the clinic diagnosis of hearing disorders.

Intracellular accumulation of mutant proteins causes proteinopathies, which lack targeted therapies. Autosomal dominant hearing loss (DFNA67) is caused by frameshift mutations in OSBPL2. Here, we show that DFNA67 is a toxic proteinopathy. Mutant OSBPL2 accumulated intracellularly and bound to macroautophagy/autophagy proteins. Consequently, its accumulation led to defective endolysosomal homeostasis and impaired autophagy. Transgenic mice expressing mutant OSBPL2 exhibited hearing loss, but osbpl2 knockout mice or transgenic mice expressing wild-type OSBPL2 did not. Rapamycin decreased the accumulation of mutant OSBPL2 and partially rescued hearing loss in mice. Rapamycin also partially improved hearing loss and tinnitus in individuals with DFNA67. Our findings indicate that dysfunctional autophagy is caused by mutant proteins in DFNA67; hence, we recommend rapamycin for DFNA67 treatment. Abbreviations: ABR: auditory brainstem response; ACTB: actin beta; CTSD: cathepsin D; dB: decibel; DFNA67: deafness non-syndromic autosomal dominant 67; DPOAE: distortion product otoacoustic emission; fs: frameshift; GFP: green fluorescent protein; HsQ53R-TG: human p.Q53Rfs*100-transgenic: HEK 293: human embryonic kidney 293; HFD: high-fat diet; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSHL: non-syndromic hearing loss; OHC: outer hair cells; OSBPL2: oxysterol binding protein-like 2; SEM: scanning electron microscopy; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: transgenic; WES: whole-exome sequencing; YUHL: Yonsei University Hearing Loss; WT: wild-type.

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2 loxP/loxP ; ROSA26 CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker’s organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker’s organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca 2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function.

Mammalian hearing involves the mechanoelectrical transduction (MET) of sound-induced fluid waves in the cochlea. Essential to this process are the specialised sensory cochlear cells, the inner (IHCs) and outer hair cells (OHCs). While genetic hearing loss is highly heterogeneous, understanding the requirement of each gene will lead to a better understanding of the molecular basis of hearing and also to therapeutic opportunities for deafness. The Neuroplastin ( Nptn ) gene, which encodes two protein isoforms Np55 and Np65, is required for hearing, and homozygous loss-of-function mutations that affect both isoforms lead to profound deafness in mice. Here we have utilised several distinct mouse models to elaborate upon the spatial, temporal, and functional requirement of Nptn for hearing. While we demonstrate that both Np55 and Np65 are present in cochlear cells, characterisation of a Np65-specific mouse knockout shows normal hearing thresholds indicating that Np65 is functionally redundant for hearing. In contrast, we find that Nptn -knockout mice have significantly reduced maximal MET currents and MET channel open probabilities in mature OHCs, with both OHCs and IHCs also failing to develop fully mature basolateral currents. Furthermore, comparing the hearing thresholds and IHC synapse structure of Nptn- knockout mice with those of mice that lack Nptn only in IHCs and OHCs shows that the majority of the auditory deficit is explained by hair cell dysfunction, with abnormal afferent synapses contributing only a small proportion of the hearing loss. Finally, we show that continued expression of Neuroplastin in OHCs of adult mice is required for membrane localisation of Plasma Membrane Ca 2+ ATPase 2 (PMCA2), which is essential for hearing function. Moreover, Nptn haploinsufficiency phenocopies Atp2b2 (encodes PMCA2) mutations, with heterozygous Nptn -knockout mice exhibiting hearing loss through genetic interaction with the Cdh23 ahl allele. Together, our findings provide further insight to the functional requirement of Neuroplastin for mammalian hearing.

Voltage-gated Ca v Ca 2+ channels are critical for the development and mature function of the nervous system. Variants in the CACNA2D4 gene encoding the α 2 δ-4 auxiliary subunit of these channels are associated with neuropsychiatric and neurodevelopmental disorders. α 2 δ-4 is prominently expressed in the retina and is crucial for vision, but extra-retinal functions of α 2 δ-4 have not been investigated. Here, we sought to fill this gap by analyzing the behavioral phenotypes of α 2 δ-4 knockout (KO) mice. α 2 δ-4 KO mice (both males and females) exhibited significant impairments in prepulse inhibition that were unlikely to result from the modestly elevated auditory brainstem response thresholds. Whereas α 2 δ-4 KO mice of both sexes were hyperactive in various assays, only females showed impaired motor learning/coordination in the rotarod assay. Female but not male α 2 δ-4 KO mice exhibited anxiolytic and anti-depressive behaviors in the elevated plus maze and tail suspension tests, respectively. Our results reveal an unexpected role for α 2 δ-4 in cognitive and motor function and identify α 2 δ-4 KO mice as a novel model for studying the pathophysiology associated with CACNA2D4 variants.

Outer hair cell (OHC) degeneration is a major cause of progressive hearing loss and presbycusis. Despite the high prevalence of these disorders, targeted therapy is currently not available. Methods: We generated a mouse model harboring Kcnq4W276S/+ to recapitulate DFNA2, a common genetic form of progressive hearing loss accompanied by OHC degeneration. After comprehensive optimization of guide RNAs, Cas9s, vehicles, and delivery routes, we applied in vivo gene editing strategy to disrupt the dominant-negative allele in Kcnq4 and prevent progressive hearing loss. Results:In vivo gene editing using a dual adeno-associated virus package targeting OHCs significantly improved auditory thresholds in auditory brainstem response and distortion-product otoacoustic emission. In addition, we developed a new live-cell imaging technique using thallium ions to investigate the membrane potential of OHCs and successfully demonstrated that mutant allele disruption resulted in more hyperpolarized OHCs, indicating elevated KCNQ4 channel activity. Conclusion: These findings can facilitate the development of targeted therapies for DFNA2 and support the use of CRISPR-based gene therapy to rectify defects in OHCs.

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo . In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

Failure in the structural maintenance of the hair cell stereocilia bundle and ribbon synapse causes hearing loss. Here, we have studied how ER stress elicits hair cell pathology, using mouse models with inactivation of Manf (mesencephalic astrocyte-derived neurotrophic factor), encoding an ER-homeostasis-promoting protein. From hearing onset, Manf deficiency caused disarray of the outer hair cell stereocilia bundle and reduced cochlear sound amplification capability throughout the tonotopic axis. In high-frequency outer hair cells, the pathology ended in molecular changes in the stereocilia taper region and in strong stereocilia fusion. In high-frequency inner hair cells, Manf deficiency degraded ribbon synapses. The altered phenotype strongly depended on the mouse genetic background. Altogether, the failure in the ER homeostasis maintenance induced early-onset stereociliopathy and synaptopathy and accelerated the effect of genetic causes driving age-related hearing loss. Correspondingly, MANF mutation in a human patient induced severe sensorineural hearing loss from a young age onward. Thus, we present MANF as a novel protein and ER stress as a mechanism that regulate auditory hair cell maintenance in both mice and humans.

Mitochondrial oxidative stress is involved in hair cell damage caused by noise-induced hearing loss (NIHL). Sirtuin-3 (SIRT3) plays an important role in hair cell survival by regulating mitochondrial function; however, the role of SIRT3 in NIHL is unknown. In this study, we used 3-TYP to inhibit SIRT3 and found that this inhibition aggravated oxidative damage in the hair cells of mice with NIHL. Moreover, 3-TYP reduced the enzymatic activity and deacetylation levels of superoxide dismutase 2 (SOD2). Subsequently, we administered adeno-associated virus-SIRT3 to the posterior semicircular canals and found that SIRT3 overexpression significantly attenuated hair cell injury and that this protective effect of SIRT3 could be blocked by 2-methoxyestradiol, a SOD2 inhibitor. These findings suggest that insufficient SIRT3/SOD2 signaling leads to mitochondrial oxidative damage resulting in hair cell injury in NIHL. Thus, ameliorating noise-induced mitochondrial redox imbalance by intervening in the SIRT3/SOD2 signaling pathway may be a new therapeutic target for hair cell injury.