Novel Pathogenic Variants in PJVK, the Gene Encoding Pejvakin, in Subjects with Autosomal Recessive Non-Syndromic Hearing Impairment and Auditory Neuropathy Spectrum Disorder.

Pathogenic variants in the PJVK gene cause the DFNB59 type of autosomal recessive non-syndromic hearing impairment (AR-NSHI). Phenotypes are not homogeneous, as a few subjects show auditory neuropathy spectrum disorder (ANSD), while others show cochlear hearing loss. The numbers of reported cases and pathogenic variants are still small to establish accurate genotype-phenotype correlations. We investigated a cohort of 77 Spanish familial cases of AR-NSHI, in whom DFNB1 had been excluded, and a cohort of 84 simplex cases with isolated ANSD in whom OTOF variants had been excluded. All seven exons and exon-intron boundaries of the PJVK gene were sequenced. We report three novel DFNB59 cases, one from the AR-NSHI cohort and two from the ANSD cohort, with stable, severe to profound NSHI. Two of the subjects received unilateral cochlear implantation, with apparent good outcomes. Our study expands the spectrum of PJVK mutations, as we report four novel pathogenic variants: p.Leu224Arg, p.His294Ilefs*43, p.His294Asp and p.Phe317Serfs*20. We review the reported cases of DFNB59, summarize the clinical features of this rare subtype of AR-NSHI and discuss the involvement of PJVK in ANSD.

Auditory Neuropathy Spectrum Disorder (ANSD)-Clinical Characteristics and Pathogenic Variant Analysis of Three Nonsyndromic Deafness Families.

OBJECTIVE: To analyze the phenotypic features and pathogenic variants of three unrelated families presenting with nonsyndromic auditory neuropathy spectrum disorder (ANSD). METHODS: Three recruited families that were affected by congenital deafness were clinically evaluated, including a detailed family history and audiological and radiological examination. The peripheral blood of all patients and their parents was collected for DNA extraction, and then, the exonic and flanking regions were enriched and sequenced using targeted capture and high-throughput sequencing technology. Bioinformatics analyses and the Sanger sequencing were carried out to screen and validate candidate pathogenic variants. The pathogenicity of candidate variants was evaluated by an approach that was based on the standards and guidelines for interpreting genetic variants as proposed by the American College of Medical Genetics and Genomics (ACMG). RESULTS: Four patients in three families were diagnosed as nonsyndromic ANSD, and all exhibited OTOF gene mutations. Among them, two individuals in family 1 (i.e., fam 1-II-2 and fam 1-II-3) carried homozygous variants c.[2688del];[2688del] (NM_194248.3). Two individuals from family 2 (fam 2-II-1) and family 3 (fam 3-II-4) carried compound heterozygous variants c.[4960G>A];[1469C>G] and c.[2675A>G];[2977_2978del], respectively. CONCLUSIONS: Three unrelated pedigrees with ANSD were caused by pathogenic variants in the OTOF gene. Five mutations were found and included c.2688del, c.2675A>G, c.2977_2978del, c.4960G>A, and c.1469C>G, of which the first two are novel and expanded mutational spectrum of the OTOF gene, thus having important implications for genetic counseling of the family.

Case studies in neuroscience: subcortical origins of the frequency-following response.

The auditory frequency-following response (FFR) reflects synchronized and phase-locked activity along the auditory pathway in response to sound. Although FFRs were historically thought to reflect subcortical activity, recent evidence suggests an auditory cortex contribution as well. Here we present electrophysiological evidence for the FFR's origins from two cases: a patient with bilateral auditory cortex lesions and a patient with auditory neuropathy, a condition of subcortical origin. The patient with auditory cortex lesions had robust and replicable FFRs, but no cortical responses. In contrast, the patient with auditory neuropathy had no FFR despite robust and replicable cortical responses. This double dissociation shows that subcortical synchrony is necessary and sufficient to generate an FFR.NEW & NOTEWORTHY The frequency-following response (FFR) reflects synchronized and phase-locked neural activity in response to sound.  The authors present a dual case study, comparing FFRs and cortical potentials between a patient with auditory neuropathy (a condition of subcortical origin) and a patient with bilateral auditory cortex lesions. They show that subcortical synchrony is necessary and sufficient to generate an FFR.

The use of animal models to study cell transplantation in neuropathic hearing loss.

Auditory neuropathy (AN) is a form of sensorineural deafness specifically affecting the conduction of the nerve impulse from the cochlear hair cells to the auditory centres of the brain. As such, the condition is a potential clinical target for 'cell replacement therapy', in which a functioning auditory nerve is regenerated by transplanting an appropriated neural progenitor. In this review, we survey the current literature and examine possible experimental models for this condition, with particular reference to their compatibility as suitable hosts for transplantation. The use of exogenous neurotoxic agents such as ouabain or ß-bungarotoxin is discussed, as are ageing and noise-induced synaptopathy models. Lesioning of the nerve by mechanical damage during surgery and the neuropathy resulting from infectious diseases may be very relevant clinically, and we discuss whether there are good models for these situations. We also address genetic models for AN, examining whether the phenotypes truly model the clinical situation in their human counterpart syndromes - we use the example of the hyperbilirubinaemic Gunn rat as a particular instance in this regard.

Neuroanatomical and resting state EEG power correlates of central hearing loss in older adults.

To gain more insight into central hearing loss, we investigated the relationship between cortical thickness and surface area, speech-relevant resting state EEG power, and above-threshold auditory measures in older adults and younger controls. Twenty-three older adults and 13 younger controls were tested with an adaptive auditory test battery to measure not only traditional pure-tone thresholds, but also above individual thresholds of temporal and spectral processing. The participants' speech recognition in noise (SiN) was evaluated, and a T1-weighted MRI image obtained for each participant. We then determined the cortical thickness (CT) and mean cortical surface area (CSA) of auditory and higher speech-relevant regions of interest (ROIs) with FreeSurfer. Further, we obtained resting state EEG from all participants as well as data on the intrinsic theta and gamma power lateralization, the latter in accordance with predictions of the Asymmetric Sampling in Time hypothesis regarding speech processing (Poeppel, Speech Commun 41:245-255, 2003). Methodological steps involved the calculation of age-related differences in behavior, anatomy and EEG power lateralization, followed by multiple regressions with anatomical ROIs as predictors for auditory performance. We then determined anatomical regressors for theta and gamma lateralization, and further constructed all regressions to investigate age as a moderator variable. Behavioral results indicated that older adults performed worse in temporal and spectral auditory tasks, and in SiN, despite having normal peripheral hearing as signaled by the audiogram. These behavioral age-related distinctions were accompanied by lower CT in all ROIs, while CSA was not different between the two age groups. Age modulated the regressions specifically in right auditory areas, where a thicker cortex was associated with better auditory performance in older adults. Moreover, a thicker right supratemporal sulcus predicted more rightward theta lateralization, indicating the functional relevance of the right auditory areas in older adults. The question how age-related cortical thinning and intrinsic EEG architecture relates to central hearing loss has so far not been addressed. Here, we provide the first neuroanatomical and neurofunctional evidence that cortical thinning and lateralization of speech-relevant frequency band power relates to the extent of age-related central hearing loss in older adults. The results are discussed within the current frameworks of speech processing and aging.

FDXR Mutations Cause Sensorial Neuropathies and Expand the Spectrum of Mitochondrial Fe-S-Synthesis Diseases.

Hearing loss and visual impairment in childhood have mostly genetic origins, some of them being related to sensorial neuronal defects. Here, we report on eight subjects from four independent families affected by auditory neuropathy and optic atrophy. Whole-exome sequencing revealed biallelic mutations in FDXR in affected subjects of each family. FDXR encodes the mitochondrial ferredoxin reductase, the sole human ferredoxin reductase implicated in the biosynthesis of iron-sulfur clusters (ISCs) and in heme formation. ISC proteins are involved in enzymatic catalysis, gene expression, and DNA replication and repair. We observed deregulated iron homeostasis in FDXR mutant fibroblasts and indirect evidence of mitochondrial iron overload. Functional complementation in a yeast strain in which ARH1, the human FDXR ortholog, was deleted established the pathogenicity of these mutations. These data highlight the wide clinical heterogeneity of mitochondrial disorders related to ISC synthesis.

Novel compound heterozygous mutations in the OTOF Gene identified by whole-exome sequencing in auditory neuropathy spectrum disorder.

BACKGROUND: Many hearing-loss diseases are demonstrated to have Mendelian inheritance caused by mutations in single gene. However, many deaf individuals have diseases that remain genetically unexplained. Auditory neuropathy is a sensorineural deafness in which sounds are able to be transferred into the inner ear normally but the transmission of the signals from inner ear to auditory nerve and brain is injured, also known as auditory neuropathy spectrum disorder (ANSD). The pathogenic mutations of the genes responsible for the Chinese ANSD population remain poorly understood. METHODS: A total of 127 patients with non-syndromic hearing loss (NSHL) were enrolled in Guangxi Zhuang Autonomous Region. A hereditary deafness gene mutation screening was performed to identify the mutation sites in four deafness-related genes (GJB2, GJB3, 12S rRNA, and SLC26A4). In addition, whole-exome sequencing (WES) was applied to explore unappreciated mutation sites in the cases with the singularity of its phenotype. RESULTS: Well-characterized mutations were found in only 8.7% (11/127) of the patients. Interestingly, two mutations in the OTOF gene were identified in two affected siblings with ANSD from a Chinese family, including one nonsense mutation c.1273C > T (p.R425X) and one missense mutation c.4994 T > C (p.L1665P). Furthermore, we employed Sanger sequencing to confirm the mutations in each subject. Two compound heterozygous mutations in the OTOF gene were observed in the two affected siblings, whereas the two parents and unaffected sister were heterozygous carriers of c.1273C > T (father and sister) and c.4994 T > C (mother). The nonsense mutation p.R425X, contributes to a premature stop codon, may result in a truncated polypeptide, which strongly suggests its pathogenicity for ANSD. The missense mutation p.L1665P results in a single amino acid substitution in a highly conserved region. CONCLUSIONS: Two mutations in the OTOF gene in the Chinese deaf population were recognized for the first time. These findings not only extend the OTOF gene mutation spectrum for ANSD but also indicate that whole-exome sequencing is an effective approach to clarify the genetic characteristics in non-syndromic ANSD patients.

Photobiomodulation by laser therapy rescued auditory neuropathy induced by ouabain.

Auditory neuropathy is a hearing disorder caused by impaired auditory nerve function. The lack of information about the pathophysiology of this disease limits early diagnosis and further treatment. Laser therapy is a novel approach to enhance nerve growth or induce axonal regeneration. We induced auditory neural degeneration sparing the sensory epithelium with local ouabain application in an animal model and observed the rescue effect of photobiomodulation (PBM), showing recovered auditory function and favorable histologic outcome. Hearing was evaluated using the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). Seven days after ouabain application, the animals were sacrificed to evaluate the morphological changes. DPOAE change was not observed in all groups after ouabain application indicating no changes of outer hair cell function. Ouabain application increased the ABR thresholds increase, while the use of ouabain plus laser produced lower threshold compared to the ouabain group. Hematoxylin and Eosin staining of cochlea mid-modiolar sections in animals treated with ouabain showed damaged spiral ganglion cells, neurofilaments, and post synaptic puncta. Ouabain plus laser group showed higher number of spiral ganglion cells, higher density of neurofilaments, and higher number post synaptic puncta counts compared with ouabain application group. Short-term application of ouabain caused spiral ganglion cell damage while sparing the inner and outer hair cells in gerbils. Photobiomodulation alleviated the hearing loss caused by ouabain induced auditory neuropathy. The results indicate the possible role of photobiomodulation therapy for inner ear diseases accompanied by spiral ganglion degeneration.

Remodeling of the Inner Hair Cell Microtubule Meshwork in a Mouse Model of Auditory Neuropathy AUNA1.

Auditory neuropathy 1 (AUNA1) is a form of human deafness resulting from a point mutation in the 5' untranslated region of the Diaphanous homolog 3 (DIAPH3) gene. Notably, the DIAPH3 mutation leads to the overexpression of the DIAPH3 protein, a formin family member involved in cytoskeleton dynamics. Through study of diap3-overexpressing transgenic (Tg) mice, we examine in further detail the anatomical, functional, and molecular mechanisms underlying AUNA1. We identify diap3 as a component of the hair cells apical pole in wild-type mice. In the diap3-overexpressing Tg mice, which show a progressive threshold shift associated with a defect in inner hair cells (IHCs), the neurotransmitter release and potassium conductances are not affected. Strikingly, the overexpression of diap3 results in a selective and early-onset alteration of the IHC cuticular plate. Molecular dissection of the apical components revealed that the microtubule meshwork first undergoes aberrant targeting into the cuticular plate of Tg IHCs, followed by collapse of the stereociliary bundle, with eventual loss of the IHC capacity to transmit incoming auditory stimuli.

SMAD4 Defect Causes Auditory Neuropathy Via Specialized Disruption of Cochlear Ribbon Synapses in Mice.

More than 100 genes have been associated with deafness. However, SMAD4 is rarely considered a contributor to deafness in humans, except for its well-defined role in cell differentiation and regeneration. Here, we report that a SMAD4 defect in mice can cause auditory neuropathy, which was defined as a mysterious hearing and speech perception disorder in human for which the genetic background remains unclear. Our study showed that a SMAD4 defect induces failed formation of cochlear ribbon synapse during the earlier stage of auditory development in mice. Further investigation found that there are nearly normal morphology of outer hair cells (OHCs) and post-synapse spiral ganglion nerves (SGNs) in SMAD4 conditional knockout mice (cKO); however, a preserved distortion product of otoacoustic emission (DPOAE) and cochlear microphonic (CM) still can be evoked in cKO mice. Moreover, a partial restoration of hearing detected by electric auditory brainstem response (eABR) has been obtained in the cKO mice using electrode stimuli toward auditory nerves. Additionally, the ribbon synapses in retina are not affected by this SMAD4 defect. Thus, our findings suggest that this SMAD4 defect causes auditory neuropathy via specialized disruption of cochlear ribbon synapses.

Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve.

The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration.

Mutations in apoptosis-inducing factor cause X-linked recessive auditory neuropathy spectrum disorder.

BACKGROUND: Auditory neuropathy spectrum disorder (ANSD) is a form of hearing loss in which auditory signal transmission from the inner ear to the auditory nerve and brain stem is distorted, giving rise to speech perception difficulties beyond that expected for the observed degree of hearing loss. For many cases of ANSD, the underlying molecular pathology and the site of lesion remain unclear. The X-linked form of the condition, AUNX1, has been mapped to Xq23-q27.3, although the causative gene has yet to be identified. METHODS: We performed whole-exome sequencing on DNA samples from the AUNX1 family and another small phenotypically similar but unrelated ANSD family. RESULTS: We identified two missense mutations in AIFM1 in these families: c.1352G>A (p.R451Q) in the AUNX1 family and c.1030C>T (p.L344F) in the second ANSD family. Mutation screening in a large cohort of 3 additional unrelated families and 93 sporadic cases with ANSD identified 9 more missense mutations in AIFM1. Bioinformatics analysis and expression studies support this gene as being causative of ANSD. CONCLUSIONS: Variants in AIFM1 gene are a common cause of familial and sporadic ANSD and provide insight into the expanded spectrum of AIFM1-associated diseases. The finding of cochlear nerve hypoplasia in some patients was AIFM1-related ANSD implies that MRI may be of value in localising the site of lesion and suggests that cochlea implantation in these patients may have limited success.

Vestibular involvement in peripheral neuropathy: a review.

OBJECTIVE: This paper reviews the current literature on involvement of the vestibular division of the eighth cranial nerve in peripheral neuropathies. The literature abounds with references to auditory neuropathy, which is frequently related to more generalized neuropathies, but there is a marked paucity of work regarding vestibular neuropathy. A brief overview of neuropathies and the anatomy of the vestibulocochlear nerve provide the background for a review of the literature of vestibular nerve involvement in a range of neuropathies. DESIGN: A literature search including textbooks, and peer-reviewed published journal articles in online bibliographic databases was conducted. STUDY SAMPLE: Two databases for medical research were included in this review. RESULTS: The review of the literature indicates that vestibular involvement is a common and consistent finding in many peripheral neuropathies of different aetiologies. Specifically, if patients present unsteadiness/ataxia out of proportion to objective signs of somatosensory loss or muscle weakness. CONCLUSION: This review concludes that vestibular neuropathy, is common in peripheral neuropathy and should be identified to optimize patient management and rehabilitation.

Mutation of Foxo3 causes adult onset auditory neuropathy and alters cochlear synapse architecture in mice.

Auditory neuropathy is a form of hearing loss in which cochlear inner hair cells fail to correctly encode or transmit acoustic information to the brain. Few genes have been implicated in the adult-onset form of this disease. Here we show that mice lacking the transcription factor Foxo3 have adult onset hearing loss with the hallmark characteristics of auditory neuropathy, namely, elevated auditory thresholds combined with normal outer hair cell function. Using histological techniques, we demonstrate that Foxo3-dependent hearing loss is not due to a loss of cochlear hair cells or spiral ganglion neurons, both of which normally express Foxo3. Moreover, Foxo3-knock-out (KO) inner hair cells do not display reductions in numbers of synapses. Instead, we find that there are subtle structural changes in and surrounding inner hair cells. Confocal microscopy in conjunction with 3D modeling and quantitative analysis show that synaptic localization is altered in Foxo3-KO mice and Myo7a immunoreactivity is reduced. TEM demonstrates apparent afferent degeneration. Strikingly, acoustic stimulation promotes Foxo3 nuclear localization in vivo, implying a connection between cochlear activity and synaptic function maintenance. Together, these findings support a new role for the canonical damage response factor Foxo3 in contributing to the maintenance of auditory synaptic transmission.

Cortical deafness: a case report and review of the literature.

OBJECTIVE: To review the literature regarding cortical hearing loss and document a case of cortical hearing loss including its presentation, diagnosis, and evolution over 32 months of follow-up. PATIENT: A 56-year-old woman with profound bilateral sensorineural hearing loss secondary to sequential hemorrhagic, temporal lobe infarctions separated in time by 8 months. INTERVENTION: Diagnostic. RESULTS: Sequential infarctions affecting the patient's auditory radiations and primary auditory cortices bilaterally combined to cause cortical hearing loss. At presentation, audiogram revealed a bilateral profound sensorineural hearing loss with no reliable responses to pure-tone or speech audiometry. She has subsequently recovered the ability to distinguish environmental sounds. At her 32-month follow-up, she had a pure-tone average (PTA) of 62 dB on the right and 70 dB on the left but continued to display a poor word recognition score (0%). A literature review was performed from the year 1891 until the present. CONCLUSION: Cortical deafness is an exceedingly rare entity. Presentation and recovery of hearing are dependent on the extent of the initial lesions. The majority of patients can expect improvements in pure-tone auditory thresholds over time; however patients should be counseled that recovery of the ability to understand speech is unlikely.

High doses of cobalt induce optic and auditory neuropathy.

The adverse biological effects of continuous exposure to cobalt and chromium have been well defined. In the past, this toxicity was largely an industrial issue concerning workers exposed in occupational setting. Nevertheless, recent reports have described a specific toxicity mediated by the high levels of cobalt and chromium released by metallic prostheses, particularly in patients who had received hip implants. Clinical symptoms, including blindness, deafness and peripheral neuropathy, suggest a specific neurotropism. However, little is known about the neuropathological basis of this process, and experimental evidence is still lacking. We have investigated this issue in an experimental setting using New Zealand White rabbits treated with repeated intravenous injections of cobalt and chromium, alone or in combination. No evident clinical or pathological alterations were associated after chromium administration alone, despite its high levels in blood and tissue while cobalt-chromium and cobalt-treated rabbits showed clinical signs indicative of auditory and optic system toxicity. On histopathological examination, the animals showed severe retinal and cochlear ganglion cell depletion along with optic nerve damage and loss of sensory cochlear hair cells. Interestingly, the severity of the alterations was related to dosages and time of exposure. These data confirmed our previous observation of severe auditory and optic nerve toxicity in patients exposed to an abnormal release of cobalt and chromium from damaged hip prostheses. Moreover, we have identified the major element mediating neurotoxicity to be cobalt, although the molecular mechanisms mediating this toxicity still have to be defined.

Persistent cortical deafness: a voxel-based morphometry and tractography study.

OBJECTIVE: In cortical deafness, no auditory signals can be perceived by the cortex despite normal peripheral hearing. Cortical deafness rarely persists, but generally evolves into other cortical auditory syndromes. In this report, we describe a patient showing a stable and persisting pattern of cortical deafness 16 months after two major ischemic strokes. METHODS: Voxel based morphometric evidence from high resolution three-dimensional MRI and data from tractography are reported for the first time, to our knowledge, in this syndrome in addition to behavioral and electrophysiological findings. RESULTS: The most remarkable findings came from the tractography data, where an asymmetric pattern was found showing severe damage of connections within the anterior right hemisphere, in regions subserving self-awareness. Frontal asymmetry, although detectable by the morphometric analysis, was less informative than that detected in the tractography data. CONCLUSION: The evidence from this case study suggests that damage to the neural systems involved in awareness may play an important role in the emergence of cortical deafness and its persistence.

Bilirubin induces auditory neuropathy in neonatal guinea pigs via auditory nerve fiber damage.

Bilirubin can cause temporary or permanent sensorineural deafness in newborn babies with hyperbilirubinemia. However, the underlying targets and physiological effects of bilirubin-induced damage in the peripheral auditory system are unclear. Using cochlear functional assays and electron microscopy imaging of the inner ear in neonatal guinea pigs, we show here that bilirubin exposure resulted in threshold elevation in both compound action potential (CAP) and auditory brainstem response (ABR), which was apparent at 1 hr and peaked 8 hr after drug administration. The threshold elevation was associated with delayed wave latencies and elongated interwave intervals in ABR and CAP. At 72 hr postinjection, these measures returned to control levels, except for the CAP amplitude. Cochlear microphonics remained unchanged during the experiment. Morphological abnormalities were consistent with the electrophysiological dysfunction, revealing fewer auditory nerve fibers (ANFs) in the basal turn, myelin sheath lesions of spiral ganglion neurons (SGNs) and ANFs, and loss of type 1 afferent endings beneath inner hair cells (IHCs) without loss of hair cells at 8 hr posttreatment. Similar to the electrophysiological findings, morphological changes were mostly reversed 10 days after treatment, except for the ANF reduction in the basal turn. These results suggest that hyperbilirubinemia in neonatal guinea pigs impaired auditory peripheral neuromechanisms that targeted mainly the IHC synapses and the myelin sheath of SGNs and their fibers. Our observations indicate a potential connection between hyperbilirubinemia and auditory neuropathy.

Inhalation of hydrogen gas attenuates ouabain-induced auditory neuropathy in gerbils.

AIM: Auditory neuropathy (AN) is a hearing disorder characterized by abnormal auditory nerve function with preservation of normal cochlear hair cells. This study was designed to investigate whether treatment with molecular hydrogen (H(2)), which can remedy damage in various organs via reducing oxidative stress, inflammation and apoptosis, is beneficial to ouabain-induced AN in gerbils. METHODS: AN model was made by local application of ouabain (1 mmol/L, 20 mL) to the round window membrane in male Mongolian gerbils. H(2) treatment was given twice by exposing the animals to H(2) (1%, 2%, and 4%) for 60 min at 1 h and 6 h after ouabain application. Before and 7 d after ouabain application, the hearing status of the animals was evaluated using the auditory brainstem response (ABR) approach, the hear cell function was evaluated with distortion product otoacoustic emissions (DPOAE). Seven days after ouabain application, the changes in the cochleae, especially the spiral ganglion neurons (SGNs), were morphologically studied. TUNEL staining and immunofluorescent staining for activated caspase-3 were used to assess the apoptosis of SGNs. RESULTS: Treatment with H(2) (2% and 4%) markedly attenuated the click and tone burst-evoked ABR threshold shift at 4, 8, and 16 kHz in ouabain-exposed animals. Neither local ouabain application, nor H(2) treatment changed the amplitude of DPOAE at 4, 8, and 16 kHz. Morphological study showed that treatment with H(2) (2%) significantly alleviated SGN damage and attenuated the loss of SGN density for each turn of cochlea in ouabain-exposed animals. Furthermore, ouabain caused significantly higher numbers of apoptotic SGNs in the cochlea, which was significantly attenuated by the H(2) treatment. However, ouabain did not change the morphology of cochlear hair cells. CONCLUSION: The results demonstrate that H(2) treatment is beneficial to ouabain-induced AN via reducing apoptosis. Thus, H(2) might be a potential agent for treating hearing impairment in AN patients.