ABR/DPOAE publications

Stereocilia are rod-like mechanosensory projections consisting of unidirectionally oriented actin filaments that extend into the inner ear hair cell cytoskeleton, forming dense rootlets. Taperin (TPRN) localizes to the narrowed-down base of stereocilia, where they pivot in response to sound and gravity. We show that TPRN-deficient mice have progressive deafness characterized by gradual asynchronous retraction and fusion of outer and inner hair cell stereocilia, followed by synaptic abnormalities. Stereocilia that lack TPRN develop warped rootlets with gradual loss of TRIOBP-5 and ANKRD24 from mechanosensory rows starting postnatally. In contrast, TPRN overexpression causes excessive F-actin bundling, extra rows, and over-elongation of stereocilia during development. Purified full-length mouse TPRN cross-links F-actin into bendable bundles reflecting in vivo data. This F-actin–bundling ability is attributed to the TPRN N-terminal region. TPRN interacts with the membrane receptor PTPRQ, connecting the F-actin core to the plasma membrane, stabilizing stereocilia. Thus, TPRN is a specialized F-actin bundler strategically located to augment stereocilia rootlet formation and their pivot point flexibility for sustained sound-induced deflections.

Introduction Tinnitus is the perception of non-meaningful sound in the absence of external stimuli. Although tinnitus behavior in animal models is associated with altered central nervous system activity, it is not currently possible to identify tinnitus using neuronal activity alone. In the mouse inferior colliculus (IC), a subpopulation of neurons demonstrates a sustained increase in spontaneous activity after a long-duration sound (LDS). Methods Here, we use the “ LDS test ” to reveal tinnitus-specific differences in sound-evoked plasticity through IC extracellular recordings and the auditory brainstem response (ABR LDS ) in CBA/CaJ mice after sound exposure and behavioral tinnitus assessment. Results Sound-exposed mice showed stronger and shorter tone-evoked responses in the IC compared to unexposed controls, but these differences were not strong predictors of tinnitus. In contrast, in the LDS test, non-tinnitus mice had a significantly stronger suppression in tone-evoked spike rate compared to tinnitus and unexposed control mice. ABR peak amplitudes also revealed robust differences between tinnitus and non-tinnitus mice, with ABR peaks from non-tinnitus mice exhibiting significantly stronger suppression in the LDS test compared to tinnitus and control mice. No significant differences were seen between cohorts in ABR amplitude, latency, wave V:I ratio, wave V:III ratio, I-V intra-peak latency, and I-VI intra-peak latency. We found high-frequency tone stimuli better suited to reveal tinnitus-specific differences for both extracellular IC and ABR recordings. Discussion We successfully used the LDS test to demonstrate that tinnitus-specific differences in sound-evoked plasticity can be shown using both multi-unit near-field recordings in the IC and non-invasive far-field recordings, providing a foundation for future electrophysiological research into the causes and treatment of tinnitus.

Exposure to traumatic noise triggers cochlear damage and consequently causes permanent sensorineural hearing loss. However, effective treatment strategies for noise-induced hearing loss (NIHL) are lacking. Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that plays a critical role in multiple physiological and pathological events. However, its role in NIHL pathogenesis remains elusive. This study revealed that SIRT1 expression in the cochlea progressively decreases in a mouse model of NIHL. Hair cell-specific knockout of SIRT1 exacerbates the noise-induced loss of outer and inner hair cell synaptic ribbons, retraction of cochlear nerve terminals, and oxidative stress, leading to more severe NIHL. Conversely, adeno-associated virus (AAV)-mediated SIRT1 overexpression effectively attenuated most noise-induced cochlear damage and alleviated NIHL. Transcriptomic analysis revealed that SIRT1 deficiency impairs glucose metabolism and inhibits antioxidant pathways in the cochlea following exposure to noise. Further investigation revealed that SIRT1 exerts an antioxidant effect, at least in part, through AMPK activation in cultured auditory HEI-OC1 cells exposed to oxidative stress. Collectively, these findings indicate that SIRT1 is essential for the maintenance of redox balance and mitochondrial function in the cochlea after traumatic noise exposure, thus providing a promising therapeutic target for NIHL treatment.

Molecular profiling of inner ear neurons has broadened the classification of the primary afferents that support neural coding for hearing and balance. To extend spatiotemporal characterization of auditory and vestibular neuron diversity, we established a transgenic reporter mouse model (Prphp-mCherry), where elements of the peripherin promoter (Prphp) drive expression of the mCherry fluorescent reporter. Type III intermediate filament protein peripherin expression is a marker for type II spiral ganglion neurons (SGN) that innervate the cochlear outer hair cells, and the small diameter 'bouton' vestibular ganglion neurons (VGN) innervating the type II vestibular hair cells. Using Nanopore genome sequencing, the integration site of the transgene construct was identified within the class III metabotropic glutamate receptor 8 gene (Grm8, chromosome 6). Use of CUBIC / PEGASOS clearing of early postnatal to adult inner ear tissues enabled in situ 3D spatial localization of a dispersed population of cochlear mCherry + ve SGN, with highest expression and density in the hook (high frequency encoding) basal region. Of these mCherry + ve SGN, type II SGN (peripherin-immunopositive) were all co-labeled in the basal region, but the majority of the overall mCherry-delineated SGN auditory subpopulation were type I SGN innervating inner hair cells. In the VGN, mCherry + ve neurons represented ~ 15% of the adult population, dispersed as a small diameter subpopulation throughout both the inferior and superior VGN regions. These findings resolve heterogeneous type I and type II cochlear SGN subpopulations, particularly in the structurally complex hook region, and further differentiate vestibular primary afferents across postnatal development.

Human cytomegalovirus (HCMV) is the most common viral infection acquired in utero and a leading cause of neurodevelopmental abnormalities, including sensorineural hearing loss (SNHL). In previous studies using a murine model of HCMV induced SNHL, hearing loss was correlated with virus-induced cochlear inflammation but not cochlear viral load. However, these previous findings were determined at the time of auditory testing, a time poiont well past critical periods of auditory development. In the current study, cochlear virus load early in auditory development could be correlated with the magnitude of virus-induced cochlear inflammation, cochlear histopathology and the development of hearing loss. Transcriptional profiling at early times after infection revealed dysregulation of multiple well described deafness-related genes (DRG). Treatment with antiviral antibodies early after infection decreased cochlear virus load, cochlear inflammation, cochlear histopathology, and normalized DRG expression arguing that virus-induced cochlear inflammation can result in pleiotropic effects on the developing auditory system. Finally, this model also demonstrated that sterilizing immunity was unnecessary for prevention of SNHL, thus providing a rationale for inteventions that could limit, but not completely prevent HCMV infection of the developing auditory system.

The homozygous Cntnap2 knockout (KO) rat is a well-established genetic model for neurodevelopmental disorders, exhibiting core features of autism spectrum disorder (ASD), including impaired sensory processing and sensorimotor gating. Recent findings indicate that the severity of ASD-like phenotypes in Cntnap2 KO offspring is influenced by the parental genotype, with more pronounced impairments observed in KO rats bred from homozygous pairs compared to heterozygous pairs ( Cntnap2 HET). However, it is unclear to what extent this is due to in utero versus postnatal effects. We, therefore, investigated how early postnatal environmental factors, shaped by differences in parental and littermate genotypes, influence auditory processing and sensorimotor gating in Cntnap2 KO rats. To examine this, we cross-fostered Cntnap2 KO pups bred from Cntnap2 KO rats to be reared with litters of Cntnap2 HET dams. Cross-fostering Cntnap2 KO rats reversed or partially reversed delayed hearing sensitivity maturation, heightened acoustic startle responses, and deficits in prepulse inhibition of the acoustic startle response. However, cross-fostering also exacerbated deficits in the neural responsiveness and conductivity in the auditory brainstem, as well as in gap-induced prepulse inhibition of the acoustic startle response. These results emphasize the importance of considering the postnatal environment and breeding strategies in preclinical genetic models of neuropsychiatric disorders. More importantly, they also demonstrate that ASD-like traits, including alterations in brainstem sensory processing, are not strictly determined by genetic factors, but remain malleable by environmental factors during early postnatal development.

FKBP5 is a 51-Da FK506-binding protein and member of the immunophilin family involved in controlling the signaling of glucocorticoid receptor from the cytosol to nucleus. Fkbp5 has previously been shown to be expressed in murine cochlear tissue, including the organ of Corti (i.e., the sensory epithelium of the cochlea). Fkbp5-/- mice as used in this study show hearing loss in the low-frequency (8-kHz) range and click-evoked auditory brainstem response (ABR) threshold compared to wild-type mice. Both Fkbp5-/- and wild-type mice showed hearing loss at all frequencies and click-ABR thresholds at 24 h and 14 days following acoustic overexposure (AO). Tissues of the organ of Corti were subjected to RNA sequencing and KEGG pathway analysis. In Fkbp5-/- mice before AO, the mitogen-activated protein kinase (MAPK) signaling pathway was dysregulated compared to wild-type mice. In wild-type mice at 12 h following AO, the most significantly modulated KEGG pathway was the TNF signaling pathway and major MAPK molecules p38 and Jun were involved in the TNF signaling pathway. In Fkbp5-/- mice at 12 h following AO, the MAPK signaling pathway was dysregulated compared to wild-type mice following AO. In conclusion, Fkbp5 interacts with MAPK signaling in the organ of Corti in mice cochleae.

Objective: High altitude hypobaric hypoxia can induce hearing impairment and hearing acclimatization, but few studies have been performed to decipher the potential transition between the two states. To decipher transition-related circular RNAs (circRNAs)-microRNAs (miRNAs)-messenger RNA (mRNAs) regulatory network. Methods: Wistar rats were airlifted from plain to high altitude and maintained for 30 days and 60 days. Hearing acclimatization was determined using the Auditory Brainstem Response (ABR) test. Cochlea tissues were isolated, and high-throughput circRNA analysis and mRNAs-sequencing were performed. Differentially Expressed circRNAs (DEcircRNAs) and Differentially Expressed mRNAs (DEmRNAs) were obtained, and circRNA-miRNA and miRNA-mRNA regulation were predicted. A circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was also constructed. The DEmRNAs in this network were functionally annotated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses based on Metascape. Results: The ABR assay indicated that hearing impairment happened on day 30 and hearing acclimatization occurred on day 60. Hearing impairment-related circRNAs (64 upregulated and 147 downregulated) and genes (572 upregulated and 757 downregulated) were identified. Hearing acclimatization-related circRNAs (79 upregulated and 142 downregulated) and genes (690 upregulated and 751 downregulated) were also identified. Hearing impairment and hearing acclimatization ceRNA networks were also constructed after integrating the predicted miRNA regulation analyses. Anterograde trans-synaptic signaling (GO:0098916) and negative regulation of cellular response to growth factor stimulus (GO:0090288) were regulated by hearing impairment ceRNA networks, and embryonic organ development (GO:0048568) was regulated by hearing acclimatization ceRNA networks. Conclusion: Hearing impairment- and hearing acclimatization-associated circRNAs and ceRNA networks were identified, which contribute new knowledge to our understanding of acclimatization transition.

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response (UPR) and is important for ER function and protein homeostasis in metazoan cells. Patients carrying loss-of-function ATF6 disease alleles develop the cone dysfunction disorder achromatopsia. The effect of loss of ATF6 function on other cell types, organs, and diseases in people remains unclear. Here, we report that progressive sensorineural hearing loss was a notable complaint in some patients carrying ATF6 disease alleles and that Atf6 –/– mice also showed progressive auditory deficits affecting both sexes. In mice with hearing deficits, we found disorganized stereocilia on hair cells and focal loss of outer hair cells. Transcriptomics analysis of Atf6 –/– cochleae revealed a marked induction of the UPR, especially through the protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm. These findings identify ATF6 as an essential regulator of cochlear health and function. Furthermore, they support the idea that ATF6 inactivation in people causes progressive sensorineural hearing loss as part of a blindness-deafness genetic syndrome targeting hair cells and cone photoreceptors. Last, our genetic findings indicate that ER stress is an important pathomechanism underlying cochlear damage and hearing loss, with clinical implications for patient lifestyle modifications that minimize environmental and physiological sources of ER stress to the ear.

Assembly of actin-based stereocilia is critical for cochlear hair cells to detect sound. To tune their mechanosensivity, stereocilia form bundles composed of graded rows of ascending height, necessitating the precise control of actin polymerization. Myosin 15 (MYO15A) drives hair bundle development by delivering critical proteins to growing stereocilia that regulate actin polymerization via an unknown mechanism. Here, we show that MYO15A is itself an actin nucleation-promoting factor. Moreover, a deafness-causing mutation in the MYO15A actin-binding interface inhibits nucleation activity but still preserves some movement on filaments in vitro and partial trafficking on stereocilia in vivo. Stereocilia fail to elongate correctly in this mutant mouse, providing evidence that MYO15A-driven actin nucleation contributes to hair bundle biogenesis. Our work shows that in addition to generating force and motility, the ATPase domain of MYO15A can directly regulate actin polymerization and that disrupting this activity can promote cytoskeletal disease, such as hearing loss.

While soundscapes shape the structure and function of auditory systems over evolutionary timescales, there is limited information regarding the adaptation of wild fish populations to their natural acoustic environments. This is particularly relevant for freshwater ecosystems, which are extremely diverse and face escalating pressures from human activities and associated noise pollution. The Siamese fighting fish Betta splendens is one of the most important cultured species in the global ornamental fish market and is increasingly recognized as a model organism for genetics and behavioural studies. This air-breathing species (Anabantoidei), characterized by the presence of a suprabranchial labyrinth organ that enhances auditory sensitivity, is native to Southeast Asia and inhabits low flow freshwater ecosystems that are increasingly threatened due to habitat destruction and pollution. We characterized the underwater soundscape, along with various ecological parameters, across five marshland habitats of B. splendens , from lentic waterbodies to small canals near a lake in Chiang Rai province (Thailand). All habitats exhibited common traits of low dissolved oxygen and dense herbaceous vegetation. Soundscapes were relatively quiet with Sound Pressure Level (SPL) around 102–105 dB re 1 µPa and most spectral energy below 1,000 Hz. Sound recordings captured diverse biological sounds, including potential fish vocalizations, but primarily insect sounds. Hearing thresholds were determined using auditory evoked potential (AEP) recordings, revealing best hearing range within 100–400 Hz. Males exhibited lower hearing thresholds than females at 400 and 600 Hz. This low-frequency tuning highlights the potential susceptibility of B. splendens to anthropogenic noise activities. This study provides first characterization of the auditory sensitivity and natural soundscape of B. splendens, establishing an important ground for future hearing research in this species. The information provided on the auditory sensory adaptation of B. splendens emphasizes the importance of preserving quiet soundscapes from lentic freshwater ecosystems.

Noise-induced hearing loss is one of the most common forms of hearing loss in adults and also one of the most common occupational diseases. Extensive previous work has shown that the highly sensitive synapses of the inner hair cells (IHCs) may be the first target for irreparable damage and permanent loss in the noise-exposed cochlea, more precisely in the cochlear base. However, how such synaptic loss affects the synaptic physiology of the IHCs in this particularly vulnerable part of the cochlea has not yet been investigated. To address this question, we exposed 3–4-week-old C57BL/6J mice to 8–16 kHz noise for 2 h under isoflurane anesthesia. We then employed hearing measurements, immunohistochemistry and patch-clamp to assess IHC synaptic function. Two noise sound pressure levels (SPLs) were used to evoke acute hearing threshold elevations with different levels of recovery 2 weeks post-exposure. Regardless of noise intensity, the exposure resulted in a loss of approximately 25–36% of ribbon synapses in the basal portions of the cochlea that persisted 2 weeks after exposure. Perforated patch-clamp recordings were made in the IHCs of the basal regions of the cochlea where the greatest synaptic losses were observed. Depolarization-evoked calcium currents in IHCs 2 weeks after exposure were slightly but not significantly smaller as compared to controls from age-matched non-exposed animals. Exocytic changes monitored as changes in membrane capacitance did not follow that trend and remained similar to controls despite significant loss of ribbons, likely reflecting increased exocytosis at the remaining synapses. Additionally, we report for the first time that acute application of isoflurane reduces IHC calcium currents, which may have implications for noise-induced IHC synaptic loss.

To encode continuous sound stimuli, the inner hair cell (IHC) ribbon synapses utilize calcium-binding proteins (CaBPs), which reduce the inactivation of their Ca V 1.3 calcium channels. Mutations in the CABP2 gene underlie non-syndromic autosomal recessive hearing loss DFNB93. Besides CaBP2, the structurally related CaBP1 is highly abundant in the IHCs. Here, we investigated how the two CaBPs cooperatively regulate IHC synaptic function. In Cabp1/2 double-knockout mice, we find strongly enhanced Ca V 1.3 inactivation, slowed recovery from inactivation and impaired sustained exocytosis. Already mild IHC activation further reduces the availability of channels to trigger synaptic transmission and may effectively silence synapses. Spontaneous and sound-evoked responses of spiral ganglion neurons in vivo are strikingly reduced and strongly depend on stimulation rates. Transgenic expression of CaBP2 leads to substantial recovery of IHC synaptic function and hearing sensitivity. We conclude that CaBP1 and 2 act together to suppress voltage- and calcium-dependent inactivation of IHC Ca V 1.3 channels in order to support sufficient rate of exocytosis and enable fast, temporally precise and indefatigable sound encoding.

Current tests of hearing fail to diagnose pathologies in ~10% of patients seeking help for hearing difficulties. Neural ensemble responses to perceptually relevant cues in the amplitude envelope, termed envelope following responses (EFR), hold promise as an objective diagnostic tool to probe these 'hidden' hearing difficulties. But clinical translation is impeded by current measurement approaches involving static amplitude modulated (AM) tones, which are time-consuming and lack optimal spectrotemporal resolution. Here we develop a framework to rapidly measure EFRs using dynamically varying AMs combined with spectrally specific analyses. These analyses offer 5x improvement in time and 30x improvement in spectrotemporal resolution, and more generally, are optimal for analyzing time-varying signals with known spectral trajectories of interest. We validate this approach across several mammalian species, including humans, and demonstrate robust responses that are highly correlated with traditional static EFRs. Our analytic technique facilitates rapid and objective neural assessment of temporal processing throughout the brain that can be applied to track auditory neurodegeneration using EFRs, as well as tracking recovery after therapeutic interventions.

The goal of the present study was to determine, through a detailed study of the auditory brainstem response (ABR) waves, the possible existence of an early functional marker for the onset of presbycusis in an animal model. Toward this goal, Wistar rats were divided into four age groups: 3-month-old (3M, n = 6, control), 9-month-old (9M, n = 6), 14-month-old (14M, n = 6), and 20-month-old (20M, n = 6). ABR recordings were performed at 0.5, 1, 2, 4, 8, 16, and 32 kHz. The novel result reported here is that wave amplitudes, particularly wave II, were significantly diminished in the 9M group, even though there was no evidence of significant age-related threshold shift at that age. A significant increase in auditory thresholds with age was first detected at 14M, which further progressed at 20M, confirming our previous findings. These findings suggest that measurable alterations in ABR waves may precede age-related threshold shift and could serve as early markers to detect the onset of age-related hearing loss. Upon translation to humans, they could be used to implement early objective diagnosis, crucial to prevent or mitigate the negative consequences of presbycusis, a common, progressive, and irreversible neurodegenerative age-related disorder. This may allow, for instance, a better preservation of residual hearing, thus delaying the progression of the disease and minimizing the impact of hearing loss, ultimately improving the quality of life for those who suffer from this neurodegenerative condition.

Neural circuits in the auditory brainstem compute interaural time and intensity differences used to determine the locations of sound sources. These circuits display features that are specialized for these functions. The projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid (MNTB) body travels along highly myelinated fibers and terminates in the calyx of Held. This monoinnervating synapse emerges during development as multiple inputs are eliminated. We previously demonstrated that elimination of microglia with a colony stimulating factor-1 inhibitor results in impaired synaptic pruning so that multiple calyceal terminals reside on principal cells of MNTB. This inhibitor also resulted in impaired auditory brainstem responses (ABRs), with elevated thresholds and increased peak latencies. Loss of the microglial fractalkine receptor, CX3CR1, decreased peak latencies in the ABR. The mechanisms underlying these effects are not known. One prominent microglial signaling pathway involved in synaptic pruning and plasticity during development and aging is the C1q-initiated compliment cascade. Here we investigated the classical complement pathway initiator, C1q, in auditory brainstem maturation. We found that C1q expression is detected in the MNTB by the first postnatal week. C1q levels increased with age and were detected within microglia and surrounding the soma of MNTB principal neurons. Loss of C1q did not affect microglia-dependent calyceal pruning. Excitatory and inhibitory synaptic markers in the MNTB and LSO were not altered with C1q deletion. ABRs showed that C1q KO mice had normal hearing thresholds but shortened peak latencies. Altogether this study uncovers the developmental time frame of C1q expression in the sound localization pathway and shows a subtle functional consequence of C1q knockdown.

Background: Preserving residual hearing after cochlear implant (CI) surgery remains a crucial challenge. The application of dexamethasone (DEX) has been proven to positively affect residual hearing. To deliver DEX in a localized and controlled way, a round window niche implant (RNI), allowing drug diffusion via the round window membrane into the cochlea, may be used. To prove this concept, an RNI for guinea pigs as a CI-trauma model was manufactured by molding and tested for its drug release in vitro and biological effects in vivo. Methods: The RNIs were molded using silicone containing 10% DEX. Release was analyzed over time using high-performance liquid chromatography (HPLC). Fourteen adult guinea pigs were randomly assigned to two groups (CI or CI + RNI group). All animals received a unilateral CI electrode insertion trauma followed by CI insertion. The CI + RNI group was additionally implanted with an RNI containing 10% DEX. Animals were followed up for 4 weeks. Acoustically evoked auditory brainstem response and impedance measurement, micro-computed tomography (µCT) imaging, and histology were performed for evaluation. Results: DEX was released for more than 250 days in vitro, with an initial burst followed by a slower release over time. Comparing the hearing threshold shift (from day 0 to day 28) of the CI and CI + RNI groups, significant differences were observed at 32 and 40 kHz. The impedance shift at basal contacts was lower in the CI + RNI group than in the CI group. Moreover, the fibrosis in the lower basal turn was reduced in the CI + RNI group in contrast to the CI group. Conclusions: The RNI containing 10% DEX has anti-inflammatory potential concerning fibrosis inhibition and has beneficial effects on hearing preservation at high frequencies.

Background Prepulse inhibition (PPI) is a phenomenon where a weak prepulse stimulus inhibits the startle reflex to a subsequent stronger stimulus, which can be induced by various sensory stimulus modalities such as visual, tactile, and auditory stimuli. Methods This study investigates the neural mechanisms underlying auditory PPI by focusing on the deep layers of the superior colliculus (deepSC) and the inferior colliculus (IC) in rats. Nineteen male Sprague-Dawley rats were implanted with electrodes in the left deepSC and the right IC, and electrophysiological recordings were conducted under anesthesia to observe the frequency following responses (FFRs) to startle stimuli with and without prepulse stimuli. Results Our results showed that in the deepSC, narrowband noise as a prepulse stimulus significantly inhibited the envelope component of the startle response, while the fine structure component remained unaffected. However, this inhibitory effect was not observed in the IC or when the prepulse stimulus was a gap. Conclusion These findings suggest that the deepSC plays a crucial role in the neural circuitry of PPI, particularly in the modulation of the envelope component of the startle response. The differential effects of narrowband noise and gap as prepulse stimuli also indicate distinct neural pathways for sound-induced PPI and Gap-PPI. Understanding these mechanisms could provide insights into sensory processing and potential therapeutic targets for disorders involving impaired PPI, such as tinnitus.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder in which changes in hearing sensitivity precede cognitive decline. Despite a well-known link between dementia and hearing loss, few AD model mouse lines have hearing characterized. We screened for hearing loss using auditory brainstem responses (ABR) in young (3–4 months) and aging (9–10 months) mice with a P301S tauopathy (PS19 mice). Compared to wild types, aging PS19 mice did not show accelerated hearing loss but did show latency differences in centrally generated ABR waveform components. These results suggest that tauopathy causes mild central auditory dysfunction in the absence of overt hearing loss.

Cisplatin is an election chemotherapeutic agent used for many cancer treatments. Its cytotoxicity against neoplastic cells is mirrored by that taking place in healthy cells and tissues, resulting in serious adverse events. A very frequent one is ototoxicity, causing hearing loss which may permanently affect quality of life after successful oncologic treatments. Exacerbated oxidative stress is a main cytotoxic mechanism of cisplatin, including ototoxicity. Previous reports have shown antioxidant protection against cisplatin ototoxicity, but there is a lack of comparative studies on the otoprotectant activity and mechanism of antioxidant formulations. Here, we show evidence that a cocktail of vitamins A, C, and E along with Mg++ (ACEMg), previously shown to protect against noise-induced hearing loss, reverses auditory threshold shifts, promotes outer hair cell survival, and attenuates oxidative stress in the cochlea after cisplatin treatment, thus protecting against extreme cisplatin ototoxicity in rats. The addition of 500 mg N-acetylcysteine (NAC), which, administered individually, also shows significant attenuation of cisplatin ototoxicity, to the ACEMg formulation results in functional degradation of ACEMg otoprotection. Mg++ administered alone, as MgSO4, also prevents cisplatin ototoxicity, but in combination with 500 mg NAC, otoprotection is also greatly degraded. Increasing the dose of NAC to 1000 mg also results in dramatic loss of otoprotection activity compared with 500 mg NAC. These findings support that single antioxidants or antioxidant combinations, particularly ACEMg in this experimental series, have significant otoprotection efficacy against cisplatin ototoxicity. However, an excess of combined antioxidants and/or elevated doses, above a yet-to-be-defined “antioxidation threshold”, results in unrecoverable redox imbalance with loss of otoprotectant activity.