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The cochlea, the sensory organ for hearing, has a protected immune environment, segregated from the systemic immune system by the blood-labyrinth barrier. Previous studies have revealed that acute acoustic injury causes the infiltration of circulating leukocytes into the cochlea. However, the molecular mechanisms controlling immune cell trafficking are poorly understood. Here, we report the role of CX3CR1 in regulating the entry of neutrophils into the cochlea after acoustic trauma. We employed B6.129P-Cx3cr1 tm1Litt /J mice, a transgenic strain that lacks the gene, Cx3cr1, for coding the fractalkine receptor. Our results demonstrate that lack of Cx3cr1 results in the augmentation of neutrophil infiltration into cochlear tissues after exposure to an intense noise of 120 dB SPL for 1 hr. Neutrophil distribution in the cochlea is site specific, and the infiltration level is positively associated with noise intensity. Moreover, neutrophils are short lived and macrophage phagocytosis plays a role in neutrophil clearance, consistent with typical neutrophil dynamics in inflamed non-cochlear tissues. Importantly, our study reveals the potentiation of noise-induced hearing loss and sensory cell loss in Cx3cr1 -/- mice. In wild-type control mice (Cx3cr1 +/+ ) exposed to the same noise, we also found neutrophils. However, neutrophils were present primarily inside the microvessels of the cochlea, with only a few in the cochlear tissues. Collectively, our data implicate CX3CR1-mediated signaling in controlling neutrophil migration from the circulation into cochlear tissues and provide a better understanding of the impacts of neutrophils on cochlear responses to acoustic injury., (© 2021 Wiley Periodicals LLC.)

Background: High doses of salicylate are known to reduce cochlear response amplitude and raise threshold. However, its effect on the cochlear forward masking, reflecting temporal resolution, is still unclear., Methods: The neural forward masking of cochlea was evaluated using double-tone stimulation. The first tone burst (5ms) was named the "masker" and the second tone burst (5 ms) was named the "probe". The frequency and intensity of the masker and probe were equal, and the masker-probe interval varied from 2 to 32 ms. The reduction (%) of the probe-evoked cochlear compound action potential caused by the addition of the masker tone was used to represent cochlear forward masking. The data obtained before and 2 h following the injection of sodium salicylate (250 mg/kg) were compared., Results: The neural forward masking of cochlea in the normal rats increased as the masker-probe interval decreased. At 16 kHz, for example, it increased from ~5% to 32ms masker-probe interval to ~85% at 2ms masker-probe interval. Two hours post salicylate injection, the neural forward masking was significantly enhanced except at 32 ms masker-probe interval. Interestingly, this enhancement was only observed in the limited frequency range of 16-30 kHz., Discussion: The enhancement of forward masking of cochlea following salicylate administration may reflect defective neurotransmitter release. This frequency-dependent injury in the cochlea may lead to the development of central plasticity observed after salicylate administration, likely through the increase in central gain, leading to perceptual consequences., (Copyright © 2021. Published by Elsevier B.V.)

The effect of intense noise on cochlear sensitivity has been extensively studied, but its influence on the temporal characteristics of the cochlear response is still unclear. This study investigated the effects of noise exposure on the latency of cochlear response and cochlear forward masking. Rats were exposed to an octave band noise (8-16 kHz) at 90 dB SPL for 5 days. Cochlear compound action potentials (CAPs) induced by single- and double-tone stimuli and distortion product otoacoustic emissions (DPOAE) were recorded 1 day or 2 months after the noise exposure. The latency of the CAP and its forward masking were compared between the noise-exposed rats and normal control rats. The noise exposure significantly reduced DPOAE and elevated CAP threshold in the noise band region, but not in the other areas. Even in the noise band area, the noise did not reduce CAP-amplitude at the high stimulation level (80 dB SPL). Correspondingly, about one-third of the outer hair cells (OHC) in the noise band area disappeared, while the inner hair cells (IHC) did not. However, the noise exposure in the frequency range of 4-24 kHz significantly prolonged CAP latency and increased its variability, while the CAP forward masking effect was significantly enhanced in the frequency range of 16-30 kHz. The frequency-dependent changes in CAP latency and forward masking after noise exposure may reflect different types of synaptic subinjury in the cochlea, which may lead to psychophysical consequences of sound localization and speech recognition., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests or conflicts of interest., (Copyright © 2021. Published by Elsevier B.V.)

Auditory brain stem response (ABR) and compound action potential (CAP) recordings have been used in animal research to determine hearing sensitivity. Because of the relative ease of testing, the ABR test has been more commonly used in assessing cochlear lesions than the CAP test. The purpose of this experiment is to examine the difference between these two methods in monitoring the dynamic changes in auditory function after cochlear damage and in detecting asymmetric hearing loss due to unilateral cochlear damage. ABR and CAP were measured in two models of cochlear damage: acoustic trauma induced by exposure to a narrowband noise centered at 4 kHz (2,800-5,600 Hz) at 105 dB sound pressure level for 5 h in chinchillas and unilateral cochlear damage induced by surgical destruction of one cochlea in guinea pigs. Cochlear hair cells were quantified after completing the evoked potential testing. In the noise-damaged model, we found different recovery patterns between ABR and CAP. At 1 day after noise exposure, the ABR and CAP assessment revealed a similar level of threshold shifts. However, at 30 days after noise exposure, ABR thresholds displayed an average of 20-dB recovery, whereas CAP thresholds showed no recovery. Notably, the CAP threshold signifies the actual condition of sensory cell pathogenesis in the cochlea because sensory cell death is known to be irreversible in mammals. After unilateral cochlear damage, we found that both CAP and ABR were affected by cross-hearing when testing the damaged ear with the testing stimuli delivered directly into the canal of the damaged ear. When cross-hearing occurred, ABR testing was not able to reveal the presence of cross-hearing because the ABR waveform generated by cross-stimulation was indistinguishable from that generated by the test ear (damaged ear), should the test ear be intact. However, CAP testing can provide a warning sign, since the typical CAP waveform became an ABR-like waveform when cross-hearing occurred. Our study demonstrates two advantages of the CAP test over the ABR test in assessing cochlear lesions: contributing evidence for the occurrence of cross-hearing when subjects have asymmetric hearing loss and providing a better assessment of the progression of cochlear pathogenesis. NEW & NOTEWORTHY Auditory brain stem response (ABR) is more commonly used to evaluate cochlear lesions than cochlear compound action potential (CAP). In a noise-induced cochlear damage model, we found that the reduced CAP and enhanced ABR caused the threshold difference. In a unilateral cochlear destruction model, a shadow curve of the ABR from the contralateral healthy ear masked the hearing loss in the destroyed ear.

In industrial and military settings, individuals who suffer from one episode of acoustic trauma are likely to sustain another episode of acoustic stress, creating an opportunity for a potential interaction between the two stress conditions. We previously demonstrated that acoustic overstimulation perturbs the cochlear immune environment. However, how the cochlear immune system responds to repeated acoustic overstimulation is unknown. Here, we used a mouse model to investigate the cochlear immune response to repeated stress. We reveal that exposure to an intense noise at 120 dB SPL for 1 h activates the cochlear immune response in a time-dependent fashion with substantial expansion and activation of the macrophage population in the cochlea at 2-days post-exposure. At 20-days post-exposure, the number and pro-inflammatory phenotypes of cochlear macrophages have significantly subsided, but have yet to return to homeostatic levels. Monocytes with anti-inflammatory phenotypes are recruited into the cochlea. With the presence of this residual immune activation, a second exposure to the same noise provokes an exaggerated inflammatory response as evidenced by exacerbated maturation of macrophages. Furthermore, the second noise causes greater sensory cell pathogenesis. Unlike the first noise-induced damage that occurs mainly between 0 and 2 days post-exposure, the second noise-induced damage occurs more frequently between 2 and 20 days post-exposure, the period when secondary damage takes place. These observations suggest that repeated acoustic overstimulation exacerbates cochlear inflammation and secondary sensory cell pathogenesis. Together, our results suggest that the cochlear immune system plays an important role in modulating cochlear responses to repeated acoustic stress., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prolonged exposure to low-level noise has often been used scientifically as well as clinically to induce neuroplastic changes within the central auditory pathway in order to reduce central gain, suppress tinnitus and hyperacusis, and modulate different features of central auditory processing. A fundamental assumption underling these studies is that the noise exposure levels are so low that they have no effect on the neural output of the cochlea. Therefore, functional changes occurring in the central auditory pathway must be the results of central rather than peripheral changes. In an attempt to identify long-term noise exposures that did not cause peripheral changes, we measured the compound action potential (CAP) input/output functions from control rats and rats exposed for 6-weeks to 18-24 kHz noise presented at 25, 45, 55, 65, 75 or 85 dB SPL. Exposures >65 dB SPL significantly increased CAP thresholds; the critical intensity (Ct) below which no threshold shift occurred was estimated to be 55 dB SPL. Exposures >55 dB SPL significantly reduced suprathreshold CAP amplitudes; the critical intensity (Ca) below which no amplitude change was predicted to occur was a remarkably low level of 19 dB SPL. These results demonstrate that even extremely low-intensity long duration exposures can disrupt the neural output of the cochlea; these peripheral modifications are likely to contribute to the extensive compensatory changes observed at multiple levels of the central auditory pathway, neural network changes aimed at re-establishing homeostasis., Competing Interests: Declaration of competing interest The authors declare no competing financial interests or conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To measure the cochlear compound action potential (CAP) and the densities of hair cells (HCs) along the whole length of the basilar membrane (BM) in adult chinchillas. And to investigate the relationship between the severity of inner hair cells (IHCs) loss and the changes of CAP by using carboplatin-cochlear lesion model. Methods: Totally 18 chinchillas were recruited after ontological evaluation. They were randomly divided into three groups (with 6 subjects in each), A: control, B and C: legion groups treated with one or two shot(s) of carboplatin respectively (76 mg/kg in one shot, i.p., one-week interval between the two shots). Endpoint tests were performed 30 days after the carboplatin treatment in groups B and C, and matched time in group A. A sliver-ball electrode was placed into round window niche via hypotympanic approach in anesthetized chinchilla. CAP was measured in response to clicks and tone burst of 0.5, 1, 2, 4, 8, 16 kHz respectively under anesthesia. CAP amplitudes and thresholds were measured and compared across the groups. After the recording, the whole cochlea surface preparation was made and the HCs were stained in histochemistry against substrate of succinate dehydrogenase (SDH). Images were taken with high-resolution digital camera under light microscope and across the whole cochlea. The length of the basilar membrane (BM) and the number of both IHCs and OHCs were counted. The HC density was calculated as the number of HCs per 10% BM length. Results: The CAP thresholds were (7.1±2.6), (25.4±5.0), (24.6±5.4), (10.4±5.0), (0.4±1.4), (4.2±6.3) and (17.1±14.1) dB SPL (from 6 subjects in group A, n= 12 ears) corresponding to stimuli of Click and 0.5, 1, 2, 4, 8, 16 kHz tone bursts respectively. The total number of cochlear HCs were measured as (8 936±643) ( x ± s ) and the average length of the BMs was (17.73±1.012) mm from the six subjects in the group A ( n= 12 ears). The HC density was found to be varied slightly across the BM. There was no significant CAP threshold difference between the control (group A) and the group B, which received one shot of carboplatin. However, the maximal CAP amplitude was reduced by 40% in the group B and compared with group A. Correspondingly, approximately 40% loss of IHCs were seen. In contrast, a significant CAP threshold shift was seen in subjects receiving two shots of carboplatin (group C), which was accompanied by a loss of 90% IHCs. Conclusions: The CAP thresholds of adult chinchillas show typical open-V shape with the lowest values at 2, 4, and 8 kHz. IHC loss by carboplatin in certain degree is well correlated with CAP amplitude reduction, but does not change the threshold when inner hair cell loss reaches 40%, however, if inner hair cell loss exceeds 80%, the threshold shift of CAP will be inevitable.

The glutathione transferase (GST) detoxification system converts exogenous and endogenous toxins into a less toxic form by conjugating the toxic compound to reduced glutathione (GSH) by a variety of GST enzymes. Of the ~20 GST isoforms, GSTA4 exhibits high catalytic efficiency toward 4-hydroxynonenal (4-HNE), one of the most abundant end products of lipid peroxidation that contributes to neurodegenerative diseases and age-related disorders. Conjugation to GSH by GSTA4 is thought to be a major route of 4-HNE elimination. In the current study, we investigated the effects of Gsta4 deficiency on age-related cochlear pathology and hearing loss using young (3-5 months old) and old (24-25 months old) Gsta4 +/+ and Gsta4 -/- mice that were backcrossed onto the CBA/CaJ mouse strain, a well-established model of age-related hearing loss (AHL). At 3-5 months of age, loss of Gsta4 resulted in decreased total GSTA activity toward 4-HNE in the inner ears of young mice. However, there were no differences in the levels of 4-HNE in the inner ears between Gsta4 +/+ and Gsta4 -/- mice at 3-5 or 24-25 months of age. No histological abnormalities were observed in the cochlea and no hearing impairments were observed in young Gsta4 -/- mice. At 24-25 months of age, both Gsta4 +/+ and Gsta4 -/- mice showed elevated ABR thresholds compared to 3-month-old mice, but there were no differences in ABR thresholds, cochlear spiral ganglion neuron densities, or stria vascularis thickness between Gsta4 +/+ and Gsta4 -/- mice. Together, these results suggest that under normal physiological conditions or during normal aging, GSTA4 is not essential for removal of 4-HNE in mouse inner ears., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A wide variety of ototoxic drugs are capable of damaging the sensory hair cells in the mammalian cochlea resulting in permanent hearing loss. However, the toxic properties of these drugs suggest that some could potentially damage cochlear support cells as well. To test the hypothesis, we treated postnatal day three rat cochlear cultures with toxic doses of gentamicin, cisplatin, mefloquine, and cadmium. Gentamicin primarily destroyed the hair cells and disrupted the intercellular connection with the surrounding support cells. Gentamicin-induced hair cell death was initiated through the caspase-9 intrinsic apoptotic pathway followed by activation of downstream executioner caspase-3. In contrast, cisplatin, mefloquine, and cadmium initially damaged the support cells and only later damaged the hair cells. Support cell death was initiated through the caspase-8 extrinsic apoptotic pathway followed later by downstream activation of caspase-3. Cisplatin, mefloquine, and cadmium significantly reduced the expression of actin and laminin, in the extracellular matrix, leading to significant disarray of the sensory epithelium.

Noise-induced hearing loss generally induces loudness recruitment, but sometimes gives rise to hyperacusis, a debilitating condition in which moderate intensity sounds are perceived abnormally loud. In an attempt to develop an animal model of loudness hyperacusis, we exposed rats to a 16-20 kHz noise at 104 dB SPL for 12 weeks. Behavioral reaction time-intensity functions were used to assess loudness growth functions before, during and 2-months post-exposure. During the exposure, loudness recruitment (R) was present in the region of hearing loss, but subtle evidence of hyperacusis (H) started to emerge at the border of the hearing loss. Unexpectedly, robust evidence of hyperacusis appeared below and near the edge of the hearing loss 2-months post-exposure. To identify the neural correlates of hyperacusis and test the central gain model of hyperacusis, we recorded population neural responses from the cochlea, auditory cortex and lateral amygdala 2-months post-exposure. Compared to controls, the neural output of the cochlea was greatly reduced in the noise group. Consistent with central gain models, the gross neural responses from the auditory cortex and amygdala were proportionately much larger than those from the cochlea. However, despite central amplification, the population responses in the auditory cortex and amygdala were still below the level needed to fully account for hyperacusis and/or recruitment. Having developed procedures that can consistently induce hyperacusis in rats, our results set the stage for future studies that seek to identify the neurobiological events that give rise to hyperacusis and to develop new therapies to treat this debilitating condition., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Prolonged noise exposures presented at low to moderate intensities are often used to investigate neuroplastic changes in the central auditory pathway. A common assumption in many studies is that central auditory changes occur independent of any hearing loss or cochlear dysfunction. Since hearing loss from a long term noise exposure can only occur if the level of the noise exceeds a critical level, prolonged noise exposures that incrementally increase in intensity can be used to determine the critical level for any given species and noise spectrum. Here we used distortion product otoacoustic emissions (DPOAEs) to determine the critical level in male, inbred Sprague-Dawley rats exposed to a 16-20 kHz noise that increased from 45 to 92 dB SPL in 8 dB increments. DPOAE amplitudes were largely unaffected by noise presented at 60 dB SPL and below. However, DPOAEs within and above the frequency band of the exposures declined rapidly at noise intensities presented at 68 dB SPL and above. The largest and most rapid decline in DPOAE amplitude occurred at 30 kHz, nearly an octave above the 16-20 kHz exposure band. The rate of decline in DPOAE amplitude was 0.54 for every 1 dB increase in noise intensity. Using a linear regression calculation, the estimated critical level for 16-20 kHz noise was remarkably low, approximately 60 dB SPL. These results indicate that long duration, 16-20 kHz noise exposures in the 65-70 dB SPL range likely affect the cochlea and central auditory system of male Sprague-Dawley rats., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Remarkably, the central auditory system can modify the strength of its sound-evoked neural response based on prior acoustic experiences, a phenomenon referred to as central gain. Gain changes are well documented following traumatic noise exposure, but much less is known about central gain dynamics following prolonged exposure to low-level noise, a common acoustic experience in many urban and work environments. We recently reported that the neural output of the cochlea is reduced, while gain was enhanced in the inferior colliculus (IC) following a 5-week exposure to 75 dB noise. To determine if similar effects were present at even lower intensities, we exposed rats to a 65 dB noise expecting to see little to no change in the cochlea or IC. The exposure had little effect on distortion product otoacoustic emissions and did not cause any hair cell loss. However, the amplitude of the CAP, which reflects the neural output of cochlea, was depressed by 50-75%. Surprisingly, neural responses from the IC were enhanced up to 70%, mainly at frequencies within the noise exposure band. One-week post-exposure, CAP amplitudes returned to normal at frequencies within or above the exposure band, whereas responses evoked by frequencies below the exposure band were enhanced by more than 80%. In contrast, IC responses below the exposure frequency were depressed 10-20% whereas responses within the exposure frequency band were enhanced 10-20%. Thus, the central auditory system dynamically up- and down-regulates its gain to maintain supra-threshold neural responses within a narrow homeostatic range; a function that likely contributes to the prevention of sounds from being perceived as muffled or too loud., (Copyright © 2018 Elsevier B.V. All rights reserved.)

There is a growing interest in the auditory community to develop novel prophylactic and therapeutic drugs to prevent permanent sensorineural hearing loss following acute cochlear injury. The jun-N-terminal protein kinase (JNK) pathway plays a crucial role in acute sensory hearing loss. Blocking the JNK pathway using the cell-penetrating peptide D-JNKI-1 (AM-111/brimapitide) has shown promise as both a prophylactic and therapeutic agent for acute cochlear injury. A number of pre-clinical and clinical studies have determined the impact of D-JNKI-1 on acute sensorineural hearing loss. Given the inner-ear selective therapeutic profile, local route of administration, and ability to diffuse across cellular membranes rapidly using both active and passive transport makes D-JNK-1 a promising oto-protective drug. In this review article, we discuss the application of D-JNKI-1 in various auditory disorders as well as its pharmacological properties and distribution in the cochlea., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paraquat (PQ), one of the most widely used herbicides, is extremely dangerous because it generates the highly toxic superoxide radical. When paraquat was applied to cochlear organotypic cultures, it not only damaged the outer hair cells (OHCs) and inner hair cells (IHCs), but also caused dislocation of the hair cell rows. We hypothesized that the dislocation arose from damage to the support cells (SCs) that anchors hair cells within the epithelium. To test this hypothesis, rat postnatal cochlear cultures were treated with PQ. Shortly after PQ treatment, the rows of OHCs separated from one another and migrated radially away from IHCs suggesting loss of cell-cell adhesion that hold the hair cells in proper alignment. Hair cells dislocation was associated with extensive loss of SCs in the organ of Corti, loss of tympanic border cells (TBCs) beneath the basilar membrane, the early appearance of superoxide staining and caspase-8 labeling in SCs below the OHCs and disintegration of E-cadherin and β-catenin in the organ of Corti. Damage to the TBCs and SCs occurred prior to loss of OHC or IHC loss suggesting a form of detachment-induced apoptosis referred to as anoikis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microglia are glial-immune cells that are essential for the function and survival of the central nervous system. Microglia not only protect neural tissues from immunological insults, but also play a critical role in neural development and repair. However, little is known about the biology of microglia in the cochlea, the auditory portion of the inner ear. In this study, we detected TMEM119+, CD11b+, CD45+ and Iba1+ populations of cells in the rat cochlea, particularly in Rosenthal's canal, inner sulcus and stria vascularis. Next, we isolated and enriched the population of CD11b+ cells from the cochlea and immortalized these cells with the 12S E1A gene of adenovirus in a replication-incompetent retroviral vector to derive a novel microglial cell line, designated Mocha (microglia of the cochlea). The resulting Mocha cells express a number of markers consistent with microglia and respond to lipopolysaccharide (LPS) stimulation by upregulation of genes (Cox2, ICAM-1, Il6r, Ccl2, Il13Ra and Il15Ra) as well as releasing cytokines (IL-1beta, IL-12, IL-13 and RANTES). As evidence of microglial function, Mocha cells phagocytose fluorescent beads at 37°C, but not at 4°C. The expression pattern of microglial markers in Mocha cells suggests that immortalization leads to a more primitive phenotype, a common phenomenon in immortalized cell lines. In summary, Mocha cells display key characteristics of microglia and are now available as a useful model system for the study of cochlear microglial behavior, both in vitro and in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sestrin 2 (SESN2) is a stress-inducible protein that protects tissues from oxidative stress and delays the aging process. However, its role in maintaining the functional and structural integrity of the cochlea is largely unknown. Here, we report the expression of SESN2 protein in the sensory epithelium, particularly in hair cells. Using C57BL/6J mice, a mouse model of age-related cochlear degeneration, we observed a significant age-related reduction in SESN2 expression in cochlear tissues that was associated with early onset hearing loss and accelerated age-related sensory cell degeneration that progressed from the base toward the apex of the cochlea. Hair cell death occurred by caspase-8 mediated apoptosis. Compared to C57BL/6J control mice, Sesn2 KO mice displayed enhanced expression of proinflammatory genes and activation of basilar membrane macrophages, suggesting that loss of SESN2 function provokes the immune response. Together, these results suggest that Sesn2 plays an important role in cochlear homeostasis and immune responses to stress., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Prolonged low-level noise exposure alters loudness perception in humans, presumably by decreasing the gain of the central auditory system. Here we test the central gain hypothesis by measuring the acute and chronic physiologic changes at the level of the cochlea and inferior colliculus (IC) after a 75-dB SPL, 10-20-kHz noise exposure for 5weeks. The compound action potential (CAP) and summating potential (SP) were used to assess the functional status of the cochlea and 16 channel electrodes were used to measure the local field potentials (LFP) and multi-unit spike discharge rates (SDR) from the IC immediately after and one-week post-exposure. Measurements obtained immediately post-exposure demonstrated a significant reduction in supra-threshold CAP amplitudes. In contrast to the periphery, sound-evoked activity in the IC was enhanced in a frequency-dependent manner consistent with models of enhanced central gain. Surprisingly, one-week post-exposure supra-threshold responses from the cochlea had not only recovered, but were significantly larger than normal, and thresholds were significantly better than controls. Moreover, sound-evoked hyperactivity in the IC was sustained within the noise exposure frequency band but suppressed at higher frequencies. When response amplitudes representing the neural output of the cochlea and IC activity at one-week post exposure were compared with control animal responses, a central attenuation phenomenon becomes evident, which may play a key role in understanding why low-level noise can sometimes ameliorate tinnitus and hyperacusis percepts., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Chronic tinnitus and hyperacusis often develop with age-related hearing loss presumably due to aberrant neural activity in the central auditory system (CAS) induced by cochlear pathologies. However, the full spectrum of physiological changes that occur in the CAS as a result age-related hearing loss are still poorly understood. To address this issue, neurophysiological measures were obtained from the cochlea and the inferior colliculus (IC) of 2, 6 and 12 month old C57BL/6J mice, a mouse model for early age-related hearing loss. Thresholds of the compound action potentials (CAP) in 6 and 12 month old mice were significantly higher than in 2 month old mice. The sound driven and spontaneous firing rates of IC neurons, recorded with 16 channel electrodes, revealed mean IC thresholds of 22.8 ± 6.5 dB (n = 167) at 2 months, 37.9 ± 6.2 dB (n = 132) at 6 months and 47.1 ± 15.3 dB (n = 151) at 12 months of age consistent with the rise in CAP thresholds. The characteristic frequencies (CF) of IC neurons ranged from 3 to 32 kHz in 2 month old mice; the upper CF ranged decreased to 26 kHz and 16 kHz in 6 and 12 month old mice respectively. The percentage of IC neurons with CFs between 8 and 12 kHz increased from 36.5% in 2 month old mice, to 48.8% and 76.2% in 6 and 12 month old mice, respectively, suggesting a downshift of IC CFs due to the high-frequency hearing loss. The average spontaneous firing rate (SFRs) of all recorded neurons in 2 month old mice was 3.2 ± 2.5 Hz (n = 167). For 6 and 12 month old mice, the SFRs of low CF neurons (<8 kHz) was maintained at 3-6 spikes/s; whereas SFRs of IC neurons with CFs > 8 kHz increased to 13.0 ± 15.4 (n = 68) Hz at 6 months of age and then declined to 4.8 ± 7.4 (n = 110) spikes/s at 12 months of age. In addition, sound-evoked activity at suprathreshold levels at 6 months of age was much higher than at 2 and 12 months of age. To evaluate the behavioral consequences of sound evoked hyperactivity in the IC, the amplitude of the acoustic startle reflex was measured at 4, 8 and 16 kHz using narrow band noise bursts. Acoustic startle reflex amplitudes in 6 and 12 month old mice (n = 4) were significantly larger than 2 month old mice (n = 4) at 4 and 8 kHz, but not 16 kHz. The enhanced reflex amplitudes suggest that high-intensity, low-frequency sounds are perceived as louder than normal in 6 and 12 month old mice compared to 2 month olds. The increased spontaneous activity, particularly at 6 months, may be related to tinnitus whereas the increase in sound-evoked activity and startle reflex amplitudes may be related to hyperacusis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP + to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd -deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP + to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1 , but not G6pd , decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea. SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP + to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea., (Copyright © 2017 the authors 0270-6474/17/375770-12$15.00/0.)

A single nucleotide variant (SNV) of the cadherin 23 gene (Cdh23 c.753A ), common to many inbred mouse strains, accelerates age-related hearing loss (AHL) and can worsen auditory phenotypes of other mutations. We used homologous recombination in C57BL/6 NJ (B6N) and 129S1/SvImJ (129S1) embryonic stem cells to engineer mouse strains with reciprocal single base pair substitutions (B6-Cdh23 c.753A>G and 129S1-Cdh23 c.753G>A ). We compared ABR thresholds and cochlear pathologies of these SNV mice with those of congenic (B6.129S1-Cdh23 Ahl+ and 129S1.B6-Cdh23 ahl ) and parental (B6N and 129S1) strain mice. Results verified the protective effect of the Cdh23 c.753G allele, which prevented high frequency hearing loss in B6 mice to at least 18 months of age, and the AHL-inducing effect of the Cdh23 c.753A allele, which worsened hearing loss in 129S1 mice. ABR thresholds differed between 129S-Cdh23 c.753A SNV and 129S1.B6-Cdh23 ahl congenic mice, and a linkage backcross involving these strains localized a Chr 10 QTL contributing to the difference. These results illustrate the large effects that strain background and congenic regions have on the hearing loss associated with Cdh23 c.753 alleles. Importantly, the B6-Cdh23 c.753G strain can be used to eliminate the confounding influence of the Cdh23 c.753A variant in hearing studies of B6 mice and mutant mice on the B6 background.

Carbaryl, a widely used carbamate-based insecticide, is a potent anticholinesterase known to induce delayed neurotoxicity following chronic exposure. However, its potential toxic effects on the cochlea, the sensory organ for hearing that contains cholinergic efferent neurons and acetylcholine receptors on the hair cells (HC) and spiral ganglion neurons has heretofore not been evaluated. To assess ototoxic potential of carbaryl, cochlear organotypic cultures from postnatal day 3 rats were treated with doses of carbaryl ranging from 50 to 500 μM for 48 h up to 96 h. Carbaryl damaged both the sensory HC and spiral ganglion neurons in a dose- and duration-dependent manner. HC and neuronal damage was observed at carbaryl concentrations as low as 50 μM after 96-h treatment and 100 μM after 48-h treatment. Hair cell was greatest in the high frequency basal region of the cochlea and progressively decreased towards the apex consistent with the majority of ototoxic drugs. In contrast, damage to the spiral ganglion neurons was of similar magnitude in the basal and apical regions of the cochlea. Carbaryl damage was characterized by soma shrinkage, nuclear condensation and fragmentation, and blebbing, morphological features of programmed cell death. Carbaryl upregulated the expression of executioner caspase-3 in HC and spiral ganglion neurons indicating that cellular damage occurred primarily by caspase-mediated apoptosis. These results suggest that chronic exposure to carbaryl and other carbamate anticholinesterases may be ototoxic. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 956-969, 2017., (© 2016 Wiley Periodicals, Inc.)

The degenerative actions of Mn caused by persistent exposure to high atmospheric levels not only provokes irreversible damage to the CNS with symptoms comparable to that of Parkinson's disease but also may have deleterious consequences to other organs including the auditory system. The putative deleterious consequences of prolonged Mn overexposure on hearing, however, is confounded by the fact that chronically-exposed individuals often work in high noise environments where noise by itself is known to cause hearing loss. Thus, the question as to whether Mn alone is actually ototoxic and whether exposure to Mn when combined with noise increases the risk of hearing loss and cochlear pathology has never been examined. To examine whether noise effects Mn ototoxicity, we exposed rats to a moderate dose of Mn (10mg MnCl2/liter water) alone, a high level of noise (octave band noise, 8-16kHz, presented at 90dB SPL for 8h/d) alone or the combination of Mn plus noise and measured the changes in auditory function and the cochlear histopathologies. Results of these studies, based on various measures of hearing including histological examination of cochlear tissue suggest that noise alone produced significant hearing deficits whereas semi-chronic exposure to moderate levels of Mn in drinking water for 90days either in the presence or absence of noise had, at best, only a minor effect on hearing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Membrane transporters can be major determinants in the targeting and effectiveness of pharmaceutical agents. A large number of biologically important membrane transporters have been identified and localized to a variety of tissues, organs and cell types. However, little is known about the expression of key membrane transporters in the inner ear, a promising site for targeted therapeutics, as well as a region vulnerable to adverse drug reactions and environmental factors. In this study, we examined the levels of endogenous membrane transporters in rat cochlea by targeted PCR array analysis of 84 transporter genes, followed by validation and localization in tissues by immunohistochemistry. Our studies indicate that several members of the SLC, VDAC and ABC membrane transporter families show high levels of expression, both at the RNA and protein levels in the rat cochlea. Identification and characterization of these membrane transporters in the inner ear have clinical implications for both therapeutic and cytotoxic mechanisms that may aid in the preservation of auditory function., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu) are essential transitions metals that are required in trace amounts, however chronic exposure to high concentrations can cause severe and irreversible neurotoxicity. Since prolonged exposure to Mn leads to manganism, a disorder exhibiting a diverse array of neurological impairments progressing to a debilitating and irreversible extrapyramidal condition symptomatically similar to Parkinson's disease, we measured the concentration of Mn as well as Fe, Zn and Cu in three region of the brain (globus pallidus, striatum and inferior colliculus) and three regions in the cochlea (stria vascularis, basilar membrane and modiolus) under normal conditions or after 30 or 60 days of oral administration of Mn (10 mg/ml ad libitum). Under normal conditions, Mn, Zn and Fe were typically higher in the cochlea than in the three brain regions whereas Cu was equal to or lower. Oral treatment with Mn for 30 or 60 days resulted in 20-75 % increases in Mn concentrations in both cochlea and brain samples, but had little effect on Cu and Fe levels. In contrast, Zn levels decreased (20-80 %) with Mn exposure. Our results show for the first time how prolonged oral Mn-ingestion affects the concentration of Mn, Cu, Zn and Fe, in the three regions of the cochlea, the inferior colliculus in auditory midbrain and the striatum and globus pallidus, two regions implicated in Parkinson's disorder. The Mn-induced changes in the concentration of Mn, Cu, Zn and Fe may provide new insights relevant to the neurotoxicity of Mn and the transport and accumulation of these metals in cochlea and brain.

Cobalt (Co) is a required divalent metal used in the production of metal alloys, batteries, and pigments and is a component of vitamin B12. Excessive uptake of Co is neurotoxic causing temporary or permanent hearing loss; however, its ototoxic effects on the sensory hair cells, neurons, and support cells in the cochlea are poorly understood. Accordingly, we treated postnatal day 3 rat cochlear organotypic cultures with various doses and durations of CoCl2 and quantified the damage to the hair cells, peripheral auditory nerve fibers, and spiral ganglion neurons (SGN). Five-day treatment with 250 μM CoCl2 caused extensive damage to hair cells and neurons which increased with dose and treatment duration. CoCl2 caused greater damage to outer hair cells than inner hair cells; damage was greatest in the base of the cochlea and decreased towards the base. CoCl2 increased expression of superoxide radical in hair cells and SGNs and SGN loss was characterized by nuclear condensation and fragmentation, morphological features of apoptosis. CoCl2 treatment increased the expression of caspase-3 indicative of caspase-mediated programmed cell death. These results identify hair cells and spiral ganglion neurons as the main targets of Co ototoxicity in vitro and implicate the superoxide radical as a trigger of caspase-mediated ototoxicity.

Sodium Salicylate (SS) reliably induces a sensorineural hearing loss and tinnitus when administered in high doses. Recent animal modeled studies indicate that potassium channel openers such as Maxipost and Retigabine (RTG) can block SS- or noise-induced tinnitus respectively; however, the origins and mechanisms are poorly understood. Since SS blocks the same potassium channels that Maxipost and RTG open, we postulated that these drugs might influence peripheral auditory function. To test this hypothesis Maxipost or RTG were administered alone or in combination with SS in rats. When administered alone, Maxipost and RTG had no effect on distortion product otoacoustic emissions (DPOAE) or compound action potentials (CAPs). However when Maxipost or RTG were administered with SS, Maxipost prevented the SS-reduced CAP amplitudes at high frequencies (≥20 kHz) and RTG prevented SS-reduced CAP amplitudes at low frequencies (≤8 kHz). These results suggest that Maxipost and RTG can protect against peripheral damage and therefore reduce the incidence of tinnitus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Trimethyltin (TMT), which has a variety of applications in industry and agricultural, is a neurotoxin that is known to affect the auditory system as well as central nervous system of humans and experimental animals. However, the mechanisms underlying TMT-induced auditory dysfunction are poorly understood. To gain insights into the neurotoxic effect of TMT on the peripheral auditory system, we treated cochlear organotypic cultures with concentrations of TMT ranging from 5 to 100 μM for 24 h. Interestingly, TMT preferentially damaged auditory nerve fibers and spiral ganglion neurons in a dose-dependent manner, but had no noticeable effects on the sensory hair cells at the doses employed. TMT-induced damage to auditory neurons was associated with significant soma shrinkage, nuclear condensation, and activation of caspase-3, biomarkers indicative of apoptotic cell death. Our findings show that TMT is exclusively neurotoxicity in rat cochlear organotypic culture and that TMT-induced auditory neuron death occurs through a caspase-mediated apoptotic pathway.

Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are essential nutrients which aid in the proper functioning of cells, but high concentrations of these metals can be toxic to various organs. Little is known about the endogenous concentrations of these metals in the cochlea, the auditory portion of the inner ear which is extremely small and difficult to access. To fill this gap, a trace quantitative digestion and inductively coupled plasma mass spectrometry method was developed to determine the concentrations of these metals in the stria vascularis, organ of Corti, and spiral ganglion, three critically important parts of the cochlea (≤ 1.5 mg); these values were compared to those in specific brain regions (≤ 20 mg) of rats. Rats were sacrificed and the cochlea and brain regions were carefully isolated, digested, and analyzed to determine baseline concentrations of Mn, Fe, Cu, and Zn. In the cochlea, Mn, Fe, Cu, and Zn concentrations ranged from 3.2-6, 73-300, non-detect, and 13-200 µg/g respectively. In the brain, Mn, Fe, Cu, and Zn concentrations ranged from 1.3-2.72, 21-120, 5.0-10.6, and 33-47 µg/g respectively. Significant differences (p < 0.05) were observed between the tissue types within the cochlea, and between the cochlea and brain. This validated method provides the first quantitative assessment of these metals in the three key subdivisions of the cochlea compared to the levels in the brain; Mn, Fe, and Zn levels were considerably higher in the cochlea than brain.

Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 μM. No obvious histopathologies were observed after 24h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 μM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 μM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cadmium (Cd), a widely used industrial metal, is extremely nephrotoxic and neurotoxic; however, its effects on the peripheral auditory system are poorly understood. To evaluate the ototoxicity of Cd, we treated cochlear organotypic cultures from postnatal day 3 rats with Cd concentrations from 10 to 500 μM for 24 or 48 h. Afterward, we evaluated the degree of damage to hair cells, auditory nerve fibers, and spiral ganglion neurons. Damage to the hair cells, auditory nerve fibers, and spiral ganglion neurons systematically increased in a dose and time-dependent manner. Exposure to Cd concentrations of 10 μM for 24 and 48 h resulted in minor inner and outer hair cell loss in the basal third of the cochlea. As Cd concentrations increased, toxicity spread toward the apex, also in a time-dependent manner. Treatment with 100 μM Cd for 48 h resulted in substantial (>30 %) hair cell loss over the entire cochlea. Cd was also toxic to auditory nerve fibers and spiral ganglion neurons; 100 μM of Cd for 24 h or 10 μM of Cd for 48 h resulted in considerable damage to auditory nerve fibers and spiral ganglion neurons. These findings are the first to demonstrate that Cd can cause significant lesions to peripheral auditory nerve fibers, spiral ganglion neurons, and sensory hair cells in organotypic cultures from postnatal cochleae.

Manganese (Mn) is an essential trace mineral for normal growth and development. Persistent exposures to high atmospheric levels of Mn have deleterious effects on CNS and peripheral nerves including those associated with the auditory system. Nicotinamide adenine dinucleotide (NAD) is a coenzyme which functions in the electron transfer system within the mitochondria. One of the most notable protective functions of NAD is to delay axonal degenerations caused by various neurodegenerative injuries. We hypothesized that NAD might also protect auditory nerve fibers (ANF) and SGN from Mn injury. To test this hypothesis, cochlear organotypic cultures were treated with different doses of Mn (0.5-3.0 mM) alone or combined with 20 mM NAD. Results demonstrate that the percentage of hair cells, ANF and SGN decreased with increasing Mn concentration. The addition of 20 mM NAD did not significantly reduce hair cells loss in the presence of Mn, whereas the density of ANF and SGN increased significantly in the presence of NAD. NAD suppressed Mn-induced TUNEL staining and caspase activation suggesting it prevents apoptotic cell death. These results suggest that excess Mn has ototoxic and neurotoxic effects on the auditory system and that NAD may prevent Mn-induced axonal degeneration and avoid or delay hearing loss caused by excess Mn exposure., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives/hypothesis: The round window acts as a vent for releasing inner ear pressure and facilitating basilar membrane vibration. Loss of this venting function affects cochlear function, which leads to hearing impairment. In an effort to identify functional changes that might be used in clinical diagnosis of round window atresia, the current investigation was designed to examine how the cochlea responds to suprathreshold stimuli following round window closure., Study Design: Prospective, controlled, animal study., Methods: A rat model of round window occlusion (RWO) was established. With this model, the thresholds of auditory brainstem responses (ABR) and the input/output (IO) functions of distortion product otoacoustic emissions (DPOAEs) and acoustic startle responses were examined., Results: Round window closure caused a mild shift in the thresholds of the auditory brainstem response (13.5 ± 9.1 dB). It also reduced the amplitudes of the distortion product otoacoustic emissions and the slope of the input/output functions. This peripheral change was accompanied by a significant reduction in the amplitude, but not the threshold, of the acoustic startle reflex, a motor response to suprathreshold sounds., Conclusions: In addition to causing mild increase in the threshold of the auditory brainstem response, round window occlusion reduced the slopes of both distortion product otoacoustic emissions and startle reflex input/output functions. These changes differ from those observed for typical conductive or sensory hearing loss, and could be present in patients with round window atresia. However, future clinical observations in patients are needed to confirm these findings., (Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects., Principal Findings: In this study, we show that the coenzyme NAD(+), known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD(+) protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD(+) reduced the levels of these oxidative stress and apoptosis markers., Conclusions/significance: Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD(+) suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.

High doses of sodium salicylate (SS) have long been known to induce temporary hearing loss and tinnitus, effects attributed to cochlear dysfunction. However, our recent publications reviewed here show that SS can induce profound, permanent, and unexpected changes in the cochlea and central nervous system. Prolonged treatment with SS permanently decreased the cochlear compound action potential (CAP) amplitude in vivo. In vitro, high dose SS resulted in a permanent loss of spiral ganglion neurons and nerve fibers, but did not damage hair cells. Acute treatment with high-dose SS produced a frequency-dependent decrease in the amplitude of distortion product otoacoustic emissions and CAP. Losses were greatest at low and high frequencies, but least at the mid-frequencies (10-20 kHz), the mid-frequency band that corresponds to the tinnitus pitch measured behaviorally. In the auditory cortex, medial geniculate body and amygdala, high-dose SS enhanced sound-evoked neural responses at high stimulus levels, but it suppressed activity at low intensities and elevated response threshold. When SS was applied directly to the auditory cortex or amygdala, it only enhanced sound evoked activity, but did not elevate response threshold. Current source density analysis revealed enhanced current flow into the supragranular layer of auditory cortex following systemic SS treatment. Systemic SS treatment also altered tuning in auditory cortex and amygdala; low frequency and high frequency multiunit clusters up-shifted or down-shifted their characteristic frequency into the 10-20 kHz range thereby altering auditory cortex tonotopy and enhancing neural activity at mid-frequencies corresponding to the tinnitus pitch. These results suggest that SS-induced hyperactivity in auditory cortex originates in the central nervous system, that the amygdala potentiates these effects and that the SS-induced tonotopic shifts in auditory cortex, the putative neural correlate of tinnitus, arises from the interaction between the frequency-dependent losses in the cochlea and hyperactivity in the central nervous system., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of Taok1, a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and Taok1, suggesting the presence of a miR-183/Taok1 target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/Taok1 target pair is likely to play a role in this regulatory process.

Cisplatin, carboplatin, nedaplatin, and oxaliplatin are widely used in contemporary oncology; however, their ototoxic and neurotoxic side effects are quite different as discussed in this review. Cisplatin is considered the most ototoxic, but despite its reputation, the magnitude of hair cell loss that occurs with a single, large drug bolus is limited and confined to the base of the cochlea. For all of these platinum compounds, a major factor limiting damage is drug uptake from stria vascularis into the cochlear fluids. Disrupting the blood-labyrinth barrier with diuretics or noise exposure enhances drug uptake and significantly increases the amount of damage. Combined treatment with ethacrynic acid (a loop diuretic) and cisplatin results in rapid apoptotic hair cell death characterized by upregulation of initiator caspase-8 and membrane death receptor, TRADD, followed by downstream executioners, caspase-3 and caspase-6. Unlike cisplatin, nedaplatin and oxaliplatin are highly neurotoxic when applied to cochlear cultures preferentially damaging auditory nerve fibers at low concentrations and hair cells at high concentrations. Carboplatin, considered far less ototoxic than cisplatin, is paradoxically highly toxic to chinchilla inner hair cells and type I spiral ganglion neurons; however, at high doses it also damages outer hair cells. Hair cell death from cisplatin and carboplatin is characterized in its early stages by upregulation of p53; blocking p53 expression with pifithrin-α prevents hair cell death. Major differences in the toxicity of these four platinum compounds may arise from several different metal transporters that selectively regulate the influx, efflux, and sequestration of these drugs., (Copyright © 2012 Wiley Periodicals, Inc.)

The biological mechanisms that give rise to age-related hearing loss (ARHL) are still poorly understood. However, there is growing recognition that oxidative stress may be an important factor. To address this issue, we measured the changes in the expression of cochlear oxidative stress and antioxidant defense-related genes in young (2 months old), middle-aged (12 months old), and old (21-25 months old) Fischer 344/NHsd (F344/NHsd) rats and compared gene expression changes with ARHL. A quantitative real-time reverse transcription polymerase chain reaction array revealed a significant age-related downregulation of only 1 gene, stearoyl-coenzyme A desaturase 1, and upregulation of 12 genes: 24-dehydrocholesterol reductase; aminoadipate-semialdehyde synthase; cytoglobin; dual oxidase 2; glutathione peroxidase 3; glutathione peroxidase 6; glutathione S-transferase, kappa 1; glutathione reductase; nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase, quinone 1; solute carrier Family 38, Member 5; thioredoxin interacting protein; and vimentin. Statistical analyses revealed significant correlations between gene expression and auditory function in 8 genes. Our results identified specific subsets of oxidative stress genes that appear to play an important role in ARHL in the Fischer 344/NHsd rat., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tyrosine nitration is an important sequel of cellular signaling induced by reactive oxygen species. Cisplatin is an anti-neoplastic agent that damages the inner ear through reactive oxygen species and by the formation of DNA adducts. This study reveals a correlation between cisplatin-mediated hearing loss and nitroxidative modification of cochlear proteins and is the first to report nitration of Lmo4. Cisplatin induced a dose-dependent increase in hearing loss in Wistar rats. A 10-15-dB decrease in distortion product amplitude and massive loss of outer hair cells at the basal turn of the cochlea was observed 3 days post-treatment after a 16 mg/kg dose. Cisplatin induced nitration of cellular proteins within the organ of Corti, spiral ganglion, and stria vascularis, which are known targets of cisplatin ototoxicity. Nitration of a 76-kDa cochlear protein correlated with cisplatin dose. The nitrated protein was identified as Lmo4 (LIM domain only 4) by MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass spectrometry and confirmed by reciprocal immunoprecipitation and immunoblotting. Co-localization of nitrotyrosine and Lmo4 was particularly high in outer hair cell nuclei after cisplatin treatment. Cochlear levels of Lmo4 were decreased in rats treated with cisplatin. In vitro studies supported the repression of Lmo4 in nitroxidative conditions and the induction of apoptosis upon repression of Lmo4. Inhibition of cochlear protein nitration prevented cisplatin-induced hearing loss. As Lmo4 is a transcriptional regulator that controls the choice between cell survival and cell death, these results support the hypothesis that nitration of Lmo4 influences cisplatin-induced ototoxicity.

Noise exposure is a major cause of hearing loss. Classical methods of studying protein involvement have provided a basis for understanding signaling pathways that mediate hearing loss and damage repair but do not lend themselves to studying large networks of proteins that are likely to increase or decrease during noise trauma. To address this issue, antibody microarrays were used to quantify the very early changes in protein expression in three distinct regions of the chinchilla cochlea 2h after exposure to a 0.5-8 kHz band of noise for 2h at 112 dB SPL. The noise exposure caused significant functional impairment 2h post-exposure which only partially recovered. Distortion product otoacoustic emissions were abolished 2h after the exposure, but at 4 weeks post-exposure, otoacoustic emissions were present, but still greatly depressed. Cochleograms obtained 4 weeks post-exposure demonstrated significant loss of outer hair cells in the basal 60% of the cochlea corresponding to frequencies in the noise spectrum. A comparative analysis of the very early (2h post-exposure) noise-induced proteomic changes indicated that the sensory epithelium, lateral wall and modiolus differ in their biological response to noise. Bioinformatic analysis of the cochlear protein profile using "The Database for Annotation, Visualization and Integrated Discovery 2008" (DAVID - http://david.abcc. ncifcrf.gov) revealed the initiation of the cell death process in sensory epithelium and modiolus. An increase in Fas and phosphorylation of FAK and p38/MAPK in the sensory epithelium suggest that noise-induced stress signals at the cell membrane are transmitted to the nucleus by Fas and focal adhesion signaling through the p38/MAPK signaling pathway. Up-regulation of downstream nuclear proteins E2F3 and WSTF in immunoblots and microarrays along with their immunolocalization in the outer hair cells supported the pivotal role of p38/MAPK signaling in the mechanism underlying noise-induced hearing loss., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Exposure to intense noise induces apoptosis in hair cells in the cochlea. To identify the molecular changes associated with noise-induced apoptosis, we used quantitative real-time PCR to evaluate the changes in 84 apoptosis-related genes in cochlear samples from the sensory epithelium and lateral wall. Sprague-Dawley rats exposed to a continuous noise at 115 dB SPL for 2 h. The exposure caused a 40-60 dB threshold shift 4 h post-exposure that decreased to 20-30 dB 7 days post-exposure. These functional changes were associated with apoptotic markers including nuclear condensation and fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Immediately after the noise exposure, 12 genes were downregulated, whereas only one gene (Traf4) was upregulated. At 4 h post-exposure, eight genes were upregulated; three (Tnrsf1a, Tnfrsf1b, Tnfrst5) belonged to the Tnfrsf family, three (Bir3, Mcl1 and Prok2) have anti-apoptotic properties and one (Gadd45a) is a target of p53. At 7 days post-exposure, all the upregulated genes returned to pre-noise levels. Interestingly, the normal control cochlea had high constitutive levels of several apoptosis-related genes. These constitutively expressed genes, together with the inducible genes, may participate in the induction of cochlear apoptotic activity.

Differences in the expression of cochlear proteins are likely to affect the susceptibility of different animal models to specific types of auditory pathology. However, little is currently known about proteins that are abundantly expressed in inner ear. Identification of these proteins may facilitate the search for biomarkers of susceptibility and intervention targets. To begin to address this issue, we analyzed cochlear protein profiles of three strains of rats, Wistar, Sprague-Dawley, and Fischer 344, using a broad spectrum antibody microarray. Normal hearing function of the animals was ascertained using distortion product otoacoustic emissions (DPOAE). Of 725 proteins screened in whole cochlea, more than 80% were detected in all three strains. However, there were striking differences in the levels at which they occur. Among 213 proteins expressed at levels>or=2 fold of actin, only 7.5% were detected at these levels in all three strains. Myosin light chain kinase (MLCK) was immunolocalized in cuticular plate of outer hair cells (OHC) while mitogen activated protein (MAP) kinase-extracellular-signal regulated kinase1/2 (ERK1/2) was detected as foci in OHC, pillar cells, strial marginal cells, and fibroblasts of spiral ligament. A review of literature indicated that the expression of 7 (44%) of these 16 proteins were detected for the first time in the inner ear, although there were implications of the presence of some of these proteins. One of these abundant, but unstudied, proteins, MAP kinase activated protein kinase2 (MAPKAPK2), shows strong immunolabeling in pillar cells and inner hair cells (IHC). There was moderate MAPKAPK2 labeling in OHC, supporting cells, neurons, and marginal, intermediate, and basal cells. The current study provides the first, large cochlear protein profile of multiple rat strains. The diversity in expression of abundant proteins in these strains may contribute to differences in susceptibility of these strains to aging, noise, or ototoxic drugs.

As previously reported [Popelar, J., Groh, D., Pelanova, J., Canlon, B., Syka, J., 2006. Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats. Neurobiol. Aging 27, 490-500; Buckiova, D., Popelar, J., Syka, J., 2007. Aging cochleas in the F344 rat: morphological and functional changes. Exp. Gerontol. 42, 629-638; Bielefeld, E.C., Coling, D., Chen, G.D., Li, M.N., Tanaka, C., Hu, B.H., Henderson, D., 2008. Age-related hearing loss in the Fischer 344/NHsd rat substrain. Hear. Res. 241, 26-33], aged Fischer 344 (F344) rats with severe hearing loss retain many outer hair cells (OHCs) especially in the middle turn of the cochlea. The current study confirmed the previous findings showing that aged OHCs were present, but dysfunctional. Distortion product otoacoustic emissions (DPOAE), which are believed to reflect in vivo OHC motility, were absent in the aged rats while the majority of OHCs (>80%) were present and morphologically intact. There was no detectable injury of OHC stereocilia as assessed by actin-staining and examination under the light microscope. Cochlear microphonics (CM) at 12kHz, recorded from the middle turn, only showed a slight age-related reduction, indicating a normal mechanoelectrical transduction apparatus in the remaining OHCs in the cochlear regions with 10-20% OHC loss. Activities of succinate dehydrogenase (SDH), an enzyme shared by the citric acid cycle and the mitochondrial electron transport chain (METC), were also at normal levels in aged OHCs. Importantly, aged OHCs showed reduced levels of prestin immunolabeling compared to young controls. Together with our previous finding showing that the stria vascularis and endocochlear potential were essentially normal in aged F344 rats [Bielefeld, E.C., Coling, D., Chen, G.D., Li, M.N., Tanaka, C., Hu, B.H., Henderson, D., 2008. Age-related hearing loss in the Fischer 344/NHsd rat substrain. Hear. Res. 241, 26-33], the results suggest that disruption of prestin is the major cause of DPOAE loss and loss of cochlear sensitivity.

Gonadectomized and intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to an augmented acoustic environment (AAE): repetitive bursts of a 70dB SPL noise band. The high-frequency AAE (HAAE) was a half-octave band centered at 20kHz; the low-frequency AAE (LAAE) was a 2-8kHz band. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (a trait of B6 mice) were evaluated by obtaining auditory brainstem response (ABR) thresholds at ages 3-, 6-, and 9-months. At 9-months of age, hair cell counts (cytocochleograms) were obtained, and morphometric measures of the anteroventral cochlear nucleus (AVCN) were obtained. LAAE treatment caused elevation in ABR thresholds (8-24kHz), with the highest thresholds occurring in intact females. LAAE treatment caused some loss of outer hair cells in the basal half of the cochlea (in addition to losses normally occurring in B6 mice), with intact females losing more cells than intact males. The loss of AVCN neurons and shrinkage of tissue volume that typically occur in 9-month-old B6 mice was lessened by LAAE treatment in intact (but not gonadectomized) male mice, whereas the degenerative changes were exacerbated in intact (but not gonadectomized) females. These LAAE effects were prominent in, but not restricted to, the tonotopic low-frequency (ventral) AVCN. HAAE treatment resulted in some loss of neurons in the high-frequency (dorsal) AVCN. In general, LAAE treatment plus male gonadal hormones (intact males) had an ameliorative effect whereas HAAE or LAAE treatment plus ovarian hormones (intact females) had a negative effect on age-related changes in the B6 auditory system.

Mutations in connexin (Cx) genes are responsible for a large proportion of human inherited prelingual deafness cases. The most commonly found human Cx mutations are either Cx26 or Cx30 deletions. Histological observations made in the organ of Corti of homozygous Cx26 and Cx30 gene knockout mice show that cochlear hair cells degenerate after the onset of hearing. However, it is unclear whether vestibular hair cells undergo similar degeneration in connexin knockout mice. To address this question, we first examined expression patterns of Cx26 and Cx30 in the saccule, utricle, and ampulla by immunolabeling. In wild-type mice, Cx26 and Cx30 immunoreactivity was found extensively in vestibular supporting cells and connective tissue cells, and the two Cxs were co-localized in most gap junction (GJ) plaques. Targeted deletion of the Cx30 gene, which caused little change in Cx26 expression pattern, resulted in a significant and age-related loss of vestibular hair cells only in the saccule. dUTP nick end labeling (TUNEL) staining also revealed on-going apoptosis specifically in saccular hair cells of Cx30(-/-) mice. These results indicated that hair cell survival in the utricle and ampulae does not require Cx30. Importantly, over-expressing the Cx26 gene from a modified bacterial artificial chromosome in the Cx30(-/-) background rescued the saccular hair cells. These results suggest that it is the reduction in the total amount of GJs rather than the specific loss of Cx30 that underlies saccular hair cell death in Cx30(-/-) mice. Hybrid GJs co-assembled from Cx26 and Cx30 were not essential for the survival of saccular hair cells.

Gonadectomized and surgically intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to a high-frequency augmented acoustic environment (AAE): repetitive bursts of a half-octave noise band centered at 20 kHz, 70 dB SPL. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (exhibited by B6 mice) were evaluated by obtaining auditory brainstem response thresholds at ages 3-, 6-, and 9-months; hair cell counts (cytocochleograms) were obtained at 9 months. A sex difference in the rate of genetic progressive hearing loss in B6 mice (observed by earlier studies) was confirmed, with females exhibiting a faster rate of threshold elevations and more severe loss of hair cells at age 9 months. Gonadectomy had no consistent effects on the rate or severity of hearing loss in non-exposed mice of either sex. An unexpected finding was that the high-frequency AAE treatment caused additional ABR threshold elevations and hair cell loss. In an earlier study, the same high-frequency AAE treatment on DBA/2J mice ameliorated hearing loss. The most severe AAE-induced losses occurred in surgically intact females, suggesting a potentiating effect of ovarian hormone(s).

Cisplatin, a commonly used antineoplastic agent, destroys the sensory hair cells in the cochlear and vestibular system leading to irreversible hearing loss and balance problems. Cisplatin-induced hair cell damage presumably occurs by apoptosis. Recent studies suggest that p53 may play an important role initiating cisplatin-induced apoptosis in some cell types. To determine if p53 plays a role in cisplatin-mediated hair cell loss, cochlear and utricular organotypic cultures were prepared from postnatal day 3-4 rats and treated with cisplatin or cisplatin plus pifithrin-alpha (PFT), a p53 inhibitor. Control cultures were devoid of p53 immunolabeling, caspase-1 and caspase-3 labeling and p53 protein was absent from Western blots. Cisplatin (1-10 microg/ml) caused a dose-dependent loss of hair cells in cochlear and utricular cultures, up-regulated phospho-p53 serine 15 immunolabeling, increased the expression of phospho-p53 serine 15 in Western blots from 6 to 48 h after the onset of cisplatin-treatment, and increased caspase-1 and caspase-3 labeling in cochlear and vestibular cultures. Addition of PFT (20-100 microM) to cisplatin-treated cochlear and utricular cultures resulted in a dose-dependent increase in hair cell survival; suppressed the expression of p53 in Western blots and eliminated caspase-1 and caspase-3 labeling in cultures. These results suggest that the tumor suppressor protein, p53, plays a critical role in initiating apoptosis in cochlear and vestibular hair cells. Temporary suppression of p53 with PFT provides significant protection against cisplatin-induced hair cell loss and offers the potential for reducing the ototoxic, vestibulotoxic and neurotoxic side effects of cisplatin.

Immunolabeling of heregulin, a growth factor that enhances cell proliferation in damaged utricles, and one of its binding receptors, ErbB-2, has been briefly described in the P3 rat cochlea and utricle [Zheng et al. (1999) J. Neurocytol. 28, 901-912]. However, little is known about the distribution of heregulin and its three binding receptors in adult animals. Here we describe the immunolabeling patterns for heregulin, ErbB-2, ErbB-3 and ErB-4 in the cochlea, spiral ganglion, utricle and saccule of the adult chinchilla using confocal microscopy. Heregulin immunolabeling was intense along the apical pole of Deiters cells and Hensen cells and along the membrane of supporting cells of the utricle and saccule; light immunolabeling was present in the outer layer of the spiral prominence and cytoplasm of spiral ganglion neurons. In the cochlea, intense to moderate ErbB-2 immunolabeling was evident in the cytoplasm of pillar cells, outer hair cells (OHCs), border cells, stria vascularis and spiral ligament; moderate ErbB-2 immunolabeling was present in the cytoplasm of the hair cell and supporting cell layers of the utricle and saccule. In the cochlea, light ErbB-3 immunolabeling was present in the inner hair cells, OHCs, marginal and intermediate cell layers of the stria vascularis and spiral ganglion neurons; moderate ErbB-3 immunolabeling was present in the cytoplasm of hair cells and supporting cells of the utricle and saccule. In the cochlea, utricle and saccule, ErbB-4 immunolabeling was intense in the nuclei and light to moderate in the cytoplasm and membrane of sensory cells and supporting cells. These results suggest that heregulin acting through ErbB receptors and various receptor complexes may play an important role in cell proliferation and survival in the cochlea and vestibular system.

Kainic acid (KA) selectively damages afferent synapses that innervate, in chickens, mainly tall hair cells. To better understand the nature of KA-induced excitotoxic damage to the cochlear afferent neurons, KA, at two different concentrations (0.3 or 5 mM), was injected directly into the inner ear of adult chickens. Pathologic changes in the afferent nerve ending and cell body were evaluated with light and transmission electron microscopy at various time points after KA application. The compound action potential (CAP) and cochlear microphonic (CM) potential were recorded to monitor the physiologic status of the afferent neurons and hair cells, respectively. Hair cell morphology and function were essentially normal after KA treatment. However, afferent synapses beneath tall hair cells were swollen within 30 minutes after KA at both low (KA-L) and high (KA-H) doses. In the KA-L group, the swelling disappeared within 1 day and the morphology of the postsynaptic region returned to near normal condition. In the KA-H group, by contrast, the vacant region beneath tall hair cells remained evident even 20 weeks after KA. The number of cochlear ganglion neurons in the KA-H group decreased progressively from 1 to 8-20 weeks, whereas hair cells in the basilar papilla remained morphologically intact out to 20 weeks after KA. There was no significant change in neuron number in the KA-L group. Temporal changes in the CAP amplitude paralleled the anatomic changes, although the CAP only partially recovered. These results suggest that KA induces partially reversible damage to cochlear afferent neurons with low KA concentration; above this level, KA triggers irreversible, progressive neurodegeneration., (Copyright 2001 Wiley-Liss, Inc.)

This paper will review some of the functional changes that occur in the central auditory pathway after the cochlea is damaged by acoustic overstimulation or by carboplatin, an ototoxic drug that selectively destroys inner hair cells (IHCs) in the chinchilla. Acoustic trauma typically impairs the sensitivity and tuning of auditory nerve fibers and reduces the neural output of the cochlea. Surprisingly, our results show that restricted cochlear damage enhances neural activity in the central auditory pathway. Despite a reduction in the auditory-nerve compound action potential (CAP), the local field potential from the inferior colliculus (IC) increases at a faster than normal rate and its maximum amplitude is enhanced at frequencies below the region of hearing loss. To determine if this enhancement was due to loss of sideband inhibition, we recorded from single neurons in the IC and dorsal cochlear nucleus before and after presenting a traumatizing above the unit's characteristic frequency (CF). Following the exposure, some neurons showed substantial broadening of tuning below CF, less inhibition, and a significant increase in discharge rate, consistent with a model involving loss of sideband inhibition. The central auditory system of the chinchilla can be deprived of some of its cochlear inputs by selectively destroying IHCs with carboplatin. Selective IHC loss reduces the amplitude of the CAP without affecting the threshold and tuning of the remaining auditory nerve fibers. Although the output of the cochlea is reduced in proportion to the amount of IHC loss, the IC response shows only a modest amplitude reduction, and remarkably, the response of the auditory cortex is enhanced. These results suggest that the gain of the central auditory pathway can be up- or down regulated to compensate for the amount of neural activity from the cochlea.