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Mitochondrial dysfunction with aging is associated with the development of age-related hearing loss. Mitophagy is a cardinal mechanism to maintain a healthy mitochondrial population through the turnover of damaged mitochondria. Declining mitophagy with age causes a buildup of damaged mitochondria, leading to sensory organ dysfunction. The effect of Urolithin A (UA), a mitophagy inducer, was investigated on age-related hearing loss in a mouse model. C57BL/6J mice were treated with UA from 6 to 10 months of age. UA attenuated an auditory brainstem responses (ABR) threshold shift at 8, 16, and 32 kHz frequencies, and improved mitochondrial DNA integrity and ATP production in the cochlea and auditory cortex. The mRNA levels of mitophagy-related genes and protein levels of PINK1, Parkin, BNIP3, and LC3B increased in the cochlea and auditory cortex. The expression of mitophagosomes and mitophagolysosomes in the cochlea, spiral ganglion, auditory cortex, and inferior colliculus increased, together with the expression of Parkin and BNIP3 in the cochlea, spiral ganglion, auditory cortex, and inferior colliculus. These results indicate that UA counteracted mitophagy decline in the auditory system and prevented age-related hearing loss. UA can be used as a potential agent to prevent age-related hearing loss., Competing Interests: Declaration of competing interest None of the authors has a conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cisplatin is a highly effective antitumor drug generally used in the treatment of solid malignant tumors. However, cisplatin causes severe side effects such as bone marrow depression, nephrotoxicity, and ototoxicity, thus limiting its clinical application. The incidence of ototoxicity induced by cisplatin ranges from 20% to 70%, and it usually manifests as a progressive, bilateral and irreversible hearing loss. Although the etiology of cisplatin-induced ototoxicity remains unclear, an increasing body of evidence suggests that the ototoxicity of cisplatin is mainly related to the production of reactive oxygen species and activation of apoptotic pathway in cochlear tissues. Many drugs have been well proved to protect cisplatin-induced hearing loss in vitro and in vivo. However, the anti-tumor effect of cisplatin is also weakened by systemic administration of those drugs for hearing protection, especially antioxidants. Therefore, establishing a local administration strategy contributes to the otoprotection without affecting the effect of cisplatin. This review introduces the pathology of ototoxicity caused by cisplatin, and focuses on recent developments in the mechanisms and protective strategies of cisplatin-induced ototoxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Inflammation is involved in noise-induced hearing loss (NIHL), but the mechanism is still unknown. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which triggers the inflammatory cascade, has been implicated in several inflammatory diseases in response to oxidative stress. However, whether the NLRP3 inflammasome is a key factor for permanent NIHL is still unknown. In this study, quantitative real-time polymerase chain reaction (qPCR), western blot, and enzyme-linked immunosorbent assays (ELISAs) demonstrated that the expression levels of activated caspase-1, interleukin (IL)-1β, IL-18, and NLRP3 were significantly increased in the cochleae of mice exposed to broadband noise (120 dB) for 4 h, compared with the control group. These results indicate that the activation of inflammasomes in the cochleae of mice during the pathological process of NIHL as well as NLRP3, a sensor protein of reactive oxygen species (ROS), may be key factors for inflammasome assembly and subsequent inflammation in cochleae. Moreover, many recent studies have revealed that NEK7 is an important component and regulator of NLRP3 inflammasomes by interacting with NLRP3 directly and that these interactions can be interrupted by oridonin. Here, we further determined that treatment with oridonin could indeed interrupt the interaction between NLRP3 and NEK7 as well as inhibit the downstream inflammasome activation in mouse cochleae after noise exposure. Furthermore, we tested anakinra, another inflammatory inhibitor, and it was shown to partially alleviate the degree of hearing impairment in some frequencies in an NIHL mouse model. These discoveries suggest that inhibiting NLRP3 inflammasomes and the downstream signaling pathway may provide a new strategy for the clinical treatment of NIHL., (Copyright © 2021 Elsevier B.V. All rights reserved.)

It is well-established that aminoglycoside antibiotics are ototoxic, and the toxicity can be drastically enhanced by the addition of loop diuretics, resulting in rapid irreversible hair cell damage. Using both electrophysiologic and morphological approaches, we investigated whether this combined treatment affected the cochlea at the region of ribbon synapses, consequently resulting in auditory synaptopathy. A series of varied gentamicin and furosemide doses were applied to C57BL/6 mice, and auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were measured to assess ototoxic damage within the cochlea. In brief, the treatment effectively induced cochlear damage and promoted a certain reorganization of synaptic ribbons, while a reduction of ribbon density only occurred after a substantial loss of outer hair cells. In addition, both the ABR wave I amplitude and the ribbon density were elevated in low-dose treatment conditions, but a correlation between the two events was not significant for individual cochleae. In sum, combined gentamicin and furosemide treatment, at titrated doses below those that produce hair cell damage, typically triggers synaptic plasticity rather than a permanent synaptic loss., (Published by Elsevier B.V.)

Epidemiological and experimental studies indicate that a number of aromatic solvents widely used in the industry can affect hearing and balance following chronic exposure. Animal studies demonstrated that long-term exposure to aromatic solvents directly damages the auditory receptor within the inner ear: the cochlea. However, no information is available on their effect on the vestibular receptor, which shares many structural features with the cochlea and is also localized in inner ear. The aim of this study was to use an in vitro approach to assess and compare the vestibular toxicity of different aromatic solvents (toluene, ethylbenzene, styrene and ortho-, meta-, para-xylene), all of which have well known cochleotoxic properties. We used a three-dimensional culture model of rat utricles ("cysts") with preserved functional sensory and secretory epithelia, and containing a potassium-rich (K + ) endolymph-like fluid for this study. Variations in K + concentrations in this model were considered as biomarkers of toxicity of the substances tested. After 72 h exposure, o-xylene, ethylbenzene and styrene decreased the K + concentration by 78 %, 37 % and 28 %, respectively. O- xylene and styrene both caused histopathological alterations in secretory and sensory epithelial areas after 72 h exposure, whereas no anomalies were observed in ethylbenzene-exposed samples. These in vitro results suggest that some widely used aromatic solvents might have vestibulotoxic properties (o-xylene, styrene and ethylbenzene), whereas others may not (p-xylene, m-xylene, toluene). Our results also indicate that variations in endolymphatic K + concentration may be a more sensitive marker of vestibular toxicity than histopathological events. Finally, this study suggests that cochleotoxic solvents might not be necessarily vestibulotoxic, and vice versa., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cisplatin is a platinum(II) chemotherapy drug that can cause the side-effect of ototoxicity and hearing loss. The monofunctional platinum(II) complexes, phenanthriplatin and pyriplatin, have recently been investigated as anti-cancer agents but their side-effects are largely unknown. Here, we used the auditory hybridoma cell line, HEI-OC1, to investigate the ototoxicity of cisplatin, phenanthriplatin and pyriplatin. The effect of these compounds against cellular viability, on reactive oxygen species (ROS) production, mitochondrial membrane polarization, caspase-3/7 activity, DNA integrity and caspase-12 expression were measured using spectrophotometric, flow cytometric and blot analyses. We found that the monofunctional complexes and cisplatin decreased cellular viability. All three compounds increased ROS yield at 24 h, but at 48 h, ROS levels returned to normal. Also, the compounds did not depolarize the mitochondrial membrane. All three compounds reduced caspase-3/7 activity at 24 h; cisplatin increased caspase-3/7 activity and caused apoptosis at 48 h. Caspase-12 expression was associated with all three compounds. In summary, the monofunctional complexes may cause ototoxicity like cisplatin. Phenanthriplatin and pyriplatin may cause ototoxicity initially by inducing ROS production, but they may also signal through distinct apoptotic pathways that do not integrate caspases-3/7, or may act at different time-points in the same pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Tsumura Suzuki Obese Diabetes (TSOD) mice exhibit early age-associated hearing loss. Histopathological analysis of these mice shows narrowing of capillaries in the stria vascularis and chronic reduction of blood flow in the cochlea. In this study, we investigated the effect of oral administration of a herbal medicine or calorie restriction on hearing in TSOD mice., Methods: TSOD mice were divided into 4 groups: CR (calorie restriction), BF and DS (treated with the herbal medicines, Bofutsushosan and Daisaikoto, respectively), and the control group. Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically., Results: Blood glucose levels were suppressed in the CR, BF, and DS groups. In addition, the elevation of ABR thresholds was inhibited in the CR, BF, and DS groups. Cochlear blood vessels remained wide in the three treatment groups compared with the control group. These results suggested that the administration of these herbal medicines improved glucose tolerance and yielded results similar to those on calorie restriction., Conclusion: Oral administration of 2 herbal medicines can prevent hearing function disorder in a model mouse of diabetes. The results may clarify the possibility of clinical application., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: The objective of this study was to investigate the influence of Ginkgo Biloba in early treatment of noise induced hearing loss on expression of IL-6, IL-1 Beta, TNF-alfa, HSP-70, HSF-1 and COX-2 in the rat cochlea., Methods: Thirty two female rats were randomly divided into four groups (Acoustic Trauma, Ginkgo Biloba, Acoustic Trauma+Ginkgo Biloba, Non Treatment). Auditory brainstem response (ABR) was applied in all the groups. At the end of the study, IL-1Beta, IL-6, TNF-alpha, HSP-70, HSF-1 and COX-2 were studied in cochlear tissue with ELISA and Western blot analysis., Results: There were significant increases in ABR values measured at days 1 and 7 compared to baseline values in Group 3. IL-1 Beta, IL-6 and TNF-alpha values were significantly higher in Group 1 than in the other groups. Whereas HSP-70 and HSF-1 values were found to be significantly lower in Group 1 compared to those in Group 2 and Group 3. COX-2 of Group 1 was significantly higher than the other groups., Conclusion: Ginkgo Biloba is helpful in the treatment of noise induced hearing loss and exerts its effect by inhibiting expression of IL-1 Beta, IL-6, TNF-alpha and COX-2 and increasing HSP-70 and HSF-1 values in rat cochlea., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chronic, low-grade inflammation, or inflammaging, is a crucial contributor to various age-related pathologies and natural processes in aging tissue, including the nervous system. Over the past two decades, much effort has been done to understand the mechanisms of inflammaging in disease models such as type II diabetes, cardiovascular disease, Alzheimer's disease, Parkinson's disease, and others. However, despite being the most prevalent neurodegenerative disorder, the number one communication disorder, and one of the top three chronic medical conditions of our aged population; little research has been conducted on the potential role of inflammation in age-related hearing loss (ARHL). Recently, it has been suggested that there is an inflammatory presence in the cochlea, perhaps involving diffusion processes of the blood-brain barrier as it relates to the inner ear. Recent research has found correlations between hearing loss and markers such as C-reactive protein, IL-6, and TNF-α indicating inflammatory status in human case-cohort studies. However, there have been very few reports of in vivo research investigating the role of chronic inflammation's in hearing loss in the aging cochlea. Future research directed at better understanding the mechanisms of inflammation in the cochlea as well as the natural changes acquired with aging may provide a better understanding of how this process can accelerate presbycusis. Animal model experimentation and pre-clinical studies designed to recognize and characterize cochlear inflammatory mechanisms may suggest novel treatment strategies for preventing or treating ARHL. In this review, we seek to summarize key research in chronic inflammation, discuss its implications for possible roles in ARHL, and finally suggest directions for future investigations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: The purpose of the study was to investigate the difference in the responses of endolymphatic hydrostatic pressure to isoproterenol, β-adrenergic receptor agonist, between pars superior and pars inferior., Methods: The hydrostatic pressure of endolymph and perilymph and endolymphatic potential in the ampulla and the cochlea during the intravenous administration of isoproterenol were recorded using a servo-null system in guinea pigs., Results: The hydrostatic pressure of endolymph and perilymph in the ampulla and cochlea was similar in magnitude. Isoproterenol significantly increased hydrostatic pressure of ampullar and cochlear endolymph and perilymph with no change in the ampullar endolymphatic potential and endocochlear potential, respectively. The isoproterenol-induced maximum change of endolymphatic hydrostatic pressure in ampulla was significantly (p<0.01) smaller than that in the cochlea. In ears with an obstructed endolymphatic sac, the action of isoproterenol on endolymphatic hydrostatic pressure in the ampulla disappeared like that in the cochlea., Conclusion: Isoproterenol elevates endolymphatic hydrostatic pressure in different manner between the vestibule and the cochlea., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In vivo studies are needed to study cisplatin ototoxicity and to evaluate candidate protective treatments. Rats and mice are the preferred species for toxicological and pharmacological pre-clinical research, but systemic administration of cisplatin causes high morbidity in these species. We hypothesized that trans-tympanic administration of cisplatin would provide a good model for studying its auditory and vestibular toxicity in the rat. Cisplatin was administered by the trans-tympanic route in one ear (50μl, 0.5-2mg/ml) of rats of both sexes and two different strains. Cochlear toxicity was corroborated by histological means. Vestibular toxicity was demonstrated by behavioral and histological analysis. Cisplatin concentrations were assessed in inner ear after trans-tympanic and i.v. administration. In all experiments, no lethality and only scant body weight loss were recorded. Cisplatin caused dose-dependent cochlear toxicity, as demonstrated by hair cell counts in the apical and middle turns of the cochlea, and vestibular toxicity, as demonstrated by behavioral analysis and hair cell counts in utricles. High concentrations of cisplatin were found in the inner ear after trans-tympanic administration. In comparison, i.v. administration resulted in lower inner ear concentrations. We conclude that trans-tympanic administration provides an easy, reproducible and safe model to study the cochlear and vestibular toxicity of cisplatin in the rat. This route of exposure may be useful to address particular questions on cisplatin induced ototoxicity and to test candidate protective treatments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Peroxiredoxin (Prx) is a new family of antioxidative proteins. Prx I is ubiquitously expressed in various tissues and is important in the defense of tissues from increases in reactive oxygen species (ROS). The present study was designed to examine the expression of Prx subtypes in the mouse cochlea and to show the possible involvement of Prx I in protecting the cochlea against cisplatin ototoxicity., Methods: Postnatal-day-3-to-5 wildtype mice and Prx I-deficient mice were used. Prx expression in the cochlea was assessed by real-time PCR assay. Prx I protein expression was examined by immunofluorescence staining. Cochlear explants were exposed to 2, 5, and 10-μM cisplatin for 48h, and the cochlear hair cell losses of the wildtype and Prx I-deficient mice were compared. In addition, the histologic features of the cochlear lateral wall were examined after cisplatin incubation., Results: mRNAs of all Prx subtypes were expressed in the mouse cochlea. Prx I was one of the abundant subtypes and was upregulated after 48-h exposure to 5-μM cisplatin. Immunofluorescence staining showed the ubiquitous expression of Prx I in the cochlea. No difference in cochlear hair cell loss induced by cisplatin was found between the wildtype mice and the Prx I-deficient mice. However, spiral ligament fibrocytes of Prx I-deficient mice were significantly sensitive to cisplatin at 20-μM or lower., Conclusion: Prx I is important for protection of at least the spiral ligament fibrocytes of the cochlear lateral wall in cisplatin ototoxicity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Age-related hearing loss (AHL) -presbycusis- is the number one neurodegenerative disorder and top communication deficit of our aged population. Experimental evidence suggests that mitochondrial dysfunction associated with reactive oxygen species (ROS) plays a central role in the aging process of cochlear cells. Dietary antioxidants, in particular polyphenols, have been found to be beneficial in protecting against the generation of ROS in various diseases associated with oxidative stress, such as cancer, neurodegenerative diseases and aging., Objectives: This study was designed to investigate the effects of polyphenols on AHL and to determine whether oxidative stress plays a role in the pathophysiology of AHL., Methods: Sprague-Dawley rats (n=100) were divided into five groups according to their age (3, 6, 12, 18 and 24months old) and treated with 100mg/kg/day body weight of polyphenols dissolved in tap water for half of the life of the animal. Auditory steady-state responses (ASSR) threshold shifts were measured before sacrificing the rats. Then, cochleae were harvested to measure total superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, reactive oxidative and nitrogen species levels, superoxide anions and nitrotyrosine levels., Results: Increased levels of ROS and RNS in cochlea observed with age decreases with polyphenol treatment. In addition, the activity of SOD and GPx enzymes in older rats recovered after the administration of polyphenols., Conclusion: The reduction in oxidative and nitrosative stress in the presence of polyphenols correlates with significant improvements in ASSR threshold shifts., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Ototoxic damage caused by aminoglycosides (AG) leads to the loss of cochlear hair cells (HCs). In mammals, mature cochlear HCs are unable to regenerate, and their loss results in permanent hearing deficits. Our objective was to protect the inner ear from damage after an AG challenge. The generation of reactive oxygen species (ROS), one of the earliest events in the process of AG ototoxicity, is considered to play a key role in the initiation of HC death. We examined whether Hangesha-shin-to (TJ-014), a traditional Japanese Kampo medicine considered to be a potent antioxidant, protects HCs from gentamicin (GM)-induced damage., Methods: Organ of Corti explants removed from postnatal day 3-5 rats were maintained in tissue culture and exposed to 50μM GM for up to 48h. The effects of TJ-014 on GM-induced ototoxicity were assessed by HC counts and immunohistochemistry against cleaved caspase-3, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and a probe reacting to mitochondrial function changes., Results: TJ-014 treatments significantly reduced GM-induced HC loss and immunoreactivities for cleaved caspase-3 and 8-OHdG; these effects were correlated with increasing TJ-014 concentrations. Moreover, TJ-014 protected the mitochondrial membrane potential from GM ototoxicity., Conclusion: These findings indicate the potential of TJ-014 to prevent GM-induced cochlear damage involving ROS., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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.)

Cochlear fibrosis is a common finding following cochlear implantation. Evidence suggests that cochlear fibrosis could be triggered by inflammation and epithelial-to-mesenchymal cell transition (EMT). In this study, we investigate the mechanisms of cochlear fibrosis and the risk/benefit ratio of local administration of the anti-inflammatory drug dexamethasone (DEX) and antimitotic drug aracytine (Ara-C). Cochlear fibrosis was evaluated in cochlear fibrosis models of rat cochlear slices in vitro and in KLH-induced immune labyrinthitis and platinum wire cochlear implantation-induced fibrosis in vivo. Cochleae were invaded with tissue containing fibroblastic cells expressing α-SMA (alpha smooth muscle actin), which along with collagen I, fibronectin, and laminin in the extracellular matrix, suggests the involvement of a fibrotic process triggered by EMT in vitro and in vivo. After perilymphatic injection of an adenoviral vector expressing GFP in vivo, we demonstrated that the fibroblastic cells derived from the mesothelial cells of the scalae tympani and vestibuli. Activation of inflammatory and EMT pathways was further assessed by ELISA analysis of the expression of IL-1β and TGF-β1. Both markers were elevated in vitro and in vivo, and DEX and Ara-C were able to reduce IL-1β and TGF-β1 production. After 5days of culture in vitro, quantification of calcein-positive cells revealed that Ara-C was 30-fold more efficient in preventing fibrosis, and provoked less sensory hair cell loss, than DEX. In KLH-induced immune labyrinthitis and platinum wire-implanted models, Ara-C was more efficient in preventing proliferation of fibrosis with less side effects on hair cells and neurons than DEX. In conclusion, DEX and Ara-C both prevent fibrosis in the cochlea. Analysis of the risk/benefit ratio favors the use of Ara-C for preventing cochlear fibrosis., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Gentamicin treatment induces hair cell death or survival in the inner ear. Besides the well-known toxic effects, the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway was found to be involved in cell protection. After gentamicin application, the spatiotemporal expression patterns of Akt and its activated form (p-Akt) were determined in male guinea pigs. A single dose of 0.1 mL gentamicin (4 mg/ear/animal) was intratympanically injected. The auditory brainstem responses (ABRs) were recorded prior to application and 1, 2 and 7 days afterward. At these three time points the cochleae (n=10 in each case) were removed, transferred to fixative and embedded in paraffin. Seven ears were used as untreated controls. Gentamicin, Akt and p-Akt were identified immunohistochemically in various regions of the cochlea and their staining intensities were quantified on sections using digital image analysis. The application of gentamicin resulted in hearing loss with a concomitant up-regulation of Akt-expression in the organ of Corti and spiral ganglion cells and an additional activation in spiral ganglion cells. At the level of individual ears, clear intracellular correlations were found between Akt- and p-Akt-expression in the stria vascularis and interdental cells and, to a minor extent, in the spiral ligament and the organ of Corti. Furthermore, statistical evidence for the connection between gentamicin up-take and hearing loss was detected. The increase in Akt- and p-Akt-expression in the organ of Corti and spiral ganglion cells indicates a selected response of the cochlea against gentamicin toxicity., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the peripheral auditory pathway in Parkinson's disease (PD) by using objective, quantitative and non-invasive audiological techniques, transient-evoked (TEOAE) and distortion product (DPOAE) otoacoustic emissions, in order to detect subclinical alterations of cochlear functioning and possible changes after dopaminergic stimulation., Methods: We enrolled 11 untreated de-novo PD patients and 11 age and sex-matched healthy controls. Subjects underwent a routine audiological evaluation and otoacoustic emission recordings. The patients were then slowly-titrated to a stable dose of 100 mg levodopa four times in a day. A post-treatment assessment was made in order to detect significant changes in audiological responses. Finally, possible associations between clinical data and hearing results were also evaluated., Results: At pure-tone audiometry, higher auditory threshold levels were observed in PD when compared to the controls. Moreover, DPOAE responses in PD patients were found low at almost all tested frequencies, suggesting subclinical cochlear damage. Interestingly, after dopaminergic treatment, a significant increase in DPOAE responses was detected. Notably, DPOAE dysfunction correlated with clinical severity, whereas high hearing thresholds appeared positively related with more prolonged disease duration., Conclusions: Our findings demonstrate that otoacoustic emission recording and pure-tone audiometry reveal levodopa-sensitive cochlear dysfunction and hearing loss in PD. A parallel improvement in subjective motor symptoms and DPOAE objective responses could help clinicians in monitoring therapeutic responses and dynamic changes during the course of the disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Noise-induced hearing loss depends on progressive increase of reactive oxygen species and lipoperoxidative damage in conjunction with the imbalance of antioxidant defenses. The redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in the regulation of cellular defenses against oxidative stress, including heme oxygenase-1 (HO-1) activation. In this work we describe a link between cochlear oxidative stress damage, induced by noise exposure, and the activation of the Nrf2/HO-1 pathway. In our model, noise induces superoxide production and overexpression of the lipid peroxidation marker 4-hydroxy-nonenals (4-HNE). To face the oxidative stress, the endogenous defense system is activated as well, as shown by the slight activation of superoxide dismutases (SODs). In addition, we observed the activation of the Nrf2/HO-1 pathway after noise exposure. Nrf2 appears to promote the maintenance of cellular homeostasis under stress conditions. However, in this model the endogenous antioxidant system fails to counteract noise-induced cell damage and its activation is not effective enough in preventing cochlear damage. The herb-derived phenol rosmarinic acid (RA) attenuates noise-induced hearing loss, reducing threshold shift, and promotes hair cell survival. In fact, RA enhances the endogenous antioxidant defenses, as shown by decreased superoxide production, reduced expression of 4-HNE, and up-regulation of SODs. Interestingly, RA potentiates the Nrf2/HO-1 signaling pathway, as shown by immunohistochemical and Western blot analyses. Thus, protective effects of RA are associated with the induction/activation of the Nrf2-ARE signaling pathway in addition to RA direct scavenging capability., (Copyright © 2015. Published by Elsevier Inc.)

Objective: This study was conducted to evaluate the relationship between hearing and cochlear histopathology after arginine vasopressin administration in rats., Methods: A total of 30 Wistar rats were injected with either 0.02 unit/g of arginine vasopressin or the same amount of isotonic saline solution. The initial auditory brain stem response threshold was recorded and additional measurements were made at 10, 30, 60, and 90 min after injection of arginine vasopressin or isotonic saline solution. The threshold for each timepoint was compared with the initial threshold. Histological quantitative assessment of endolymphatic hydrops in the cochlea was performed using light microscopy and assessment of the basal, intermediate, and marginal cells of the stria vascularis was performed with electron microscopy., Results: The auditory brain stem threshold 60 min after arginine vasopressin injection increased significantly in comparison with the initial threshold (P<0.05). Although the index for endolymphatic hydrops in rats administered arginine vasopressin was not different from that in controls (P>0.05), vacuoles in the intermediate cells were increased significantly in the treated rats (P<0.01)., Conclusion: Hearing impairment was detected without endolymphatic hydrops in rats administered arginine vasopressin. An increase of vacuoles in the intermediate cells may account for the hearing impairment induced by arginine vasopressin injection., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Sensorineural hearing losses (SNHLs; e.g., ototoxicant- and noise-induced hearing loss or presbycusis) are among the most frequent sensory deficits, but they lack effective drug therapies. The majority of recent therapeutic approaches focused on the trials of antioxidants and reactive oxygen species (ROS) scavengers in SNHLs. The rationale for these studies was the prominent role of disturbed redox homeostasis and the consequent ROS elevation. Although the antioxidant therapies in several animal studies seemed to be promising, clinical trials have failed to fulfill expectations. We investigated the potential of rasagiline, an FDA-approved monomanine oxidase type B inhibitor (MAO-B) inhibitor type anti-parkinsonian drug, as an otoprotectant. We showed a dose-dependent alleviation of the kanamycin-induced threshold shifts measured by auditory brainstem response (ABR) in an ototoxicant aminoglycoside antibiotic-based hearing loss model in mice. This effect proved to be statistically significant at a 6-mg/kg (s.c.) dose. The most prominent effect appeared at 16kHz, which is the hearing sensitivity optimum for mice. The neuroprotective, antiapoptotic and antioxidant effects of rasagiline in animal models, all targeting a specific mechanism of aminoglycoside injury, may explain this otoprotection. The dopaminergic neurotransmission enhancer effect of rasagiline might also contribute to the protection. Dopamine (DA), released from lateral olivocochlear (LOC) fibers, was shown to exert a protective action against excitotoxicity, a pathological factor in the aminoglycoside-induced SNHL. We have shown that rasagiline enhanced the electric stimulation-evoked release of DA from an acute mouse cochlea preparation in a dose-dependent manner. Using inhibitors of voltage-gated Na(+)-, Ca(2+) channels and DA transporters, we revealed that rasagiline potentiated the action potential-evoked release of DA by inhibiting the reuptake. The complex, multifactorial pathomechanism of SNHLs most likely requires drugs acting on multiple targets for effective therapy. Rasagiline, with its multi-target action and favorable adverse effects profile, might be a good candidate for a clinical trial testing the otoprotective indication., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.)

Ototoxicity induced by aminoglycoside antibiotics appears to occur both in hair cells (HCs) and the cochlear nerves that innervate them. Although HC loss can be easily quantified, neuronal lesions are difficult to quantify because two types of afferent dendrites and two types of efferent axons are tangled beneath the hair cells. In the present study, ototoxicity was induced by gentamicin in combination with the diuretic agent furosemide. Neuronal lesions were quantified in cochlear whole-mount preparations combined with microsections across the habenular perforate (HP) openings to achieve a clear picture of the topographic relationship between neuronal damage and HC loss. Multiple immunostaining methods were employed to differentiate the two types of afferent dendrites and two types of efferent axons. The results show that co-administration of gentamicin and furosemide resulted in a typical dynamic pattern of HC loss that spread from the basal turn to the outer hair cells to the apex and inner hair cells, depending on the dose and survival time after drug administration. Lesions of the innervation appeared to occur at two stages. At the early stage (2-4 days), the loss of labeling of the two types of afferent dendrites was more obvious than the loss of labeled efferent axons. At the late stage (2-4 weeks), the loss of labeled efferent axons was more rapid. In the high-dose gentamicin group, the loss of outer HCs was congruent with afferent dendrite loss at the early stage and efferent axon loss at the late stage. In the low-dose gentamicin group, the loss of labeling for cochlear innervation was more severe and widespread. Thus, we hypothesize that the gentamicin-induced damage to cochlear innervation occurs independently of hair cell loss., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The present study aimed to examine whether an E-prostanoid receptor 4 (EP4) agonist has superior protective effects to those of prostaglandin E1 (PGE1) in a guinea pig model of noise trauma., Methods: Drugs were locally applied on the round window membrane of guinea pig cochleae, followed by exposure of the test animals to intense noise. Protective effects mediated by an EP4 agonist were compared with those mediated by PGE1. Auditory function was monitored by measurements of the auditory brainstem response (ABR), and histological damage was assessed by immunohistochemical analysis of cochlear specimens., Results: Animals treated with an EP4 agonist exhibited significantly better hearing recovery than those pretreated with PGE1. Histologically, the numbers of remaining outer hair cells in cochleae treated with the EP4 agonist were significantly higher than in those treated with PGE1., Conclusion: The selective activation of EP4 has a stronger protective effect on cochleae against noise trauma than does the broad activation of EPs by PGE1., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

This study was designed to investigate the impact of lead (Pb(2+)) on the auditory system and its molecular mechanisms. Pb(AC)2 was administrated to male SD rats aged 21-22 d for 8 weeks at a dose of 300ppm. Male guinea pigs were also administrated with 50mg/kg Pb(AC)2 two times a week for 8 weeks. The auditory nerve-brainstem evoked responses (ABR) was recorded and the morphological changes of the outer hair cells (OHCs) were observed with Phallodin-FITC staining. In addition, the integrity of the blood-labyrinth barrier was observed by TEM and the expression of tight junction proteins (TJPs) in the cochlear stria vascularis was determined by immunofluorescence. Our results showed that Pb(2+) exposure resulted in increased ABR threshold in both rats and guinea pigs. Abnormal shapes and loss of OHCs were found in the cochlear basilar membrane following the Pb(2+) exposure. TEM study showed that the tight junctions between the endothelial cells and the border cells were lost and disrupted. Down-regulation of the occludin, ZO-1 and claudin-5 in the stria vascularis suggested that the increased permeability of the blood-labyrinth barrier may attribute to the Pb(2+)-induced decrease of TJPs' expression. Additionally, Fe(2+) supplement partly reversed the Pb(2+)-induced hearing loss and down-regulation of TJPs. Taken together, these data indicate that the disruption of blood-labyrinth barrier by down-regulating the expression of TJPs plays a role in the Pb(2+)-induced hearing loss, and Fe(2+) supplement protects the auditory system against Pb(2+)-induced toxicity and may have significant clinical implications., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Cdh23(erl/erl) mice are a novel mouse model for DFNB12 and are characterized by progressive hearing loss. In this study, erythropoietin (EPO) was given to the Cdh23(erl/erl) mice by intraperitoneal injection every other day from P7 for 7 weeks. Phosphate-buffered saline-treated or untreated Cdh23(erl/erl) mice were used as controls. Auditory-evoked brainstem response (ABR) thresholds and distortion product oto-acoustic emission (DPOAE) were measured in the mouse groups at the age of 4, 6 and 8 weeks. The results show that EPO can significantly decrease the ABR thresholds in the Cdh23(erl/erl) mice as compared with those of the untreated mice at stimulus frequencies of click, 8-, 16- and 32-kHz at three time points. Meanwhile, DPOAE amplitudes in the EPO-treated Cdh23(erl/erl) mouse group were significantly higher than those of the untreated groups at f2 frequency of 15383 Hz at the three time points. Furthermore, the mean percentage of outer hair cell loss at middle through basal turns of cochleae was significantly lower in EPO-treated Cdh23(erl/erl) mice than in the untreated mice (P<0.05). This is the first report that EPO acts as an otoprotectant in a DFNB12 mouse model with progressive hearing loss., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: To evaluate the effect and safety of intratympanic dexamethasone administration on cisplatin-induced ototoxicity in adult male guinea pigs and to assess the differences between early and late protection from this ototoxicity., Methods: Forty eight adult male guinea pigs were divided as follows: group I served as control group. Group II was subjected to intratympanic saline (subgroup IIa) or dexamethasone (subgroup IIb) injection. Group III was intraperitoneally injected with cisplatin. Groups IV and V were subjected first to intratympanic dexamethasone administration in both ears for 5 days starting 1 day and 1h - respectively - before cisplatin intraperitoneal injection., Results: Dexamethasone intratympanic injection revealed similar functional and structural results compared with control. Cisplatin intraperitoneal injection resulted in a profound cochlear functional and structural damage in group III. Non-significant otoprotection resulted from intratympanic dexamethasone administration one day before cisplatin. Intratympanic dexamethasone injection 1h before cisplatin treatment resulted in a significant preservation of the functional and structural properties of the cochlea., Conclusion: Intratympanic dexamethasone administration is a safe, easy and efficient way to protect from cisplatin ototoxicity especially when administered 1h before cisplatin treatment., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

3-Nitropropionic acid (3-NP), a mitochondrial toxin, has been reported to induce an acute cochlear damage. Korean red ginseng (KRG) is known to have protective effects from some types of hearing loss. This study aimed to observe the protective effect of KRG in an ototoxic animal model using 3-NP intratympanic injection. BALB/c mice were classified into 5 groups (n=15) and dose-dependent toxic effects after intratympanic injection with 3-NP (300-5000 mM) on the left ear were investigated to determine the appropriate toxicity level of 3-NP. For observation of the protective effects of KRG, 23 mice were grouped into 3-NP (500 mM, n=12) and KRG+3-NP groups (300 mg/kg KRG for 7 days before 500 mM 3-NP administration, n=11). Auditory brain response (ABR) and cochlear morphological evaluations were performed before and after drug administration. The ABR thresholds in the 800-5000 mM groups exceeded the maximum recording limit at 16 and 32 kHz 1 day after 3-NP administration. The ABR threshold in the 500 mM 3-NP+KRG group was significantly lower than that in the 500 mM 3-NP group from post 1 week to 1 month. The mean type II fibrocyte counts significantly differed between the control and 3-NP groups and between the 3-NP and 3-NP+KRG groups. Spiral ganglion cell degeneration in the 3-NP group was more severe than that in the 3-NP+KRG group. This animal model exhibited a dose-dependent hearing loss with histological changes. KRG administration ameliorated the deterioration of hearing by 3-NP., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Aims: Disturbances of microcirculation and hemorheological changes in the inner ear are the results of noise-induced hearing loss (NIHL). Both the disturbances of microcirculation and hemorheological changes are the etiologies of NIHL development, but they are also the results. Although previous reports that inhalation of high concentration of CO(2) may increase cochlear blood flow (CoBF), the effects of carbogen on the cochlear microcirculation and NIHL remain unclear., Methods: Changes induced by noise, carbogen and pure oxygen within the cochlear lateral wall microvasculature and in hearing thresholds were observed in guinea pigs using intravital microscopy and the auditory brainstem response. At the same time, arterial oxygen saturation and morphologic changes of cochlear hair cells were observed., Results: Carbogen inhalation increased vessel diameters and blood flow velocities. Hearing thresholds elevation in the carbogen group was smaller than those in the control and oxygen group (p <0.05). Carbogen inhalation produced a trend toward less threshold shift after noise exposure, which reached statistical significance after day 3 (p <0.01). Respiratory acidosis was not found in our study. The segmented basal membranes of Corti in three groups indicated that no losses or discorders of hair cells were found., Conclusions: Carbogen inhalation can preserve hearing in animal models after acute acoustic trauma., (Copyright © 2012 IMSS. All rights reserved.)

Objective: Heat shock transcription factor 1 (HSF1) is a master regulator of heat shock response, and also inhibits expression of inflammatory cytokines directly or indirectly. Here, we examined effects of HSF1 activation on the expression of proinflammatory cytokines in mouse cochlea after exposure to noise., Methods: Male CBA/N mice with normal Preyer's reflex were exposed to intense noise for 3h. Three hours after noise exposure, bilateral cochleae were removed and expression of major inflammatory cytokines was examined., Results: We found that interleukin-6 (IL-6) and interleukin-1β (IL-1β) expression increased significantly after noise exposure, and the expression was suppressed significantly in mice administered with geranylgeranylacetone (GGA), which activates HSF1. Seven days after noise exposure, thresholds for auditory brainstem response were elevated, and GGA administration significantly suppressed this elevation., Conclusion: These results suggest that HSF1-mediated suppression of proinflammatory cytokines in the cochlea by GGA administration could be an important means of inner ear protection., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The production of otoacoustic emissions (OAEs) by the cochlea is a sexually dimorphic trait. Although often hypothesized to be influenced by testosterone in utero, little attention has been devoted to the possibility that levels of circulating sex steroids in adulthood might modulate the sex difference in OAE production. The purpose of the current study was to investigate whether oral contraceptive (OC) use affects OAE production in women, revisiting a question originally posed by McFadden [(2000) Hearing Research 142:23-33]. Forty-five males and 50 females were tested. The women were retrospectively classified based on whether or not they were using OCs at present. Two types of OAEs were quantified: those produced spontaneously (spontaneous otoacoustic emissions or SOAEs) and those produced in response to click stimuli (click-evoked otoacoustic emissions or CEOAEs). Women currently using OCs showed a defeminized pattern of OAE production: they produced fewer SOAEs, SOAEs with significantly less power, and smaller CEOAE response amplitudes compared with naturally cycling women who were tested irrespective of phase of the menstrual cycle. It is proposed that the observed group difference may be mediated by the interaction of circulating estradiol with estrogen receptor alpha (ERα) or estrogen receptor beta (ERβ) receptors in the cochlea., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Gentamicin is an effective and powerful antibiotic. Extended use or excessive dosages of which can result in irreversible damage to the inner ear. The development of otoprotective strategies is a primary and urgent goal in research of gentamicin ototoxicity. Ginkgo biloba leaves and their extracts are among the most widely used herbal products and/or dietary supplements in the world. We investigated the protection of EGb 761 (a standardized preparation of EGb) on gentamicin ototoxicity and the involvement of reactive oxygen species (ROS) and nitric oxide (NO)-related mechanisms using in vitro organ cultures and an in vivo animal model. Gentamicin induced hair cell damage in cochlear cultures that could be prevented by EGb 761. EGb 761 also significantly reduced gentamicin-induced ROS and NO production. Furthermore, EGb 761 inhibited cellular apoptosis in cultured cochleae treated with gentamicin. In guinea pigs with gentamicin application onto the round window membrane, the mean auditory brain stem response threshold, ratio of cochlear hair cell damage and apoptosis were significantly elevated compared with those in the control group, and this could be prevented by oral administration of EGb 761. Individual EGb 761 components quercetin, bilobalide, ginkgolide A and ginkgolide B, but not kaempferol, significantly prevented gentamicin-induced hair cell damage. These results indicate that EGb 761 has a protective effect against gentamicin ototoxicity through a reduction in the formation of ROS and NO and subsequent inhibition of hair cell apoptosis in the cochlea., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The aim of the study was: (a) to test whether short duration (6 ms) 500 Hz bone-conducted vibration (BCV) of the skull in alert head free guinea pigs would elicit eye movements; (b) to test whether these eye movements were vestibular in origin; and (c) to determine whether they corresponded to human eye movements to such stimuli. In this way we sought to establish the guinea pig as an acceptable model for testing the mechanism of the effect BCV on the vestibulo-ocular reflex. Consistent short-latency stimulus-locked responses to BCV were observed. The magnitude of eye displacement was directly related to stimulus intensity as recorded by accelerometers cemented onto the animal's skull. The strongest and most consistent response component was intorsion of both eyes. In lateral-eyed animals intorsion is produced by the combined contraction of the inferior rectus and superior oblique muscles. In humans the same pair of muscles acts to cause depression of the eye. To test whether the movements were vestibular we selectively ablated the vestibular endorgans: 3 of the 8 animals underwent a bilateral intratympanic injection of gentamicin, an ototoxic aminoglycoside antibiotic, to ablate their vestibular receptors. After ablation there was an overall reduction in the magnitude of eye displacement, as well as a reduction in the effectiveness of the BCV stimulus to elicit eye movements. The animals' hearing, as measured by the threshold for auditory brainstem responses, remained unchanged after gentamicin, confirming that the cochlea was not affected. The reduced magnitude of responses after vestibular receptor ablation demonstrates that the eye-movement responses to BCV are probably caused by the stimulation of vestibular receptors, which in turn activate the vestibulo-ocular reflex., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The matrix metalloproteinases (MMPs) are a family of proteins involved in the remodelling and homeostasis of the extracellular matrix. These proteases have been well studied in the retina and the brain, marking their importance in neuronal cell survival and death [Chintala (2006) Exp Eye Res 82:5-12; Candelario-Jalil et al. (2009) Neuroscience 158:983-994]. The neuroepithelia of the eye and the inner ear share common characteristics. Therefore, we hypothesized that MMPs could play a similar role in the cochlea as described in the retina. We focused on the localization and function of MMP-2 and MMP-9 in the cochlea, by determining their expression and activity under normal conditions and after cochlear damage via aminoglycoside exposition. We examined their expression in 5-day-old Wistar rat cochleas by RT-PCR, real-time PCR, and Western blot. We used immunohistochemistry to investigate their location in the cochleas of adult C57BL/6 mice. We also determined whether or not the exposure of the organs of Corti to aminoglycosides would change MMP-2 and MMP-9 expression patterns. Western blotting identified MMP-2 and MMP-9 in neonatal spiral ganglion, stria vascularis, and to a lesser extent the organ of Corti. Neonatal mRNA expression of MMP-2 was approximately equivalent in all three tissues, while MMP-9 mRNA was highest in spiral ganglion. Immunohistochemistry showed MMP-2 primarily in adult spiral ganglion neurons and inner hair cells, while MMP-9 was found mainly in spiral ganglion neurons, inner hair cells and supporting cells. Organs of Corti treated with gentamicin for 24 h showed an upregulation of MMP-2 and MMP-9 proteins, but did not show a significant upregulation of mRNA expression 3, 6, 12, 24, and 36 h after gentamicin exposure. Inhibition of MMP activity in organs of Corti incubated with an MMP inhibitor in organotypic cultures resulted in hair cell death-suggesting that a basal level of MMP activity is required for hair cell survival., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Occupational exposure to high atmospheric levels of Mn produces a severe and debilitating disorder known as manganism characterized by extrapyramidal disturbances similar to that seen in Parkinson's disease. Epidemiological and case studies suggest that persistent exposures to Mn may have deleterious effects on other organs including the auditory system and hearing. Mn accumulates in the inner ear following acute exposure raising the possibility that it can damage the sensory hair cells that convert sound into neural activity or spiral ganglion neurons (SGN) that transmit acoustic information from the hair cells to the brain via the auditory nerve. In this paper we demonstrate for first time that Mn causes significant damage to the sensory hair cells, peripheral auditory nerve fibers (ANF) and SGN in cochlear organotypic cultures isolated from postnatal day three rats. The peripheral ANF that make synaptic contact with the sensory hair cells were particularly vulnerable to Mn toxicity; damage occurred at concentrations as low 0.01 mM and increased with dose and duration of Mn exposure. Sensory hair cells, in contrast, were slightly more resistant to Mn toxicity than the ANF. Mn induced an atypical pattern of sensory cell damage; Mn was more toxic to inner hair cells (IHC) than outer hair cells (OHC) and in addition, IHC loss was relatively uniform along the length of the cochlea. Mn also caused significant loss and shrinkage of SGN soma. These findings are the first to demonstrate that Mn can produce severe lesions to both neurons and hair cells in the postnatal inner ear., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A major challenge in the inner ear research field is to restore hearing loss of both non-genetic and genetic origin. A large effort is being made to protect hair cells from cell death after exposure to noise or drugs that can cause hearing loss. Our research focused on protecting hair cells from cell death occurring in a genetic model for human deafness. POU4F3 is a transcription factor associated with human hearing impairment. Pou4f3 knockout mice (Pou4f3(-/-)) have no cochlear hair cells, resulting in complete deafness. Although the hair cells appear to form properly, they progressively degenerate via apoptosis. In order to rescue the hair cells in the knockout mice, we produced explant cultures from mouse cochleae at an early embryonic stage and treated the cells with z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a general caspase inhibitor. Hair cell numbers in the knockout mice treated with z-VAD-fmk were significantly higher than in the untreated mice. We found that the time window that z-VAD-fmk has a protective effect is between E14.5 (P=0.001) to E16.5 (P=0.03), but not after E18.5. The source of the surviving hair cells is not due to proliferation, as measured by 5-bromo-2-deoxyuridine (BrdU) labeling, or to supporting cell transdifferentiation to hair cells, since there was no change in supporting cell numbers. Instead, the survival appears to be a direct effect of the anti-apoptotic agent on the dying hair cells with an early developmental window. These results help towards providing a comprehensive understanding of the molecular mechanisms of hair cell death, which might lead to the development of new therapeutic anti-apoptotic agents to alleviate hereditary hearing loss (HL)., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

This study evaluated the protective role of p38 mitogen-activated protein kinase (p38 MAPK) inhibitors and sequestosome 1 (Sqstm1/A170/p62), a stress-induced signal modulator, in acoustic injury of the cochlea in mice. Two weeks after the exposure of mice to acoustic stress, threshold shifts of the auditory brainstem response (ABR) from the pre-exposure level and hair cell loss were evaluated. The activation of p38 MAPK was observed in cochlea by immunostaining 4 h after acoustic stress. To examine the role of p38 MAPK in tissue injury, its inhibitors were i.p. injected into male wild-type C57BL mice before the acoustic overexposure. The inhibitors SB202190 and SB203580 but not the inactive analogue SB202474 dose-dependently decreased the auditory threshold shift and outer hair cell loss induced by acoustic overexposure, suggesting the involvement of p38 MAPK in ototoxicity. We found that acoustic overexposure induced the up-regulation of Sqstm1 mRNA expression in the cochlea of wild-type mice and that SQSTM1-deficient mice exhibited an enhanced ABR threshold shift and hair cell loss, suggesting a role of SQSTM1 in the protection of tissue from acoustic stress., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Prostaglandin E(1) is frequently used for the clinical treatment of acute sensorineural hearing loss. However, the mechanisms underlying the effects of prostaglandin E(1) on the inner ear have not yet been elucidated. The physiological effects of prostaglandin E(1) are mediated by the prostanoid receptors prostaglandin I receptor and the prostaglandin E receptor subtypes EP1, EP2, EP3, and EP4, the respective agonists for which have been purified. In the current study, we examined the efficacy of a local EP4 agonist application for the treatment of sensorineural hearing loss. We examined EP4 expression in the mouse cochlea using the reverse transcription-polymerase chain reaction and immunohistochemistry. The protective effects of local EP4 agonist treatment before or after noise exposure were tested in guinea pigs using measurements of auditory brain-stem responses and histological analysis. The results demonstrated EP4 expression in the cochlea, and showed that pre- and post-treatment with an EP4 agonist significantly attenuated threshold shifts of auditory brain stem responses, and significant attenuation in the loss of outer hair cells was found in local EP4 agonist treatment before noise exposure. These findings indicate that EP4 is involved in mechanisms for prostaglandin E(1) actions on the cochlea, and local EP4 agonist treatment could attenuate acute sensorineural hearing loss.

Objectives: The aim of this study is to investigate the salicylate-induced morphological changes of cochlear inner hair cells (IHCs) and outer hair cells (OHCs)., Methods: IHCs and OHCs were acutely isolated from the guinea-pig cochlea. Cells were observed under the inverted microscope and 10mmol/L sodium salicylate solutions or 0.01mmol/L dexamethasone-plus-salicylate solutions were applied. The cell length or the ratio between the length and width was the indices of the morphological changes in cells., Results: Isolated IHCs did not demonstrate any significant changes in sodium salicylate solutions in 20min and in 40min, whereas OHCs were shortened by the 10mmol/L sodium salicylate to 83% in 20min and 75% in 40min. There were no significant differences between in the dexamethasone-plus-salicylate solutions and in the control solutions after 20min and 40min both in IHCs and OHCs., Conclusions: Although salicylate affected the isolated OHCs from guinea-pig cochlea, IHCs were not changed morphologically by sodium salicylate applications. Dexamethasone inhibited the salicylate-induced morphological changes of OHCs.

High doses of salicylate, the anti-inflammatory component of aspirin, induce transient tinnitus and hearing loss. Systemic injection of 250 mg/kg of salicylate, a dose that reliably induces tinnitus in rats, significantly reduced the sound evoked output of the rat cochlea. Paradoxically, salicylate significantly increased the amplitude of the sound-evoked field potential from the auditory cortex (AC) of conscious rats, but not the inferior colliculus (IC). When rats were anesthetized with isoflurane, which increases GABA-mediated inhibition, the salicylate-induced AC amplitude enhancement was abolished, whereas ketamine, which blocks N-methyl-d-aspartate receptors, further increased the salicylate-induced AC amplitude enhancement. Direct application of salicylate to the cochlea, however, reduced the response amplitude of the cochlea, IC and AC, suggesting the AC amplitude enhancement induced by systemic injection of salicylate does not originate from the cochlea. To identify a behavioral correlate of the salicylate-induced AC enhancement, the acoustic startle response was measured before and after salicylate treatment. Salicylate significantly increased the amplitude of the startle response. Collectively, these results suggest that high doses of salicylate increase the gain of the central auditory system, presumably by down-regulating GABA-mediated inhibition, leading to an exaggerated acoustic startle response. The enhanced startle response may be the behavioral correlate of hyperacusis that often accompanies tinnitus and hearing loss.

Oxidative stress in the cochlea is considered to play an important role in noise-induced hearing loss. This study determined changes in superoxide dismutase (SOD), catalase, lipid peroxidation (LPO) and the auditory brainstem response (ABR) in the cochlea of C57BL/6 mice prior to and immediately, 1, 3, 7, 10, 14 and 21 days after noise exposure (4 kHz octave band at the intensity of 110 dB SPL for 4 h). A significant increase in SOD activity immediately and on 1st day after noise exposure, without a concomitant increase in catalase activity suggested a difference in the time dependent changes in the scavenging enzymes, which facilitates the increase in LPO observed on day 7. The ABR indicated significant noise-induced functional deficits which stabilized in 2 weeks with a permanent threshold shift (PTS) of 15 dB at both 4 kHz and 8 kHz. The antioxidant D-methionine (D-Met) reversed the noise-induced changes in LPO levels and enzyme activities. It also significantly reduced the PTS observed on the 14th day from 15 dB to 5 dB for 4 kHz. In summary, the findings indicate that time-dependent alterations in scavenging enzymes facilitate the production of reactive oxygen species and that D-met effectively attenuates noise-induced oxidative stress and the associated functional loss in the mouse cochlea.

Dynorphins, glutamate, and glutamate-sensitive N-Methyl-D-Aspartate (NMDA) receptors exist in the mammalian cochlea. Dynorphins produce neural excitation and excitotoxic effects in the spinal cord through a kappa-opioid facilitation of NMDA receptor-sensitivity to glutamate. The kappa-opioid receptor drug agonists N-dimethylallyl-normetazocine [(-)-pentazocine (50 mmol)] and trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide [U-50488H (100 mmol)] were administered across the cochlear round window membrane in the chinchilla. Each drug produced significant post-baseline amplitude changes in the click-evoked auditory nerve compound action potential. Amplitude changes at threshold amounted to increases in sensitivity that ranged from 4-8 decibels, measured in sound pressure level (dB SPL). The large neural amplitude increases at threshold were accompanied by progressively smaller amplitude changes at 5 and 10 dB above threshold (dB SL). However, at stimulus intensities > or =20 dB SL, post-baseline neural amplitudes were suppressed to levels below baseline and control values. These bi-phasic intensity-dependent neural amplitude changes have never before been observed following i.v. administered (-)-pentazocine in this species. Finally, the bi-phasic neural amplitude changes in U-50488H-treated (100 mmol) animals were partially blocked (except at 20 dB SL), following a round window pre-treatment with the NMDA receptor drug antagonist, dizocilpine hydrogen maleate [(+)-MK-801 (8 mmol)]. Our data suggests that endogenous dynorphins within lateral efferent olivocochlear neurons differentially modulate auditory neural excitation, possibly through cochlear NMDA receptors and glutamate. The role played by lateral efferent opioid neuromodulation at cochlear NMDA receptors, is discussed.

Aminoglycoside antibiotic derivatives such as neamine, methyl neobiosaminide B, 2-deoxystreptamine, tetra-azidoneamine, tetra-N-acetylneamine, tetra-N-carboxy-benzylneamine, tetra-N-carboxy-methylneamine and tetra-p-methoxy-benzyliminoneamine were prepared and evaluated as to their cochlear and vestibular toxicity. Methyl neobiosaminide B, the most promising derivative in the series showed selective, cochlea-dissociated vestibulotoxic activity and was considered to be a potential lead compound for the treatment of Ménière's disease. Antimicrobial properties of the compounds, qualitatively evaluated against a group of pathogenic bacteria, indicated that neomycin B sulfate, neamine as a free base and methyl-neobiosaminide B dihydrochloride show a broader range of activity when compared to the other derivatives.

Objective: Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis., Methods: We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5-60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy., Results: Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed., Conclusion: The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

Cisplatin is a widely used chemotherapeutic agent whose dose-limiting side effects include ototoxicity and nephrotoxicity. Recent evidence indicates that cisplatin induces the expression of a novel protein, kidney injury molecule-1, in the renal proximal tubular epithelium to aid in regeneration. In this study, we determined whether kidney injury molecule-1 is expressed in the cochlea and is induced by cisplatin. Using reverse transcriptase polymerase chain reaction techniques, we have now identified kidney injury molecule-1 in the rat cochlea and in three different mouse transformed hair cell lines. Administration of cisplatin to rats produced hearing loss and induced kidney injury molecule-1 mRNA in the rat cochlea. Pretreatment of rats with lipoic acid, a scavenger of reactive oxygen species, significantly reduced cisplatin-induced hearing loss and kidney injury molecule-1 expression. Cisplatin also increased the expression of cochlear NOX3 mRNA, a member of the superoxide generating NADPH oxidase family of proteins recently identified in the cochlea, inhibition of which decreased kidney injury molecule-1 expression. Polymerase chain reaction performed on different regions of the cochlea indicated the presence of kidney injury molecule-1 mRNA in the lateral wall, organ of Corti and spiral ganglion. This distribution was confirmed by immunocytochemistry. Taken together, these data identify kidney injury molecule-1 as a novel cochlear injury molecule, whose expression is regulated by reactive oxygen species generated via the NADPH oxidase pathway.

This study examined the localization and functional expression of ryanodine receptors (RyR) within the cochlea using a combination of reverse transcription-polymerase chain reaction, immunolabeling techniques, and confocal Ca2+ imaging. All three RyR isoform mRNA transcripts were detected in the adult rat cochlea. Immunoperoxidase and immunofluorescence labeling showed that the three isoforms were differentially expressed. The most pronounced RyR protein expression, involving all three isoforms, occurred in the cell bodies of the spiral ganglion neurons. RyR3 labeling extended to the synaptic terminals innervating the inner and outer hair cells. RyR2 expression also occurred in the inner hair cells and supporting cells of the organ of Corti, while cells associated with ion homeostasis in the cochlea, such as the interdental cells of the spiral limbus (RyR1), and the epithelial cells of the spiral prominence and basal cells of the stria vascularis (RyR2 and RyR3), were also immunopositive. The functionality of RyR-gated Ca2+ stores in the spiral ganglion neurons was shown by confocal calcium imaging of fluo-4 fluorescence in rat cochlear slices. Caffeine (5 mM) evoked an increase in intracellular Ca2+ concentration in the cell bodies of the spiral ganglion neurons which occurred inthe absence of external Ca2+. Ryanodine (50 nm-1 microM) evoked comparable increases in intracellular Ca2+ concentration. These findings suggest that RyR-mediated Ca2+ release may be involved in auditory neurotransmission, sound transduction, and cochlear electrochemical homeostasis.

The role of calcium- and voltage-dependent big conductance potassium channels in regulating apoptosis was investigated in cultured type I spiral ligament fibrocytes. Incubation of type I spiral ligament fibrocytes derived from gerbil cochlea with cisplatin induced dose- and time-dependent apoptosis as demonstrated by annexin V conjugated to fluorescein isothiocyanate/prodidium iodide assays. The average voltage activation threshold of whole cell current was sharply shifted to -40 mV in the cisplatin-treated cells as compared with a value of 40 mV in control cells. The average whole-cell current of cisplatin-treated cells induced by a depolarization voltage step from -80 to -10 mV was increased significantly to 1.2+/-0.4 nA as compared with 0.08+/-0.1 nA in control cells. Coincubation with tetraethylammonium and cisplatin retained the whole cell current in the normal range (0.12+/-0.2 nA). The increment of cisplatin-induced whole-cell current was inhibited (97+/-5%) by a specific calcium- and voltage-dependent big conductance potassium channel blocker iberiotoxin. Consistent with this, co-incubation with tetraethylammonium significantly attenuated cisplatin-induced apoptosis in type I spiral ligament fibrocytes by more than 50%. We conclude that the activation of BK channels is an early event associated with cisplatin-induced apoptosis in type I spiral ligament fibrocytes. These findings also point to the calcium- and voltage-dependent big conductance potassium channels as a potential pharmacological target for manipulating cisplatin ototoxicity.

Aminoglycosides are commonly used antibiotics that often induce ototoxicity leading to permanent hair cell loss and hearing impairment. We hereby examined whether minocycline protects hair cells from gentamicin-induced hair cell damage. Two millimolar gentamicin significantly induced outer hair cell damage and the addition of minocycline to gentamicin-treated explants significantly increased hair cell survival in a dose-dependent manner. Additionally, we demonstrated that gentamicin induced p38 MAPK phosphorylation, cytochrome c release, and caspase 3 activation in these cells and these remarkable changes were blocked by minocycline treatments. Furthermore, we showed that the inhibitor of p38 MAPK or the inhibitor of caspase 3 only partially blocked gentamicin-induced hair cell damage, and the pretreatment of explants with the inhibitor of p38 MAPK and the inhibitor of caspase 3 together exerted a synergic protective effect against gentamicin-induced hair cell damage. Our results suggest that minocycline blocks gentamicin-induced hair cell loss possibly by inhibition of three mechanisms: p38 MAPK phosphorylation, cytochrome c release, and caspase 3 activation. This finding may explain why minocycline has protective activity in a variety of apoptotic models. Therapeutic intervention by using minocycline or related drugs may be a novel means for preventing inner ear injury following the use of aminoglycoside.

Objective: The steroid drugs are used for the standard treatment of sudden sensorineural hearing loss. However, clinical results on the effect of glucocorticoids in acoustic trauma have not yet been understood well. The effects of glucocorticoid receptor (GR) antagonist, mifepristone, on the cochlea sensitivity loss due to short-term sound exposure were studied in the guinea pig., Methods: Mifepristone (20 mg/kg) was injected subcutaneously immediately after the noise exposure to 4 kHz pure tone of 100 or 120 dB SPL for 10 min and also at 1 day and 3 days later. Seven days after the sound exposure, the compound action potentials (CAPs) of the cochlear nerve and the 2f(1)-f(2) distortion product oto-acoustic emissions (DPOAEs) were recorded., Results: No significant CAP threshold losses were observed in either mifepristone or saline administration after the exposure at 100 dB SPL. After the exposure at 120 dB SPL, administration of mifepristone elevated the CAP threshold at 5-8 kHz significantly as compared with the saline administration. The DPOAE output shifts of both saline and mifepristone groups were similar to each other., Conclusion: Mifepristone may influence inner hair cells (IHCs) and afferent nerve fibers beneath the IHC without having influence on outer hair cells (OHCs). It is suggested that glucocorticoid plays an important role in the improvement of hearing impairment after loud sound exposure.

Objective: Although there have been numerous reports on the relationship between the period of aminoglycoside administration and cochlear damage in animals, to date there have been no such studies in humans. The purpose of this study is to observe the early and late cochlear effects of aminoglycoside administration on hair cells, spiral ganglion cells, stria vascularis, and spiral ligament., Methods: Specimens were divided into three groups. Group I included "normal" temporal bones with no histopathologic findings of otitis media and no history of otologic or ototoxic drug administration. Group II consisted of temporal bones that received aminoglycosides within 2 weeks before death and group III of temporal bones that had aminoglycosides from 2 weeks to 6 months prior to death. Patients in groups II and III received gentamycin, kanamycin or tobramycin. Temporal bones were excluded from groups II and III if patients had a history of otologic disease or other ototoxic drugs. All temporal bones were examined under light microscopy. Standard cytocochleograms and spiral ganglion cell reconstructions were done on all temporal bones. Morphometric measurements of areas of stria vascularis were made in all turns of the cochlea on mid-modiolar sections. Spiral ligament was divided into four segments according to the locations of different types of fibrocytes. The mean loss of fibrocytes in each segment was estimated., Results: The percentages of intact outer hair cells in the basal turn were significantly greater in group I compared to groups II and III. The mean area of the stria vascularis in the apical turn was significantly less in groups II and III compared to group I., Conclusion: This study demonstrates that in a short period (within 2 weeks) after aminoglycoside administration, a decrease in hair cells and in the area of the stria vascularis occurred.