Idgenes are required for morphogenesis and cellular patterning in the developing mammalian cochlea.

Inhibitor of differentiation and DNA-binding (Id) proteins, Id1 to Id4, function in the regulation of cellular proliferation and differentiation. Id proteins have been shown to interact with bHLH proteins and other proteins involved in regulating cellular proliferation and differentiation, suggesting a widespread regulatory function. Id1-3 are known to be expressed in the prosensory domain of developing cochlea. However, the roles of Id genes in cochlear development are not fully elucidated. The deficiency of any of the Id1-3 genes individually has little effect on the cochlear development, and therefore the functional redundancy among these genes have been presumed to explain the absence of phenotype. Here, we show that conditional knockout of Id1/2/3 genes (Id TKO) causes major defects in morphogenesis and cellular patterning in the development of mammalian cochlea. Id TKO cochlea was 82% shorter than control, and both decreased proliferation and increased cell death caused the hypomorph. Sox2-positive prosensory domain was formed in Id TKO cochlea, but the formation of the medial-lateral (central-peripheral) axis was disturbed; the boundary between the medial and lateral compartments in the prosensory domain was partially doubled; the number of inner hair cells per unit length increased, and the number of outer hair cells decreased. Furthermore, the lateral non-sensory compartment expressing Bmp4 and Lmo3 was missing. Thus, the patterning of the lateral epithelium was more affected than the medial epithelium. These results suggested that Id genes are crucial for morphogenesis of the cochlea duct and patterning of the lateral epithelium in the developing cochlea. Further analyses by quantitative RT-PCR and immunostaining using cochlear explants with a Bmp pathway inhibitor revealed that the Bmp-Id pathway originates from the lateral non-sensory compartment and promotes outer hair cell differentiation.

Elmod3 knockout leads to progressive hearing loss and abnormalities in cochlear hair cell stereocilia.

ELMOD3, an ARL2 GTPase-activating protein, is implicated in causing hearing impairment in humans. However, the specific role of ELMOD3 in auditory function is still far from being elucidated. In the present study, we used the CRISPR/Cas9 technology to establish an Elmod3 knockout mice line in the C57BL/6 background (hereinafter referred to as Elmod3-/- mice) and investigated the role of Elmod3 in the cochlea and auditory function. Elmod3-/- mice started to exhibit hearing loss from 2 months of age, and the deafness progressed with aging, while the vestibular function of Elmod3-/- mice was normal. We also observed that Elmod3-/- mice showed thinning and receding hair cells in the organ of Corti and much lower expression of F-actin cytoskeleton in the cochlea compared with wild-type mice. The deafness associated with the mutation may be caused by cochlear hair cells dysfunction, which manifests with shortening and fusion of inner hair cells stereocilia and progressive degeneration of outer hair cells stereocilia. Our finding associates Elmod3 deficiencies with stereocilia dysmorphologies and reveals that they might play roles in the actin cytoskeleton dynamics in cochlear hair cells, and thus relate to hearing impairment.

GSTA4 mediates reduction of cisplatin ototoxicity in female mice.

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Expression and localization of diacylglycerol kinase ζ in guinea pig cochlea and its functional implication under noise-exposure stress conditions.

Cochlear hair cells are essential for the mechanotransduction of hearing. Sensorineural hearing loss can be irreversible because hair cells have a minimal ability to repair or regenerate themselves once damaged. In order to develop therapeutic interventions to prevent hair cell loss, it is necessary to understand the signaling pathway operating in cochlear hair cells and its alteration upon damage. Diacylglycerol kinase (DGK) regulates intracellular signal transduction through phosphorylation of lipidic second messenger diacylglycerol. We have previously reported characteristic expression and localization patterns of DGKs in various organs under pathophysiological conditions. Nevertheless, little is known about morphological and functional aspects of this enzyme family in the cochlea. First RT-PCR analysis reveals predominant mRNA expression of DGKα, DGKε and DGKζ. Immunohistochemical analysis shows that DGKζ localizes to the nuclei of inner hair cells (IHCs), outer hair cells (OHCs), supporting cells and spiral ganglion neurons in guinea pig cochlea under normal conditions. It is well known that loud noise exposure induces cochlear damage, thereby resulting in hair cell loss. In particular, OHCs are highly vulnerable to noise exposure than IHCs. We found that after 1 week of noise exposure DGKζ translocates from the nucleus to the cytoplasm in damage-sensitive OHCs and gradually disappears thereafter. In sharp contrast, DGKζ remains to the nucleus in damage-resistant IHCs. These results suggest that DGKζ cytoplasmic translocation is well correlated with cellular damage under noise-exposure stress conditions and is involved in delayed cell death in cochlear outer hair cells.

Preclinical and clinical otoprotective applications of cell-penetrating peptide D-JNKI-1 (AM-111).

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.

Effects of surgical lesions on choline acetyltransferase activity in the cat cochlea.

Although it is well established that the choline acetyltransferase (ChAT, the enzyme for acetylcholine synthesis) in the mammalian cochlea is associated with its olivocochlear innervation, the distribution of this innervation in the cochlea varies somewhat among mammalian species. The quantitative distribution of ChAT activity in the cochlea has been reported for guinea pigs and rats. The present study reports the distribution of ChAT activity within the organ of Corti among the three turns of the cat cochlea and the effects of removing olivocochlear innervation either by a lateral cut aimed to totally transect the left olivocochlear bundle or a more medial cut additionally damaging the superior olivary complex on the same side. Similarly to results for guinea pig and rat, the distribution of ChAT activity in the cat outer hair cell region showed a decrease from base to apex, but, unlike in the guinea pig and rat, the cat inner hair cell region did not. As in the rat, little ChAT activity was measured in the outer supporting cell region. As previously reported for whole cat cochlea and for rat cochlear regions, transection of the olivocochlear bundle resulted in almost total loss of ChAT activity in the hair cell regions of the cat cochlea. Lesions of the superior olivary complex resulted in loss of ChAT activity in the inner hair cell region of all cochlear turns only on the lesion side but bilateral losses in the outer hair cell region of all turns. The results are consistent with previous evidence that virtually all cholinergic synapses in the mammalian cochlea are associated with its olivocochlear innervation, that the olivocochlear innervation to the inner hair cell region is predominantly ipsilateral, and that the olivocochlear innervation to the outer hair cells is bilateral.

Moderate O3/O2 therapy enhances enzymatic and non-enzymatic antioxidant in brain and cochlear that protects noise-induced hearing loss.

Mitochondrial damage and oxidative stress are known to contribute to the pathogenesis of noise-induced hearing loss (NIHL). In this study, we examined the protective effect of O2/O3 mixture (ozone/oxygen) therapy against mitochondrial induced damage and oxidative stress by noise exposure in rat brain and cochlear. For this purpose, rats were divided into four groups: 1 - control group; 2 - noise-exposed group (100 dB); 3 - noise + O2/O3, and 4 - O2/O3 (30 µg/ml). After 14 d, animals were anesthetised. Rat brain and cochlear tissue were removed for evaluation of the histopathological damages, oxidative stress, and mitochondrial dysfunction in both tissues. Our findings indicated that noise caused pathological damage, oxidative stress, and mitochondrial dysfunction in rat brain and cochlear. Also, daily administration of an O2/O3 therapy (30 µg/ml intravenous) efficiently increased enzymatic and non-enzymatic antioxidant in brain and cochlear that this action led to inhibition of pathological damages, oxidative stress, reactive oxygen species formation, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release resulting from noise. These findings suggest that the moderate O2/O3 therapy enhances the capacity of enzymatic and non-enzymatic antioxidant in brain and cochlear that protects against NIHL.

Effects of obesity on protein kinase C, brain creatine kinase, transcription, and autophagy in cochlea.

Diet-induced obesity (DIO) has been shown to exacerbate hearing degeneration via increased hypoxia, inflammatory responses, and cell loss via both caspase-dependent and caspase-independent apoptosis signaling pathways. This study aimed to investigate the effects of DIO on the mRNA expressions of protein kinase c-ß (PKC-ß), brain creatine kinase (CKB), transcription modification genes, and autophagy-related genes in the cochlea of CD/1 mice. Sixteen 4-week-old male CD/1 mice were randomly divided into 2 groups. For 16 weeks, the DIO group was fed a high fat diet (60% kcal fat) and the controls were fed a standard diet. Morphometry, biochemistry, auditory brainstem response thresholds, omental fat, and histopathology of the cochlea were compared. Results showed that body weight, body length, body-mass index, omental fat, plasma triglyceride, and auditory brainstem response thresholds were significantly elevated in the DIO group compared with those of the control group. The ratio of vessel wall thickness to radius in the stria vascularis was significantly higher in the DIO group. The cell densities in the spiral ganglion, but not in the spiral prominence, of the cochlea were significantly lower in the DIO group. The expression of histone deacetylation gene 1 (HDAC1) was significantly higher in the DIO group than the control group. However, the expressions of PKC-ß, CKB, HDAC3, histone acetyltransferase gene (P300), lysosome-associated membrane protein 2 (Lamp2), and light chain 3 (Lc3) genes were not significantly different between two groups. These results suggest that DIO might exacerbate hearing degeneration possibly via increased HDAC1 gene expression in the cochlea of CD/1 mice.

Clinical and molecular findings in three Moroccan families with distal renal tubular acidosis and deafness: Report of a novel mutation of ATP6V1B1 gene.

BACKGROUND: Primary distal renal tubular acidosis (dRTA) is a rare genetic condition characterized by an impaired acid excretion by the intercalated cells in the renal collecting duct. Recessive forms of this disease are caused by mutations in tow major genes: ATP6V1B1 and ATP6V0A4. Causal mutations in ATP6V1B1 gene are classically associated with early sensorineural hearing loss, however cases of tubular acidosis with early deafness have also been described in patients with mutations in the ATP6V0A4 gene. METHODS: The phenotype and genotype of three Moroccan consanguineous families with dRTA and deafness were assessed. Molecular analysis was performed by PCR amplification and direct sequencing of exon 12 of ATP6V1B1 gene. RESULTS: A novel c.1169dupC frameshift mutation of ATP6V1B1 gene was identified in one family and the c.1155dupC North African mutation in the tow other families. DISCUSSION AND CONCLUSION: In this report, we propose first line genetic testing based on screening of these two mutations both located in exon 12 of ATP6V1B1 gene in Moroccan patients with recessive form of dRTA associated to precocious hearing loss. Molecular diagnosis of dRTA leads to appropriate treatment and prevention of renal failure in affected individuals and to provide genetic counseling for families at risk.

Effect of transtympanic betamethasone delivery to the inner ear.

To investigate the effect of transtympanic betamethasone administration on hearing function with histologic correlation, rats were divided into three transtympanic treatment groups: isotonic saline (group I, n = 10), gentamicin (group II, n = 10) and betamethasone (group III, n = 10). Distortion product otoacoustic emission thresholds were compared on day 10. Also histological effects on cellular apoptosis in both the inner and outer hair cells in organ of Corti and spiral ganglion neurons were evaluated. Distortion product otoacoustic emission thresholds were comparable (p > 0.05) between group I and group III in all measurements. Distortion product otoacoustic emission thresholds of group II were significantly elevated in all measurements when compared with group I (p < 0.05) and group III (p < 0.05). In the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL), Caspase-3, Caspase-8 and Caspase-9 staining method the amount of apoptotic cells in group II were significantly elevated in all measurements compared with group I (p < 0.05). In the TUNEL staining method the amount of apoptotic cells in Group III were significantly elevated compared with group I in both the organ of Corti and spiral ganglion neurons (p < 0.05). The overall histological results revealed that the severity of cellular apoptosis caused by betamethasone was somewhere between isotonic saline and gentamicin. Transtympanic betamethasone does not affect inner ear function as measured by distortion product otoacoustic emission responses, but some increase in cellular apoptosis in the organ of Corti and spiral ganglion neurons was observed. These findings suggest that transtympanic betamethasone may have mild ototoxic effects. Further studies are needed to obtain precise results for transtympanic application of betamethasone.

Decreased Poly(ADP-Ribose) Polymerase 1 Expression Attenuates Glucose Oxidase-Induced Damage in Rat Cochlear Marginal Strial Cells.

Oxidative damage to the inner ear is responsible for several types of sensorineural deafness. Cochlear stria marginal cells (MCs) are thought to be vulnerable to such oxidative stress. Activated poly(ADP-ribose) polymerase 1 (PARP1) has been implicated in several diseases, but the effect of PARP1 on MCs subjected to oxidative stress remains elusive. In this study, we established an in vitro cellular oxidative stress model using glucose oxidase (GO) and attempted to explore the role that PARP1 plays in the oxidative damage of MCs. In this study, PARP1 and poly-ADP-ribose (PAR) were highly expressed in GO-treated MCs, and this was accompanied by loss of MC viability, excessive generation of reactive oxygen species (ROS), collapse of mitochondria membrane potential (ΔΨm), and redistribution of the mitochondrial downstream pathway-related molecules Bax and cytochrome c, eventually causing MC death. These effects were almost completely counteracted by suppressing PARP1 expression with small interfering RNA (siRNA). We also found that caspase-3 activation was a downstream event of PARP activation and that apoptosis of MCs was suppressed, although not completely, by pretreatment with the pan-caspase inhibitor z-VAD-fmk. The suppression was less than that when PARP1 expression was inhibited. We conclude that GO treatment induces activation of PARP1, which causes MC damage via mitochondrial mediation. PARP1 plays a pivotal role in GO-induced MC death, at least in part, via the caspase-3 cascade. Our study might provide a new cellular and molecular approach for the treatment of oxidative stress-related sensorineural deafness.

Rosmarinic acid up-regulates the noise-activated Nrf2/HO-1 pathway and protects against noise-induced injury in rat cochlea.

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.

Identification of multiple metabolic enzymes from mice cochleae tissue using a novel functional proteomics technology.

A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.

Protection from noise-induced hearing loss with Src inhibitors.

Noise-induced hearing loss is a major cause of acquired hearing loss around the world and pharmacological approaches to protecting the ear from noise are under investigation. Noise results in a combination of mechanical and metabolic damage pathways in the cochlea. The Src family of protein tyrosine kinases could be active in both pathways and Src inhibitors have successfully prevented noise-induced cochlear damage and hearing loss in animal models. The long-term goal is to optimize delivery methods into the cochlea to reduce invasiveness and limit side-effects before human clinical testing can be considered. At their current early stage of research investigation, Src inhibitors represent an exciting class of compounds for inclusion in a multifaceted pharmacological approach to protecting the ear from noise.

[Age-related expression of plasma membrane Ca(2+) -ATPase isoform 2 in the cochleas of C57BL/6J mice].

OBJECTIVE: To investigate the location and distribution of plasma membrane Ca²âº -ATPase isoform 2(PMCA2) in the cochleas of C57BL/6J mice at various ages (4w, 14w, 22w, 45w), and to reveal the relationship of PMCA2 and age-related hearing loss (AHL). METHODS: The distribution of PMCA2 in the cochleas of C57BL/6J mice was detected by immunohistochemistry at various ages (4w, 14w, 22w, 45w). Real-time polymerase chain reaction (Rt-PCR) was used to detect the level of PMCA2 mRNA in the cochleas of C57BL/6J mice at the ages of 4, 14, 22 and 45 weeks old respectively. Using SPSS17.0 software for statistical analysis. RESULTS: PMCA2 was mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 was also observed in spiral ligament. Hair cells missed and the number of spiral ganglion cells reduced with age. Expression of PMCA2 in the cochleas of C57BL/6J mice also showed age-related decreasing. The results of Rt-PCR demonstrated the expression of mRNA of gene (Atp2b2) at 14 weeks age was significantly less than 4 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 22 weeks age was significantly less than 14 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 45 weeks age was significantly less than 14 week-old mice cochlears (P<0.01). CONCLUSIONS: PMCA2 is mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 is also observed in spiral ligament. The expression of PMCA2 demonstrates an age-related decrease with age. The mRNA expression level of PMCA2 gene(Atp2b2) in the cochleas of C57BL/6J mice displayed an age-related decrease. PMCA2 transporters may play a critical role in maintaining the normal morphology of the inner ear and it may be related to AHL.

[The changes of carbonic anhydrase activity and its mRNA expression in rats cochlea after noise exposure].

OBJECTIVE: To investigate the changes of carbonic anhydrase (CA) activity and its mRNA expression in rats cochlea after noise exposure. METHOD: Twenty-four healthy Sprague Dawley rats were randomly allocated into 4 groups including 1 control group without contact of noise and 3 experimental groups exposed to a narrowband white noise of 4 kHz, 110 dB SPL 4h/d for 1 day, 1 week and 3 weeks, respectively. ABR thresholds were tested for each group at the time points described above. An immunohistochemical method was used to detect the expression of CA in the cochlea tissue. Differences in CAII mRNA expressions with and without noise exposure were examined using RT-PCR. RESULT: The thresholds of ABR in noise exposed cochlea were increased compared with the control cochlea (P < 0.01). CA activity and the CAII mRNA expression in noise exposed cochlea were decreased compared with the control cochlea (P < 0.01). The increase of ABR thresholds and decreases of CA activity and the CAII mRNA expression showed a time dependent pattern as the extension of duration exposed to noise. CONCLUSION: Noise exposure could downregulate the CA activity and CAII mRNA expression in the cochlea. CA was proably involved in the pathogenesis of noise-induced hearing loss.

The protective effect of Salvia miltiorrhiza on gentamicin-induced ototoxicity.

OBJECTIVE: The clinical use of aminoglycoside antibiotics is limited in most countries because of auditory toxicity side effects. However, their use is common in developing countries because they are inexpensive and convenient. Salvia miltiorrhiza extracts are used clinically in China for their antioxidant properties. We investigated the effect of a clinically approved injectable S. miltiorrhiza solution on inducible nitric oxide synthase (iNOS) generation induced by the aminoglycoside antibiotic gentamicin and an ototoxicity protective mechanism. METHODS: Sixty adult guinea pigs were used in this study and divided into four groups. Auditory brainstem response (ABR) testing was performed before and after treatments and animals were sacrificed for morphological and immunostaining assays after determining threshold shifts in ABR. The cochleae were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to observe ultrastructural changes. In addition, hair cell loss, iNOS and caspase-3 expression, and apoptosis were measured. RESULTS: The result showed that hearing loss, iNOS overexpression accompanied with disorganization in the cochlea, and terminal deoxynucleotidyl transferase- mediated dUTP- biotin nick end labeling (TUNEL)-stained positive cells in animals treated with gentamicin. However, pretreatment with S. miltiorrhiza (3g/kg/day for 10 days) decreased gentamicin-induced hearing loss, attenuated iNOS and caspase-3 expression, and decreased the number of apoptotic cells. Furthermore, it also reduced the ultrastructural damage due to ototoxicity as observed by SEM and TEM. CONCLUSIONS: These findings indicate that S. miltiorrhiza protects against gentamicin-induced ototoxicity and could apply to the protection of ototoxicity.

Addition of exogenous NAD+ prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.

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.

Noise-induced changes in expression levels of NADPH oxidases in the cochlea.

UNLABELLED: NADPH oxidases are enzymes that transport electrons across the plasma membrane and generate superoxide radical from molecular oxygen. The current study investigated the expression and distribution of NOX/DUOX members of the NADPH oxidase family (NOX1-5 and DUOX1-2) in the rat cochlea and their regulation in response to noise. Wistar rats (8-10 weeks) were exposed for 24 h to band noise (8-12 kHz) at moderate (100 dB) or traumatic (110 dB) sound pressure levels (SPL). Animals exposed to ambient noise (45-55 dB SPL) served as controls. Immunohistochemistry demonstrated predominant expression of all NOX/DUOX isoforms in the sensory and supporting cells of the organ of Corti, with very limited immunoexpression in the lateral wall tissues and spiral ganglion neurons. Noise exposure induced up-regulation of NOX1 and DUOX2 in the cochlea, whereas NOX3 was down-regulated. A significant reduction in the intensity of NOX3 immunolabeling was observed in the inner sulcus region of the cochlea after exposure to noise. Post-exposure inhibition of NADPH oxidases by Diphenyleneiodonium (DPI), a broadly selective NADPH oxidase inhibitor, mitigated noise-induced hearing loss. CONCLUSION: Noise-induced up-regulation of NOX1 and DUOX2 could be linked to cochlear injury. In contrast, down-regulation of NOX3 may represent an endogenous protective mechanism to reduce oxidative stress in the noise-exposed cochlea. Inhibition of NADPH oxidases is potentially a novel pathway for therapeutic management of noise-induced hearing loss.

Reduction in impulse noise-induced permanent threshold shift with intracochlear application of an NADPH oxidase inhibitor.

BACKGROUND: Toxic levels of reactive oxygen species are key contributors to the lesion of dead outer hair cells (OHCs) seen in the cochlea after noise exposure. The current study follows previous work in which paraquat was used to demonstrate that NADPH oxidase is active in the cochlea and can contribute to cochlear reactive oxygen species formation and hair cell loss. PURPOSE: The current study was undertaken to test whether pharmacological blockade of NADPH oxidase in the cochlea would lead to reduced noise-induced hearing loss and OHC death. STUDY SAMPLE: A total of 18 chinchillas (36 ears) were assessed in the study. INTERVENTION: AEBSF (4-[2-aminoethyl]benzenesulfonyl fluoride), an inhibitor of NADPH oxidase activation, was dissolved in distilled water and delivered into the cochlea via diffusion across the round window membrane. The contralateral ears received distilled water as a vehicle control. Following treatment, chinchillas were exposed to one of two noises: a 4 kHz octave band noise at 106 dB SPL for 6 hr or an impulse noise that consisted of 75 pairs of 155 dB pSPL impulses. DATA COLLECTION AND ANALYSIS: Pre- and post-noise exposure, thresholds of the auditory brainstem response at 2-8 kHz were measured. Postmortem OHC counts were conducted at the conclusion of the study. Two- and three-factor ANOVAs were used for statistical analysis of the OHC losses and ABR threshold shifts induced by the noise exposures. RESULTS: Permanent threshold shift from the impulse noise was reduced in the ears treated with the NADPH oxidase inhibitor, but no differences were found in the groups exposed to the continuous noise. OHC losses were not statistically different between the treated and untreated ears for either noise exposure. CONCLUSIONS: The results suggest that NADPH oxidase-mediated superoxide has a role in cochlear damage from impulse noise, and pharmacologic inhibition of NADPH oxidase can reduce cochlear susceptibility to noise damage. The lack of protection from the longer-duration continuous noise can be attributed to a number of possibilities related to dose level and delivery schedule.