Audiogenic Seizures in the Fmr1 Knock-Out Mouse Are Induced by Fmr1 Deletion in Subcortical, VGlut2-Expressing Excitatory Neurons and Require Deletion in the Inferior Colliculus.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. One symptom of FXS and autism is sensory hypersensitivity (also called sensory over-responsivity). Perhaps related to this, the audiogenic seizure (AGS) is arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knock-out (KO) mouse. Therefore, the AGS may be considered a mouse model of sensory hypersensitivity. Hyperactive circuits are hypothesized to underlie dysfunction in a number of brain regions in patients with FXS and Fmr1 KO mice, and the AGS may be a result of this. But the specific cell types and brain regions underlying AGSs in the Fmr1 KO are unknown. We used conditional deletion or expression of Fmr1 in different cell populations to determine whether Fmr1 deletion in those cells was sufficient or necessary, respectively, for the AGS phenotype in males. Our data indicate that Fmr1 deletion in glutamatergic neurons that express vesicular glutamate transporter 2 (VGlut2) and are located in subcortical brain regions is sufficient and necessary to cause AGSs. Furthermore, the deletion of Fmr1 in glutamatergic neurons of the inferior colliculus is necessary for AGSs. When we demonstrate necessity, we show that Fmr1 expression in either the larger population of VGlut2-expressing glutamatergic neurons or the smaller population of inferior collicular glutamatergic neurons-in an otherwise Fmr1 KO mouse-eliminates AGSs. Therefore, targeting these neuronal populations in FXS and autism may be part of a therapeutic strategy to alleviate sensory hypersensitivity.SIGNIFICANCE STATEMENT Sensory hypersensitivity in fragile X syndrome (FXS) and autism patients significantly interferes with quality of life. Audiogenic seizures (AGSs) are arguably the most robust behavioral phenotype in the FXS mouse model-the Fmr1 knockout-and may be considered a model of sensory hypersensitivity in FXS. We provide the clearest and most precise genetic evidence to date for the cell types and brain regions involved in causing AGSs in the Fmr1 knockout and, more broadly, for any mouse mutant. The expression of Fmr1 in these same cell types in an otherwise Fmr1 knockout eliminates AGSs indicating possible cellular targets for alleviating sensory hypersensitivity in FXS and other forms of autism.

Lateralization of virtual sound sources with a binaural cochlear-implant sound coding strategy inspired by the medial olivocochlear reflex.

Many users of bilateral cochlear implants (BiCIs) localize sound sources less accurately than do people with normal hearing. This may be partly due to using two independently functioning CIs with fixed compression, which distorts and/or reduces interaural level differences (ILDs). Here, we investigate the potential benefits of using binaurally coupled, dynamic compression inspired by the medial olivocochlear reflex; an approach termed "the MOC strategy" (Lopez-Poveda et al., 2016, Ear Hear 37:e138-e148). Twelve BiCI users were asked to localize wideband (125-6000 Hz) noise tokens in a virtual horizontal plane. Stimuli were processed through a standard (STD) sound processing strategy (i.e., involving two independently functioning sound processors with fixed compression) and three different implementations of the MOC strategy: one with fast (MOC1) and two with slower contralateral control of compression (MOC2 and MOC3). The MOC1 and MOC2 strategies had effectively greater inhibition in the higher than in the lower frequency channels, while the MOC3 strategy had slightly greater inhibition in the lower than in the higher frequency channels. Localization was most accurate with the MOC1 strategy, presumably because it provided the largest and less ambiguous ILDs. The angle error improved slightly from 25.3° with the STD strategy to 22.7° with the MOC1 strategy. The improvement in localization ability over the STD strategy disappeared when the contralateral control of compression was made slower, presumably because stimuli were too short (200 ms) for the slower contralateral inhibition to enhance ILDs. Results suggest that some MOC implementations hold promise for improving not only speech-in-noise intelligibility, as shown elsewhere, but also sound source lateralization.

Disruption of SorCS2 reveals differences in the regulation of stereociliary bundle formation between hair cell types in the inner ear.

Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC) in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein) locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed by SorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia) of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC) lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle.

Tetrabromobisphenol-A induces apoptotic death of auditory cells and hearing loss.

Phenolic tetrabromobisphenol-A (TBBPA) and its derivatives are commonly used flame-retardants, in spite of reported toxic effects including neurotoxicity, immunotoxicity, nephrotoxicity, and hepatotoxicity. However, the effects of TBBPA on ototoxicity have not yet been reported. In this study, we investigated the effect of TBBPA on hearing function in vivo and in vitro. Auditory Brainstem Response (ABR) threshold was markedly increased in mice after oral administration of TBBPA, indicating that TBBPA causes hearing loss. In addition, TBBPA induced the loss of both zebrafish neuromasts and hair cells in the rat cochlea in a dose-dependent manner. Mechanistically, hearing loss is largely attributed to apoptotic cell death, as TBBPA increased the expression of pro-apoptotic genes but decreased the expression of anti-apoptotic genes. We also found that TBBPA induced oxidative stress, and importantly, pretreatment with NAC, an anti-oxidant reagent, reduced TBBPA-induced reactive oxygen species (ROS) generation and partially prevented cell death. Our results show that TBBPA-mediated ROS generation induces ototoxicity and hearing loss. These findings implicate TBBPA as a potential environmental ototoxin by exerting its hazardous effects on the auditory system.

Decreased perception of high frequency sound in severe obstructive sleep apnea.

Obstructive sleep apnea (OSA) is characterized by recurrent episodes of upper airway obstruction during sleep, which is manifested by apnea or hypopnea. Decreased blood oxygen saturation, changes in heart rate, fluctuations in brain perfusion, changes in intracranial pressure, snoring and vibration are factors that may potentially affect hearing in patients with OSA. The aim of the present study was to test the hypothesis that hearing is affected in OSA. 43 males aged 34-74 years (mean 48.2) with suspected sleep-disordered breathing without other comorbidity or medication that may affect sleep or hearing were included. Nocturnal polysomnography, pure tone audiometry (PTA), transient evoked otoacoustic emissions (TEOAE) and brainstem auditory evoked potentials (BAEP) were evaluated. The severity of OSA was indicated by the number of apneas and hypopneas per hour of sleep (apnoe/hypopnoe index - AHI). OSA (AHI>/=5) was detected in 28 patients by polysomnography. Mild OSA (AHI 5-15) was confirmed in 11 patients, severe OSA (AHI>/=30) in 17 patients. Simple snoring (AHI<5) was diagnosed in 15 males. In patients suffering from severe OSA, tone audiometry demonstrated higher auditory threshold at frequencies of 4000 and 8000 Hz than in patients with AHI<15 (p<0.005). Auditory threshold values correlated with age in all groups. At a frequency of 8000 Hz, auditory threshold additionally correlated with BMI, AHI, oxygen desaturation index and decreased oxygen saturation. No differences were detected in TEOAE and BAEP between subjects with OSA and snoring. PTA and TEOAE decreased with increasing age. The present results show decreased perception of high frequency sound in severe OSA.

The brainstem efferent acoustic chiasm in pigmented and albino rats.

The present study examined whether structural peculiarities in the brain-efferent pathway to the organ of Corti may underlie functional differences in hearing between pigmented and albino individuals of the same mammalian species. Pigmented Brown-Norway rats and albino Wistar rats received unilateral injections of an aqueous solution of the retrograde neuronal tracer Fluorogold (FG) into the scala tympani of the cochlea to identify olivocochlear neurons (OCN) in the brainstem superior olivary complex. After five days, brains were perfusion-fixed and brainstem sections were cut and analyzed with respect to retrogradely labeled neurons. Intrinsic neurons of the lateral system were located exclusively in the ipsilateral lateral superior olive (LSO) in both groups. Shell neurons surrounding the LSO and in periolivary regions, which made up only 5-8% of all OCN, were more often contralaterally located in albino than in pigmented animals. A striking difference was observed in the laterality of neurons of the medial olivocochlear (MOC) system, which provided more than one third of all OCN. These neurons, located in the rostral periolivary region and in the ventral nucleus of the trapezoid body, were observed contralateral to 45% in pigmented and to 68% in albino animals. Our study, the first to compare the origin of the olivocochlear bundle in pigmented and albino rats, provides evidence for differences in the crossing pattern of the olivocochlear pathway. These were found predominantly in the MOC system providing the direct efferent innervation of cochlear outer hair cells. Our findings may account for the alterations in auditory perception observed in albino mammals including man.

Perspectivas para el tratamiento de la hipoacusia neurosensorial mediante regeneración celular del oído interno / Perspectives for the treatment of sensorineural hearing loss by cellular regeneration of the inner ear

La hipoacusia neurosensorial es un problema que se debe principalmente a la pérdida de células ciliadas cocleares, con la consecuente desaferenciación de las neuronas del ganglio espiral. En los humanos no existe regeneración celular endógena en el oído interno, ni una terapia exógena que permita la sustitución de las células dañadas. El tratamiento actual se basa en las prótesis auditivas y los implantes cocleares. Estos dispositivos presentan resultados variables entre pacientes, con limitaciones en la discriminación auditiva y una vida útil limitada. La tecnología, cada vez más avanzada, está limitada por la capacidad funcional de las neuronas restantes del ganglio espiral. Las terapias emergentes, con células madre y reprogramación celular, han desarrollado varias posibilidades para inducir la regeneración endógena o para trasplantar células madre que puedan sustituir las células dañadas y restaurar la función auditiva. El conocimiento de la biología celular y molecular del oído interno y su desarrollo embrionario permite plantear el uso de células madre inducidas como modelos in vitro de enfermedad y terapia celular sustitutiva. La investigación traslacional en la hipoacusia neurosensorial está orientada al desarrollo de una terapia celular con aplicación clínica para el tratamiento de la hipoacusia neurosensorial profunda (AU)

Sensorineural hearing loss is a caused by the loss of the cochlear hair cells with the consequent deafferentation of spiral ganglion neurons. Humans do not show endogenous cellular regeneration in the inner ear and there is no exogenous therapy that allows the replacement of the damaged hair cells. Currently, treatment is based on the use of hearing aids and cochlear implants that present different outcomes, some difficulties in auditory discrimination and a limited useful life. More advanced technology is hindered by the functional capacity of the remaining spiral ganglion neurons. The latest advances with stem cell therapy and cellular reprogramming have developed several possibilities to induce endogenous regeneration or stem cell transplantation to replace damaged inner ear hair cells and restore hearing function. With further knowledge of the cellular and molecular biology of the inner ear and its embryonic development, it will be possible to use induced stem cells as in vitro models of disease and as replacement cellular therapy. Investigation in this area is focused on generating cellular therapy with clinical use for the treatment of profound sensorineural hearing loss (AU)

The paracrine effect of mesenchymal human stem cells restored hearing in ß-tubulin induced autoimmune sensorineural hearing loss.

The aim of this study was to examine the activities of hASCs (Human Adipose tissue Derived Stem Cells) on experimental autoimmune hearing loss (EAHL) and how human stem cells regenerated mouse cochlea cells. We have restored hearing in 19 years old white female with autoimmune hearing loss with autologous adipose tissue derived stem cells and we wish to understand the mechanism of restoration of hearing in animal model. BALB/c mice underwent to develop EAHL; mice with EAHL were given hASCs intraperitoneally once a week for 6 consecutive weeks. ABR were examined over time. The helper type 1 autoreactive responses and T-reg cells were examined. H&E staining or immunostaining with APC conjugated anti-HLA-ABC antibody were conducted. The organ of Corti, stria vascularis, spira ligament and spiral ganglion in stem cell group are normal. In control group, without receiving stem cells, the organ of Corti is replaced by a single layer of cells, atrophy of stria vascularis. Systemic infusion of hASCs significantly improved hearing function and protected hair cells in established EAHL. The hASCs decreased the proliferation of antigen specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine interleukin10 in splenocytes. They also induced the generation of antigen specific CD4(+)CD25(+)Foxp3(+)T-reg cells. The experiment showed the restoration is due to the paracrine activities of human stem cells, since there are newly regenerated mice spiral ganglion cells, not human mesenchymal stem cells derived tissue given by intraperitoneally.

All Three Rows of Outer Hair Cells Are Required for Cochlear Amplification.

In the mammalian auditory system, the three rows of outer hair cells (OHCs) located in the cochlea are thought to increase the displacement amplitude of the organ of Corti. This cochlear amplification is thought to contribute to the high sensitivity, wide dynamic range, and sharp frequency selectivity of the hearing system. Recent studies have shown that traumatic stimuli, such as noise exposure and ototoxic acid, cause functional loss of OHCs in one, two, or all three rows. However, the degree of decrease in cochlear amplification caused by such functional losses remains unclear. In the present study, a finite element model of a cross section of the gerbil cochlea was constructed. Then, to determine effects of the functional losses of OHCs on the cochlear amplification, changes in the displacement amplitude of the basilar membrane (BM) due to the functional losses of OHCs were calculated. Results showed that the displacement amplitude of the BM decreases significantly when a single row of OHCs lost its function, suggesting that all three rows of OHCs are required for cochlear amplification.

Reduction in noise-induced functional loss of the cochleae in mice with pre-existing cochlear dysfunction due to genetic interference of prestin.

Various cochlear pathologies, such as acoustic trauma, ototoxicity and age-related degeneration, cause hearing loss. These pre-existing hearing losses can alter cochlear responses to subsequent acoustic overstimulation. So far, the knowledge on the impacts of pre-existing hearing loss caused by genetic alteration of cochlear genes is limited. Prestin is the motor protein expressed exclusively in outer hair cells in the mammalian cochlea. This motor protein contributes to outer hair cell motility. At present, it is not clear how the interference of prestin function affects cochlear responses to acoustic overstimulation. To address this question, a genetic model of prestin dysfunction in mice was created by inserting an internal ribosome entry site (IRES)-CreERT2-FRT-Neo-FRT cassette into the prestin locus after the stop codon. Homozygous mice exhibit a threshold elevation of auditory brainstem responses with large individual variation. These mice also display a threshold elevation and a shift of the input/output function of the distortion product otoacoustic emission, suggesting a reduction in outer hair cell function. The disruption of prestin function reduces the threshold shifts caused by exposure to a loud noise at 120 dB (sound pressure level) for 1 h. This reduction is positively correlated with the level of pre-noise cochlear dysfunction and is accompanied by a reduced change in Cdh1 expression, suggesting a reduction in molecular responses to the acoustic overstimulation. Together, these results suggest that prestin interference reduces cochlear stress responses to acoustic overstimulation.

Renexin as a rescue regimen for noise-induced hearing loss.

Renexin, a compound of cilostazol and ginkgo biloba extract, has been reported to produce neuroprotective effects through antioxidant, antiplatelet, and vasodilatory mechanisms. This study was designed to investigate the protective effects of renexin on hearing, the organ of Corti (OC), and medial olivocochlear efferents against noise-induced damage. C57BL/6 mice were exposed to 110 dB SPL white noise for 60 min and then randomly divided into three groups: high- and low-dose renexin-treated groups and noise only group. Renexin were administered for 7 days: 90 mg/kg to the low-dose, and 180 mg/kg to the high-dose groups. All mice, including the controls underwent hearing tests on postnoise day 8 and were killed for cochlear harvest. We compared the hearing thresholds and morphology of the OC and cochlear efferents across the groups. The renexin-treated groups recovered from the immediate threshold shifts in a dose-dependent manner, while the noise group showed a permanent hearing loss. The renexin-treated ears demonstrated less degeneration of the OC. The diameters of the efferent terminals labeled with α-synuclein were preserved in the high-dose renexin-treated group. In the western blot assay of the cochlear homogenates, the treated groups displayed stronger expressions of α-synuclein than the noise and control groups, which may indicate that noise-induced enhanced activity of the cochlear efferent system was protected by renexin. Our results suggest that pharmacologic treatment with renexin is hopeful to reduce or prevent noise-induced hearing loss as a rescue regimen after noise exposure.

Adiponectin deficiency exacerbates age-related hearing impairment.

Obesity-related disorders are closely associated with the development of age-related hearing impairment (ARHI). Adiponectin (APN) exerts protective effects against obesity-related conditions including endothelial dysfunction and atherosclerosis. Here, we investigated the impact of APN on ARHI. APN-knockout (APN-KO) mice developed exacerbation of hearing impairment, particularly in the high frequency range, compared with wild-type (WT) mice. Supplementation with APN prevented the hearing impairment in APN-KO mice. At 2 months of age, the cochlear blood flow and capillary density of the stria vascularis (SV) were significantly reduced in APN-KO mice as compared with WT mice. APN-KO mice also showed a significant increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the organ of Corti in the cochlea at 2 months of age. At the age of 6 months, hair cells were lost at the organ of Corti in APN-KO mice. In cultured auditory HEI-OC1 cells, APN reduced apoptotic activity under hypoxic conditions. Clinically, plasma APN levels were significantly lower in humans with ARHI. Multiple logistic regression analysis identified APN as a significant and independent predictor of ARHI. Our observations indicate that APN has an important role in preventing ARHI.

Dominant negative connexin26 mutation R75W causing severe hearing loss influences normal programmed cell death in postnatal organ of Corti.

BACKGROUND: The greater epithelial ridge (GER) is a developmental structure in the maturation of the organ of Corti. Situated near the inner hair cells of neonatal mice, the GER undergoes a wave of apoptosis after postnatal day 8 (P8). We evaluated the GER from P8 to P12 in transgenic mice that carry the R75W + mutation, a dominant-negative mutation of human gap junction protein, beta 2, 26 kDa (GJB2) (also known as connexin 26 or CX26). Cx26 facilitate intercellular communication within the mammalian auditory organ. RESULTS: In both non-transgenic (non-Tg) and R75W + mice, some GER cells exhibited apoptotic characteristics at P8. In the GER of non-Tg mice, both the total number of cells and the number of apoptotic cells decreased from P8 to P12. In contrast, apoptotic cells were still clearly evident in the GER of R75W + mice at P12. In R75W + mice, therefore, apoptosis in the GER persisted until a later stage of cochlear development. In addition, the GER of R75W + mice exhibited morphological signs of retention, which may have resulted from diminished levels of apoptosis and/or promotion of cell proliferation during embryogenesis and early postnatal stages of development. CONCLUSIONS: Here we demonstrate that Cx26 dysfunction is associated with delayed apoptosis of GER cells and GER retention. This is the first demonstration that Cx26 may regulate cell proliferation and apoptosis during development of the cochlea.

Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.

Ouabain application to the round window can selectively destroy type-I spiral ganglion cells, producing an animal model of auditory neuropathy. To assess the long-term effects of this deafferentation on synaptic organization in the organ of Corti and cochlear nucleus, and to ask whether surviving cochlear neurons show any post-injury plasticity in the adult, we quantified the peripheral and central synapses of type-I neurons at posttreatment times ranging from 1 to 3 months. Measures of normal DPOAEs and greatly reduced auditory brainstem responses (ABRs) confirmed the neuropathy phenotype. Counts of presynaptic ribbons and postsynaptic glutamate receptor patches in the inner hair cell area decreased with post-exposure time, as did counts of cochlear nerve terminals in the cochlear nucleus. Although these counts provided no evidence of new synapse formation via branching from surviving neurons, the regular appearance of ectopic neurons in the inner hair cell area suggested that neurite extension is not uncommon. Correlations between pathophysiology and histopathology showed that ABR thresholds are very insensitive to even massive neural degeneration, whereas the amplitude of ABR wave 1 is a better metric of synaptic degeneration.

A causative link between inner ear defects and long-term striatal dysfunction.

There is a high prevalence of behavioral disorders that feature hyperactivity in individuals with severe inner ear dysfunction. What remains unknown is whether inner ear dysfunction can alter the brain to promote pathological behavior. Using molecular and behavioral assessments of mice that carry null or tissue-specific mutations of Slc12a2, we found that inner ear dysfunction causes motor hyperactivity by increasing in the nucleus accumbens the levels of phosphorylated adenosine 3',5'-monophosphate response element-binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase (pERK), key mediators of neurotransmitter signaling and plasticity. Hyperactivity was remedied by local administration of the pERK inhibitor SL327. These findings reveal that a sensory impairment, such as inner ear dysfunction, can induce specific molecular changes in the brain that cause maladaptive behaviors, such as hyperactivity, that have been traditionally considered exclusively of cerebral origin.

Gingko biloba extracts protect auditory hair cells from cisplatin-induced ototoxicity by inhibiting perturbation of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) may play an important role in the hearing process. Cisplatin is an anticancer drug that causes hearing loss and Gingko biloba extracts (EGb 761) have been used as an antioxidant and enhancer for GJIC. The purpose of this study was to examine the efficiency of EGb 761 in protecting against cisplatin-induced apoptosis and disturbance of GJIC. House Ear Institute-Organ of Corti 1 auditory cells were cultured and treated with cisplatin (50 µM) and EGb (300 µg/ml) for 24h, and then analyzed by immunocytochemistry (Annexin V/propidium iodide) and Western blots. GJIC was evaluated by scrape-loading dye transfer (SLDT). Basal turn organ of Corti (oC) explants from neonatal (p3) rats were exposed to cisplatin (1-10 µM) and EGb (50-400 µg/ml). The number of intact hair cells was counted by co-labeling with phalloidin and MyoVIIa. EGb prevented cisplatin-induced apoptosis in immunostaining and decreased caspase 3 and poly-ADP-ribose polymerase bands, which were increased in cisplatin-treated cells in Western blots. EGb prevented abnormal intracellular locations of connexin (Cx) 26, 30, 31, and 43 in cells treated with cisplatin and increased quantities of Cx bands. EGb also prevented cisplatin-induced disturbance of GJIC in SLDT. In oC explants, EGb significantly prevented hair cell damage induced by cisplatin. In animal studies, EGb significantly prevented cisplatin-induced hearing loss across 16 and 32 kHz. These results show that cisplatin induces ototoxicity including hearing loss as well as down-regulation of GJIC and inhibition of Cxs in auditory cells. EGb prevents hearing loss in cisplatin-treated rats by inhibiting down-regulation of Cx expression and GJIC. The disturbance of GJIC or Cx expression may be one of the important mechanisms of cisplatin-induced ototoxicity.

Changes in the adult vertebrate auditory sensory epithelium after trauma.

Auditory hair cells transduce sound vibrations into membrane potential changes, ultimately leading to changes in neuronal firing and sound perception. This review provides an overview of the characteristics and repair capabilities of traumatized auditory sensory epithelium in the adult vertebrate ear. Injured mammalian auditory epithelium repairs itself by forming permanent scars but is unable to regenerate replacement hair cells. In contrast, injured non-mammalian vertebrate ear generates replacement hair cells to restore hearing functions. Non-sensory support cells within the auditory epithelium play key roles in the repair processes.

A novel synthetic compound, 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione, inhibits cisplatin-induced hearing loss by the suppression of reactive oxygen species: in vitro and in vivo study.

Cisplatin, a chemotherapeutic agent for treating various solid tumors, produces hearing loss in approximately half a million cancer patients annually in the United States. In the course of developing a new protective agent against cisplatin-induced ototoxicity, we have been interested in a novel synthetic compound, 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR-22332). The effect of KR-22332 on cisplatin-induced cytotoxicity was analyzed in vitro in an organ of Corti-derived cell line (HEI-OC1), and in vivo in a zebrafish and rat model. Cisplatin-induced apoptosis, reactive oxygen species (ROS) generation and altered mitochondrial membrane potential (MMP) in HEI-OC1 cells were observed. KR-22332 significantly inhibited cisplatin-induced apoptosis, change of MMP, and intracellular ROS generation. KR-22332 markedly attenuated the cisplatin-induced loss and changes of auditory neuromasts in the zebrafish. Transtympanic administration of KR-22332 in a rat model was protective against cisplatin-induced hearing loss, as determined by click-evoked auditory brainstem response (p<0.01). Tissue terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of rat cochlea demonstrated that KR-22332 blocked cisplatin-induced apoptosis. In addition, transtympanic administration of KR-22332 inhibited cisplatin-induced nicotinamide adenine dinucleotide phosphate-oxidase 3 (NOX3) overexpression in the rat cochlea. KR-22332 significantly reduced the expression of p-53, mitogen-activated protein kinases, caspase 3, and tumor necrosis factor-α compared to their significant increase after cisplatin treatment. The results of this study suggest that KR-22332 may prevent ototoxicity caused by the administration of cisplatin through the inhibition of mitochondrial dysfunction and the suppression of ROS generation. These novel findings implicate KR-22332 as a potential candidate for protective agent against cisplatin-induced ototoxicity.

Cdc42-dependent structural development of auditory supporting cells is required for wound healing at adulthood.

Cdc42 regulates the initial establishment of cytoskeletal and junctional structures, but only little is known about its role at later stages of cellular differentiation. We studied Cdc42's role in vivo in auditory supporting cells, epithelial cells with high structural complexity. Cdc42 inactivation was induced early postnatally using the Cdc42(loxP/loxP);Fgfr3-iCre-ER(T2) mice. Cdc42 depletion impaired elongation of adherens junctions and F-actin belts, leading to constriction of the sensory epithelial surface. Fragmented F-actin belts, junctions containing ectopic lumens and misexpression of a basolateral membrane protein in the apical domain were observed. These defects and changes in aPKCλ/ι expression suggested that apical polarization is impaired. Following a lesion at adulthood, supporting cells with Cdc42 loss-induced maturational defects collapsed and failed to remodel F-actin belts, a process that is critical to scar formation. Thus, Cdc42 is required for structural differentiation of auditory supporting cells and this proper maturation is necessary for wound healing in adults.

Electrophysiological monitoring of hearing function during cochlear perilymphatic perfusions.

CONCLUSION: The cochlear perilymphatic perfusion produces, by itself, significant effects in the cochlear physiology that could be associated with the surgical procedure. These effects need to be well characterized to allow a reliable quantification of the effects of the experimental agent being tested. OBJECTIVES: The study focused on the accurate description of the electrophysiological effects on the cochlear potential recordings of perilymphatic perfusions. METHODS: Two successive cochlear perilymphatic perfusions were carried out. The first used artificial perilymph. The second used artificial perilymph alone or a kainic acid (KA) solution in artificial perilymph. The compound action potential of the auditory nerve (CAP-AN) was recorded: (1) before the first perfusion, (2) after the first perfusion and (3) after the second perfusion, and compared between groups. RESULTS: The first intracochlear perfusion with artificial perilymph produced significant effects in the CAP-AN that could be related to the surgical procedure. These effects were analysed separately from the effects produced by the KA. In particular, the KA administered intracochlearly produced a significant increase in the latency and a decrease in the amplitude of the CAP-AN N1 wave compared with the controls that were perfused twice with artificial perilymph.