Radixin modulates the function of outer hair cell stereocilia.

The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.

Differential Effects of Low- and High-Dose Dexamethasone on Electrically Induced Damage of the Cultured Organ of Corti.

An increased number of patients with residual hearing are undergoing cochlear implantation. A subset of these experience delayed hearing loss post-implantation, and the aetiology of this loss is not well understood. Our previous studies suggest that electrical stimulation can induce damage to hair cells in organ of Corti (OC) organotypic cultures. Dexamethasone has the potential to protect residual hearing due to its multiple effects on cells and tissue (e.g., anti-inflammatory, free radical scavenger). We therefore hypothesized that dexamethasone treatment could prevent electrical stimulation induced changes in the OC. Organ of Corti explants from neonatal rats (P2-4) were cultured for 24 h with two different concentrations of dexamethasone. Thereafter, OC were subjected to a charge-balanced biphasic pulsed electrical stimulation (0.44-2 mA) for a further 24 h. Unstimulated dexamethasone-treated OC served as controls. Outcome analysis included immunohistochemical labelling of ribbon synapses, histochemical analysis of free reactive oxygen species and morphological analysis of stereocilia bundles. Overall, the protective effects of dexamethasone on electrically induced damage in cochlear explants were moderate. High-dose dexamethasone protected bundle integrity at higher current levels. Low-dose dexamethasone tended to increase ribbon density in the apical region.

Protective effect of N-acetylcysteine against cisplatin ototoxicity in rats: a study with hearing tests and scanning electron microscopy / Efeito protetor da N-acetilcisteína na ototoxicidade por cisplatina em ratos: um estudo com testes auditivos e microscopia eletrônica de varredura

Abstract Introduction Ototoxicity is a health problem appearing after powerful treatments in serious health conditions. It is sometimes inevitable when treatment of the serious disease is required. Cisplatin is an antineoplastic agent which was investigated previously to reveal increased nitrogen and reactive oxygen radicals that damages hair cells, resulting in ototoxicity. N-acetylcysteine, previously shown to decrease ototoxicity caused by different agents, is known to be a powerful in vitro antioxidant. Probably N-acetylcysteine, in addition to its antioxidant effect, blocks a cascade where reactive oxygen species result in apoptosis in the cochlea. Objectives The possible preventive effect of N-acetylcysteine in cisplatin ototoxicity was studied with auditory brain stem responses, otoacoustic emissions, and histopathological investigation of the cochlea in a scanning electron microscopy. Methods This study was conducted on 21 Wistar Albino rats in four groups. 1 mL/kg/day three times in total intraperitoneal (i.p.) Saline (n = 5), 500 mg/kg/day i.p. three times in total N-acetylcysteine (n = 5), i.p. 15 mg/kg cisplatin alone (single dose) (n = 5) and i.p. 15 mg/kg cisplatin plus 500 mg/kg/day N-acetylcysteine (n = 6) were administered. The rats were anesthetized to study the hearing tests before and after the experiment. The rats were sacrificed to investigate the cochleas by scanning electron microscopy. Results Auditory brain stem responses and otoacoustic emissions values were attenuated in the cisplatin group. The group that received N-acetylcysteine in addition to cisplatin had better auditory brain stem responses thresholds and otoacoustic emissions. The samples obtained from the cisplatin group showed surface irregularities, degeneration areas, and total or partial severe stereocilia losses. The changes were milder in the cisplatin + N-acetylcysteine group. Conclusion Cisplatin ototoxicity can be detected by auditory brain stem responses and otoacoustic emissions testing in rats. N-acetylcysteine may protect the cochlear cells from histopathological changes. We concluded that N-acetylcysteine given 4 h after cisplatin injection has a potential otoprotective effect against cisplatin ototoxicity. which suggests it could be used in clinical trials.

Resumo Introdução A ototoxicidade é um problema que pode ocorrer após certos tipos de tratamentos para condições graves de saúde. Às vezes é inevitável quando o tratamento da doença é necessário. A cisplatina é um agente antineoplásico cujo uso em pesquisas anteriores demonstrou aumentar os radicais livres de nitrogênio e espécies reativas de oxigênio que danificam as células ciliadas e resultam em ototoxicidade. Por outro lado, a N-acetilcisteína, que já demonstrou diminuir a ototoxicidade causada por diferentes agentes, é conhecida por ser um potente antioxidante in vitro. Provavelmente a N-acetilcisteína, além de seu efeito antioxidante, bloqueia uma cascata onde espécies reativas de oxigênio resultam em apoptose na cóclea. Objetivos Estudar o possível efeito preventivo da N-acetilcisteína na ototoxicidade por cisplatina por meio de potencial evocado auditivo de tronco encefálico, emissões otoacústicas e investigação histopatológica da cóclea por microscopia eletrônica de varredura. Método Este estudo foi realizado em 21 ratos albinos Wistar, separados em quatro grupos. Foram administrados: 1 mL/kg/dia intraperitoneal (i.p.) de solução salina (n = 5), três vezes no total; 500 mg/kg/dia i.p. de N-acetilcisteína (n = 5), três vezes no total; 15 mg/kg i.p. (dose única) somente de cisplatina (n = 5) e 15 mg/kg i.p. de cisplatina e 500 mg/kg/dia i.p. de N-acetilcisteína (n = 6). Os ratos foram anestesiados para estudo dos testes auditivos antes e depois do experimento. Os ratos foram sacrificados para investigação da cóclea por microscopia eletrônica de varredura. Resultados Os potenciais evocados auditivos de tronco encefálico e os valores das emissões otoacústicas estavam atenuados no grupo cisplatina. O grupo que recebeu N-acetilcisteína além da cisplatina apresentou melhores limiares de respostas auditivas do tronco encefálico e emissões otoacústicas. As amostras obtidas do grupo cisplatina apresentaram irregularidades de superfície, áreas de degeneração, com perdas graves totais ou parciais de estereocílios. As alterações foram mais leves no grupo cisplatina + N-acetilcisteína. Conclusão A ototoxicidade por cisplatina pode ser detectada por meio de potenciais evocados auditivos de tronco encefálico e pelo teste de emissões otoacústicas em ratos. A N-acetilcisteína pode proteger as células cocleares contra alterações histopatológicas. Concluímos que a N-acetilcisteína administrada 4 horas após a injeção de cisplatina tem potencial efeito otoprotetor contra a ototoxicidade por cisplatina e pode ser utilizada em ensaios clínicos.

Protective effect of N-acetylcysteine against cisplatin ototoxicity in rats: a study with hearing tests and scanning electron microscopy.

INTRODUCTION: Ototoxicity is a health problem appearing after powerful treatments in serious health conditions. It is sometimes inevitable when treatment of the serious disease is required. Cisplatin is an antineoplastic agent which was investigated previously to reveal increased nitrogen and reactive oxygen radicals that damages hair cells, resulting in ototoxicity. N-acetylcysteine, previously shown to decrease ototoxicity caused by different agents, is known to be a powerful in vitro antioxidant. Probably N-acetylcysteine, in addition to its antioxidant effect, blocks a cascade where reactive oxygen species result in apoptosis in the cochlea. OBJECTIVES: The possible preventive effect of N-acetylcysteine in cisplatin ototoxicity was studied with auditory brain stem responses, otoacoustic emissions, and histopathological investigation of the cochlea in a scanning electron microscopy. METHODS: This study was conducted on 21 Wistar Albino rats in four groups. 1mL/kg/day three times in total intraperitoneal (i.p.) Saline (n=5), 500mg/kg/day i.p. three times in total N-acetylcysteine (n=5), i.p. 15mg/kg cisplatin alone (single dose) (n=5) and i.p. 15mg/kg cisplatin plus 500mg/kg/day N-acetylcysteine (n=6) were administered. The rats were anesthetized to study the hearing tests before and after the experiment. The rats were sacrificed to investigate the cochleas by scanning electron microscopy. RESULTS: Auditory brain stem responses and otoacoustic emissions values were attenuated in the cisplatin group. The group that received N-acetylcysteine in addition to cisplatin had better auditory brain stem responses thresholds and otoacoustic emissions. The samples obtained from the cisplatin group showed surface irregularities, degeneration areas, and total or partial severe stereocilia losses. The changes were milder in the cisplatin+N-acetylcysteine group. CONCLUSION: Cisplatin ototoxicity can be detected by auditory brain stem responses and otoacoustic emissions testing in rats. N-acetylcysteine may protect the cochlear cells from histopathological changes. We concluded that N-acetylcysteine given 4h after cisplatin injection has a potential otoprotective effect against cisplatin ototoxicity. which suggests it could be used in clinical trials.

A Model for Link Pruning to Establish Correctly Polarized and Oriented Tip Links in Hair Bundles.

Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious. In particular, it is unclear how tip links are strung between stereocilia so that they are oriented parallel to a single axis; why their polarity is uniform despite their constituent molecules' intrinsic asymmetry; and why only a single tip link is present at each tip-link position. We present here a series of simple rules that reasonably explain why these phenomena occur. In particular, our model relies on each of the two ends of the tip link having distinct Ca2+-dependent stability and being connected to different motor complexes. A simulation employing these rules allowed us to explore the parameter space for the model, demonstrating the importance of the feedback between transduction channels and angled links, links that are 60° off-axis with respect to mature tip links. We tested this key aspect of the model by examining angled links in chick cochlea hair cells. As implied by the assumptions used to generate the model, we found that angled links were stabilized if there was no tip link at the tip of the upper stereocilium, and appeared when transduction channels were blocked. The model thus plausibly explains how tip-link formation and pruning can occur.

Potential protective effect of N-acetyl cysteine in acoustic trauma: An experimental study using scanning electron microscopy.

BACKGROUND: Oxidative stress has been associated with pathological processes involved in acoustic trauma. OBJECTIVES: In this prospective experimental study, we investigated the potential preventive effect of N-acetyl cysteine (NAC) in rats exposed to acoustic trauma (AT). Light microscopic and scanning electron microscopic (SEM) evaluations were performed. MATERIAL AND METHODS: Healthy Wistar albino rats (n = 18) were divided into 3 groups: group 1 (control group, n = 6), group 2 (acoustic trauma group, n = 6), and group 3 (AT+NAC group, n = 6). The rats in group 2 were exposed to AT. The rats in group 3 received NAC at a dose of 100 mg/kg/day by gavage for 7 days, and then 10 min after the 7th-day dose, they were exposed to AT. RESULTS: From light and scanning electron microscopy evaluations in the control group, the cochlear structure and epithelium were normal. In group 2 (AT group), extensive hair cell loss was observed in the cochlea by light microscopy evaluation. In the SEM evaluation, various epithelial damage and loss of stereocilia were also observed. In group 3 (AT+NAC group), decreased damage with preserved cochlear structures was seen by light microscopy. In the SEM evaluation, although stereocilia loss was also seen, nearly normal cell structures and vertical and symmetrical alignment of stereocilia structures were observed compared to the AT group. CONCLUSIONS: NAC reduced cochlear damage due to acoustic trauma. Because NAC has antioxidant capacity, AT mat have caused an increase in free radicals and death of outer hair cells. NAC is an antioxidant agent and it prevented cochlear damage due to AT in rats.

Unitary ototoxic gentamicin exposure may not disrupt the function of cochlear outer hair cells in mice.

BACKGROUND: Previous study showed that mild ototoxic exposure could induce a reversible hearing impairment, and the loss and secondary incomplete recovery of cochlear ribbon synapses could be responsible for the hearing loss. However, it remains unclear whether cochlear outer hair cells' (OHCs) functions are affected. OBJECTIVE: To verify whether the function of OHCs are also affected significantly after the ototoxic exposure. METHODS: Mice were injected intraperitoneally with 100 mg/kg concentration of gentamicin daily for 14 days. Distortion Product of Oto-acoustic Emission (DPOAE) was detected at control (pre-treatment), Day 0, day 4, day 7, day 14 and day 28 after the ototoxic exposure, respectively. In addition, the morphology of OHCs was observed by electron microscopy, OHCs has been counted by light microscopy, and the hearing thresholds were detected by auditory brain response (ABR). RESULTS: No significant changes have been found in OHC and OHC stereocilia among the experimental groups (p > .05). Further, no significant changes or loss was found in the morphology of OHCs either. However, we found ABR threshold elevations occurred after ototoxic exposure. CONCLUSIONS: Unitary ototoxic gentamicin exposure may not disrupt the function of cochlear OHCs in mice, regardless of hearing loss identified in this ototoxic exposure.

Tumor Necrosis Factor-α-Induced Ototoxicity in Mouse Cochlear Organotypic Culture.

Tumor necrosis factor (TNF)-α is a cytokine involved in acute inflammatory phase reactions, and is the primary upstream mediator in the cochlear inflammatory response. Treatment of the organ of Corti with TNF-α can induce hair cell damage. However, the resulting morphological changes have not been systematically examined. In the present study, cochlear organotypic cultures from neonatal mice were treated with various concentrations and durations of TNF-α to induce inflammatory responses. Confocal microscopy was used to evaluate the condition of hair cells and supporting cells following immunohistochemical staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy. TNF-α treatment resulted in a fusion and loss of stereocilia bundles in hair cells, swelling of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. Disruption of tight junctions between hair cells and supporting cells was also observed at high concentrations. Hair cell loss was preceded by apoptosis of Deiters' and pillar cells. Taken together, these findings detail the morphological changes in the organ of Corti after TNF-α treatment, and provide an in vitro model of inflammatory-induced ototoxicity.

Effects of salicylate on sound-evoked outer hair cell stereocilia deflections.

Hearing depends on sound-evoked deflections of the stereocilia that protrude from the sensory hair cells in the inner ear. Although sound provides an important force driving stereocilia, forces generated through mechanically sensitive ion channels and through the motor protein prestin have been shown to influence stereocilia motion in solitary hair cells. While a possible influence of prestin on mechanically sensitive ion channels has not been systematically investigated, a decrease in transducer currents is evident in solitary hair cells when prestin is blocked with salicylate, raising the question of whether a reduced prestin activity or salicylate itself affected the mechanotransduction apparatus. We used two- and three-dimensional time-resolved confocal imaging to visualize outer hair cell stereocilia during sound stimulation in the apical turn of cochlear explant preparations from the guinea pig. Surprisingly, following application of salicylate, outer hair cell stereocilia deflections increased, while cochlear microphonic potentials decreased. However, when prestin activity was altered with the chloride ionophore tributyltin, both the cochlear microphonic potential and the stereocilia deflection amplitude decreased. Neither positive nor negative current stimulation abolished the bundle movements in the presence of salicylate, indicating that the observed effects did not depend on the endocochlear potential. These data suggest that salicylate may alter the mechanical properties of stereocilia, decreasing their bending stiffness.

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells.

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca(2+) entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca(2+)-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca(2+) reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca(2+) concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca(2+) buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca(2+), intracellular Ca(2+) buffering, and membrane potential, by their common effect on intracellular free Ca(2+).

Effects of DAPT and Atoh1 overexpression on hair cell production and hair bundle orientation in cultured Organ of Corti from neonatal rats.

BACKGROUND: In mammals, hair cells do not undergo spontaneous regeneration when they are damaged and result in permanent hearing loss. Previous studies in cultured Organ of Corti dissected from neonatal animals have shown that both DAPT (r-secretase inhibitor in the Notch signal pathway) treatment and Atoh1 overexpression can induce supernumerary hair cells. The effects of simultaneous DAPT treatment and Atoh1 over expression in the cells of cultured Organ of Corti from neonatal rats are still obscure. PRINCIPAL FINDINGS: In this study, we set out to investigate the interaction of DAPT treatment and Atoh1 overexpression as well as culture time and the location of basilar fragment isolated form neonatal rat inner ear. Our results showed that DAPT treatment induced more hair cells in the apical turn, while Atoh1 overexpression induced more extra hair cells in the middle turn of the cultured Organ of Corti. When used together, their effects are additive but not synergistic. In addition, the induction of supernumerary hair cells by both DAPT and Atoh1 overexpression is dependent on the treatment time and the location of the cochlear tissue. Moreover, DAPT treatment causes dramatic changes in the orientation of the stereociliary bundles of hair cells, whereas Atoh1 overexpression didn't induce drastic change of the polarity of stereociliary bundles. CONCLUSIONS/SIGNIFICANCE: Taken together, these results suggest that DAPT treatment are much more potent in inducing supernumerary hair cells than Atoh1 overexpression and that the new hair cells mainly come from the trans-differentiation of supporting cells around hair cells. The orientation change of stereociliary bundle of hair cells may be attributed to the insertion of the newly formed hair cells. The immature hair bundles on the newly formed hair cells may also contribute to the overall chaos of the stereociliary bundle of the sensory epithelia.

Divalent counterions tether membrane-bound carbohydrates to promote the cohesion of auditory hair bundles.

The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg(2+) was a more potent mediator of attachment than was Ca(2+). The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing.