The protective role of Wnt3a in peroxynitrite-induced damage of cochlear hair cells in vitro

Abstract Objective To investigate the effect of peroxynitrite on the cultured cochlear hair cells of C57BL/6 P3 mice in vitro as well as the role of Wnt3a, as an activator of the canonical Wnt signaling pathway, underlying the action of such an oxidative stress. Methods The in vitro primary cultured cochlear hair cells were subjected to l00 μM peroxynitrite and l00 μM peroxynitrite +25 ng/mL Wnt3a for 24 h, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy. Results The number of surviving hair cells was significantly reduced in the 100 μM peroxynitrite group, while it was significantly higher in the Wnt3a + peroxynitrite treated group compared with the peroxynitrite treated group. The transmission electron microscopy showed that exposure to peroxynitrite induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, while Wnt3a clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. Conclusion These results indicated that peroxynitrite could cause oxidative damage to the cochlear hair cells, and low concentrations of Wnt3a has a protective effect against oxidative damage. Level of evidence: Level 2.

Melatonin prevents age-related hearing loss in the murin experimental model

Abstract Objective: The present study aimed to perform a morphological and morphometric analysis of cochlear structures of C57BL/6J mice receiving oral melatonin for a 12-month period. Methods: 32 male C57BL/6J were divided into control and melatonin groups. Control received saline and ethanol solution and melatonin group, 50 μL of 10 mg of melatonin/kg/day orally for a 12-month period. After de experiment the animals were sacrificed into a 40% concentration of CO2 chamber, and the blades were morphological and morphometrically analyzed. Results: The melatonin group revealed a higher median density of viable cells (45 ± 10.28 cells/100 μm2, 31-73, vs. 32 ± 7.47 cells/100 μm2, 25-48). The median area of stria vascularis was 55.0 ± 12.27 cells/100 μm2 (38-80) in the control, and 59.0 ± 16.13 cells/100 μm2 (40-134) in the melatonin group. The morphometric analysis of the spiral ligament reveals a higher median of total viable neurons in the melatonin (41 ± 7.47 cells/100 μm2, 27-60) than in the control group (31 ± 5.68 cells/100 μm2, 21-44). Conclusion: Although melatonin is a potent antioxidant, it does not completely neutralize the occurrence of presbycusis; however, it may delay the onset of this condition. Level of evidence: 3.

Reliability of Contralateral Suppression in Evoked Distortion Product Otoacoustic Emissions

Abstract Introduction Distortion product otoacoustic emissions (DPOAE) and their suppression may be considered useful in monitoring cochlear function and the efferent auditory pathway inhibitory effect. Nonetheless, the establishment of reliable parameters of response variations is of great importance. Objectives To verify the replicability of test and retest in the research of the inhibitory effect of the efferent pathway using contralateral suppressing stimulus during DPOAE recording for clinical applicability. Methods Cross-sectional study with 48 volunteers, aged 18 to 30 years, with normal audiometric thresholds. The procedures included were audiometric and immittance measures to overrule any conductive or sensorineural conditions and DPOAE recordings without and with contralateral suppression with a 60 dBHL white noise. Distortion product otoacoustic emissions amplitudes were analyzed and compared in both conditions with Wilcoxon test, and the Spearman correlation test was used to assess test-retest reliability. Results The comparative analysis showed differences between amplitudes in test and retest conditions only in 1,500 Hz for DPOAE measures with all other tested frequencies showing no differences, and no difference was observed in all recorded frequencies in the test and retest comparison for DPOAE suppression. The degree of correlation between test and retest of DPOAE amplitude was good at 6,000 Hz and strong (r > 0.880) at the other frequencies. For DPOAE with suppression, all frequencies presented strong correlation between test and retest: 1,500 Hz (r = 0.880), 2,000 Hz (r = 0.882), 3,000 Hz (r = 0.940), and 6,000 Hz (r = 0.957). Conclusions The study found good replicability in contralateral suppression of DPOAE with potential clinical applicability, and we recommend conducting the test from 2000Hz to higher frequencies for more reliable results.

Distortion Product Otoacoustic Emissions (DPOAEs) In Tinnitus Patients

Abstract Introduction Tinnitus is the perception of sound in the absence of external sound stimulation. There is a general agreement that it is a direct consequence of irreversible and permanent cochlear damage. Objectives The present work is designed to study the distortion product otoacoustic emissions (DPOAEs) in tinnitus patients with normal hearing in comparison with normal hearing control and to study any possible correlation between DPOAEs recording and patients' complaints. Methods The present study included 80 subjects divided into 2 groups: Control group: consisted of 30 normal-hearing adults not complaining of tinnitus and Study group: consisted of 50 normal-hearing adults complaining of tinnitus. The methodology includes full audiological history, otoscopic examination, basic audiological evaluation, DPOAEs including both DP-gram and DPOAEs input/output functions. Results Basic audiological evaluation showed within normal hearing sensitivity in both groups, however, with significant higher hearing thresholds in tinnitus patients at all frequency ranges. The Tinnitus Handicap Inventory Questionnaire showed mean scores of 35.2 ± 16.9 in the study group. The DP-gram showed higher amplitudes in the control group when compared with tinnitus patients. The DPOAEs input-output functions at different frequencies (1, 2, 4 and 6kHz) also showed higher amplitudes at all frequencies and different input levels. The slope of the I/O function tends to be steeper in tinnitus cases. Conclusion Patients with tinnitus might have neural dysfunction at either the level of the cochlea, as shown in reduced DPOAE levels, and changes in the normal DP-I/O function recorded in the present work.

Estudo das emissões otoacústicas residuais nas perdas auditivas neurossensoriais com diferenciação de artefatos e respostas fisiológicas / Study of residual otoacoustic emissions in hearing loss with artifact differentiation and physiological responses

RESUMO Objetivo avaliar o efeito da variação da intensidade de estimulação sobre as respostas das emissões otoacústicas produto de distorção em indivíduos com perda auditiva neurossensorial, utilizando um protocolo de gradiente de fase das emissões. Métodos estudo observacional transversal. Participaram 38 indivíduos com diagnóstico de perda auditiva neurossensorial de grau leve, moderado ou severo. Foram realizadas anamnese, meatoscopia, audiometria tonal liminar, logoaudiometria, imitanciometria, emissões otoacústicas produto de distorção e emissões otoacústicas residuais. As emissões otoacústicas residuais foram coletadas com o equipamento Echodia, modelo Elios®. O protocolo utilizado permite a variação dos parâmetros frequência e intensidade e as respostas são analisadas por meio do teste do Gradiente de Fase. As respostas registradas nas emissões residuais foram consideradas como "presente", "ausente" e "artefato", considerando a variação da fase em função de f1. Resultados Foram incluídas 72 orelhas. Houve diferença estatisticamente significativa nas frequências de 1300 Hz e 2000 Hz, ao comparar os resultados das emissões residuais. Ao correlacionar o resultado da audiometria e a intensidade de estimulação que evocou a emissão residual, houve correlação positiva para as frequências de 1000 Hz e 4000Hz. O "artefato" foi registrado, principalmente, nas frequências mais agudas: 56,2% em 3000 Hz e 58,2% em 4000 Hz. A emissão otoacústica residual presente foi registrada em 18,6% em 1000 Hz, 13,4% em 2000 Hz, 6,3% em 3000 Hz e 7,5% em 4000 Hz. Conclusão o aumento da intensidade de estimulação no exame de emissões pode auxiliar no estudo das células ciliadas residuais, desde que seja utilizado um protocolo capaz de diferenciar respostas fisiológicas de artefatos.

ABSTRACT Purpose To study the effect of stimulation intensity variation on the responses of distortion products in subjects with sensorineural hearing loss using a new protocol to register the otoacoustic emissions. Methods This is a cross-sectional observational study. The following procedures were performed: anamnesis, otoscopy, pure tone audiometry, speech audiometry, tympanometry, distortion product and residual otoacoustic emissions. The residual DPOAE were collected with the Echodia equipment, Elios®. The protocol that was developed allows the variation of frequency and intensity parameters and the responses are analyzed by phase gradient test. Responses recorded in residual otoacoustic emissions were considered "present", "absent" or "artifact". Results The total included ears was 72. On residual otoacoustic emissions test, at a frequency of 1300Hz and 2000Hz, there was statistically significant difference. By analyzing the average found in the audiometry and the results of residual emissions, only the frequency of 1300Hz showed a statistically significant association in all groups. By correlating the results of the audiometry and the stimulation intensity used to evoke the residual emission, there was positive correlation for the frequencies of 1000Hz and 4000Hz. The "artifact" was mostly recorded in the higher frequencies: 56.2% in 3000Hz and 58.2% in 4000 Hz. Residual EOAPD present was recorded as 18.6% at 1000Hz, 13.4% at 2000Hz, 6.3% at 3000Hz and 7.5% at 4000Hz. Conclusion The increased stimulation intensity in the otoacoustic emissions test can aid in the study of residual outer hair cells, as long as a protocol is used to check the correctness of the responses.

Prestin-Mediated Frequency Selectivity Does not Cover Ultrahigh Frequencies in Mice / 神经科学通报·英文版

In mammals, the piezoelectric protein, Prestin, endows the outer hair cells (OHCs) with electromotility (eM), which confers the capacity to change cellular length in response to alterations in membrane potential. Together with basilar membrane resonance and possible stereociliary motility, Prestin-based OHC eM lays the foundation for enhancing cochlear sensitivity and frequency selectivity. However, it remains debatable whether Prestin contributes to ultrahigh-frequency hearing due to the intrinsic nature of the cell's low-pass features. The low-pass property of mouse OHC eM is based on the finding that eM magnitude dissipates within the frequency bandwidth of human speech. In this study, we examined the role of Prestin in sensing broad-range frequencies (4-80 kHz) in mice that use ultrasonic hearing and vocalization (to >100 kHz) for social communication. The audiometric measurements in mice showed that ablation of Prestin did not abolish hearing at frequencies >40 kHz. Acoustic associative behavior tests confirmed that Prestin-knockout mice can learn ultrahigh-frequency sound-coupled tasks, similar to control mice. Ex vivo cochlear Ca2+ imaging experiments demonstrated that without Prestin, the OHCs still exhibit ultrahigh-frequency transduction, which in contrast, can be abolished by a universal cation channel blocker, Gadolinium. In vivo salicylate treatment disrupts hearing at frequencies <40 kHz but not ultrahigh-frequency hearing. By pharmacogenetic manipulation, we showed that specific ablation of the OHCs largely abolished hearing at frequencies >40 kHz. These findings demonstrate that cochlear OHCs are the target cells that support ultrahigh-frequency transduction, which does not require Prestin.

Estudo comparativo das emissões otoacústicas evocadas em trabalhadores marítimos Offshore e Onshore / Comparative Study of Evoked Otoacoustic Emissions in Offshore and Onshore Seafaring Workers

RESUMO Objetivo Avaliar a função coclear de trabalhadores marítimos Offshore e Onshore de uma empresa naval da cidade do Rio de Janeiro e estimar a magnitude de associação entre a exposição ocupacional ao ruído e/ou substâncias químicas e alterações na função coclear. Método Neste estudo, foram avaliados trabalhadores marítimos entre 20-49 anos, de ambos os gêneros, sem queixas auditivas, distribuídos em dois grupos: o Grupo Offshore, que operam em alto mar com exposição ocupacional; e o Grupo Onshore, que operam em escritórios sem exposição ocupacional. Para avaliação da função coclear, foram realizados os exames de emissões otoacústicas evocadas por estímulo transiente (EOAT) e por produto de distorção (EOAPD). Resultados As respostas das EOAT e EOAPD foram, em média, menores no Grupo Offshore, para todas as frequências analisadas. A proporção de falhas observadas também foi maior no grupo de exposição (Offshore), tanto no critério geral quanto por frequência específica, principalmente para as frequências mais agudas de cada teste, 4 kHz para EOAT e 6 kHz para EOAPD. Conclusão Os resultados sugerem que a exposição a ruído e/ou a substâncias químicas pode contribuir significativamente para alterações da função coclear de trabalhadores marítimos, mesmo antes de manifestarem queixas auditivas.

ABSTRACT Purpose To evaluate the cochlear function of offshore and onshore seafaring workers of a naval company in the city of Rio de Janeiro and to estimate the degree of association between occupational exposure to noise and/or chemical substances and alteration in cochlear function. Methods This study evaluated seafaring workers aged 20 to 49, of both genders, without auditory symptoms, divided into two groups: the Offshore Group, operating in the high seas with occupational exposure; and the Onshore Group, operating in offices without occupational exposure. Exams were performed to evaluate cochlear function, including transient evoked otoacoustic emissions (TEOAE) and distortion product otoacoustic emissions (DPOAE). Results The TEOAE and DPOAE responses were on average lower in the Offshore Group, for all frequencies analyzed. The proportion of failures observed was also higher in the exposure group (Offshore), for general response and specific frequency, mainly for the frequencies of 4 kHz for TEOAE and 6 kHz for DPOAE. Conclusion The results suggest that exposure to noise and/or chemical substances can contribute to alterations in cochlear function in seafarers even without manifesting auditory symptoms.

Cochlea cell-specific marker expression upon in vitro Hes1 knockdown

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

Electroacoustic and electrophysiological auditory assessment in aphasic individuals / Avaliação eletroacústica e eletrofisiológica da audição em indivíduos afásicos

ABSTRACT Purpose: to verify the functioning of the outer hair cells and the medial efferent olivocochlear system, and the integrity of the auditory pathways in the brainstem up to the auditory cortex, in aphasic individuals. Methods: the sample comprised 20 individuals - 10 without aphasia and 10 with it, aged from 21 to 58 years. The procedures used were the research of the otoacoustic emissions by a transient stimulus with and without noise, and the cognitive potential (tone-burst and speech stimuli). The findings were analyzed based on descriptive statistics. Results: the suppression effect was more present in individuals without aphasia when compared with the aphasic ones. In the cognitive potential, the mean latency values of P3 was within normality standards, with a higher latency in the individuals presented with aphasia for the tone-burst stimulus in both ears. A statistically significant difference of the P3-N2 amplitude was observed for the tone-burst stimulus, comparing the ears in both groups, and for speech stimulus only to the left ear in both groups. Conclusions: aphasic individuals did not present significant differences regarding suppression of the otoacoustic emissions. As for the cognitive potential, the aphasic individuals presented higher latency values when compared to those with no aphasia.

RESUMO Objetivos: verificar o funcionamento das células ciliadas externas e do sistema olivococlear eferente medial e a integridade das vias auditivas no tronco encefálico até o córtex auditivo, em indivíduos afásicos. Métodos: a amostra foi composta por 20 indivíduos, sendo 10 indivíduos sem afasia e 10 indivíduos afásicos, com idades entre 21 e 58 anos. Os procedimentos utilizados foram: pesquisa das emissões otoacústicas por estímulo transiente sem e com ruído, e potencial cognitivo (estímulo toneburst e de fala). Os achados foram analisados a partir da estatística descritiva. Resultados: o efeito de supressão esteve mais presente nos indivíduos sem afasia quando comparado com os afásicos. No potencial cognitivo, o valor médio das latências P3, mostrou-se dentro dos padrões da normalidade com maior latência nos indivíduos com afasia para o estímulo toneburst em ambas as orelhas. Observou-se diferença estatisticamente significante da amplitude P3-N2 para o estímulo toneburst, comparando as orelhas em ambos os grupos, e para o estímulo de fala apenas à orelha esquerda em ambos os grupos. Conclusões: indivíduos afásicos não apresentaram diferenças significantes quanto à supressão das emissões otoacústicas. Quanto ao potencial cognitivo, os indivíduos afásicos apresentaram maior valor de latência em relação aos indivíduos sem afasia.

Stem-cell therapy for hearing loss: are we there yet? / Terapia com células-tronco para perda auditiva: já chegamos lá?

Abstract Introduction: Mammalian hair cells and auditory neurons do not show regenerative capacity. Hence, damage to these cell types is permanent and leads to hearing loss. However, there is no treatment that re-establishes auditory function. Regenerative therapies using stem cells represent a promising alternative. Objective: This article aims to review the current literature about the main types of stem cells with potential for application in cell therapy for sensorineural hearing loss, the most relevant experiments already performed in animals, as well as the advances that have been recently made in the field. Methods: Research included the databases PubMed/MEDLINE, Web of Science, Science Direct and SciELO, as well as gray literature. Search strategy included the following main terms: "stem cells", "hair cells" and "auditory neurons". Additionally, the main terms were combined with the following secondary terms: "mesenchymal", "iPS", "inner ear", "auditory". The research was conducted independently by three researchers. Results: Differentiation of stem cells into hair cells and auditory neurons has a high success rate, reaching up to 82% for the first and 100% for the latter. Remarkably, these differentiated cells are able to interact with hair cells and auditory neurons of cochlear explants through formation of new synapses. When transplanted into the cochlea of animals with hearing loss, auditory restoration has been documented to date only in deafferented animals. Conclusion: Advances have been more prominent in cases of auditory neuropathy, since partial improvement of auditory nerve conditions through cell-based therapy may increase the number of patients who can successfully receive cochlear implants.

Resumo Introdução: Nos mamíferos, as células ciliadas e os neurônios auditivos não apresentam capacidade regenerativa. Assim, os danos a esses tipos celulares são permanentes e levam à perda auditiva. Contudo, como não há tratamento que restabeleça a função auditiva, as terapias regenerativas utilizando células-tronco representam uma alternativa promissora. Objetivo: Este artigo tem como objetivo revisar a literatura atual sobre os principais tipos de células-tronco com potencial para aplicação em terapia celular para perda auditiva sensorioneural, os experimentos mais relevantes já realizados em animais, bem como os avanços obtidos recentemente nessa área. Método: As pesquisas incluíram as bases de dados PubMed/MEDLINE, Web of Science, Science Direct e SciELO, além da literatura cinza. A estratégia de busca incluiu os seguintes termos principais: "stem cells", "hair cells" e "auditory neurons". Além disso, os termos principais foram combinados com os seguintes termos secundários: "mesenchymal", "iPS", "inner ear" e "auditory". A pesquisa foi realizada de forma independente por três pesquisadores. Resultados: A diferenciação de células-tronco em células ciliadas e neurônios auditivos têm alta taxa de sucesso, chegando a 82% para o primeiro caso e 100% para o segundo. Notavelmente, essas células diferenciadas são capazes de interagir com células ciliadas e neurônios auditivos de explantes cocleares através da formação de novas sinapses. Quando transplantadas para a cóclea de animais com perda auditiva, a restauração da função auditiva foi observada, até o momento, apenas em animais com ablação do VIII nervo craniano. Conclusão: Os avanços têm sido mais proeminentes em casos de neuropatia auditiva. A melhora parcial das condições do nervo auditivo por meio de terapia baseada em células-tronco pode aumentar o número de pacientes candidatos a receber implantes cocleares com sucesso.

The molecular mechanisms underpinning auditory neuropathy / 中华耳鼻咽喉头颈外科杂志

Auditory neuropathy (AN) is a hearing disorder where cochlear inner hair cell and/or the auditory nerve function is disrupted while outer hair cell function is normal. It can affect people of all ages, from infancy to adulthood. People with auditory neuropathy may have normal hearing threshold, or hearing loss ranging from mild to severe; they always have poor speech-perception abilities. It is a heterogeneous disorder which can have either congenital or acquired causes. AN may result from specific loss of cochlear inner hair cells, disordered release of neurotransmitters by inner hair cell ribbon synapses, deafferentation accompanying loss of auditory nerve fibers, neural dys-synchrony or conduction block as a result of demyelination of nerve fibers and auditory nerve hypoplasia. Although the definition of AN includes the central part, its incidence is low, and the etiology and pathology are not clear. The present review aimed to provide an overview of the genetic conditions associated with AN and highlight the neural and synaptic mechanism of AN. Possible strategy for treatments of AN was also discussed.

Influence of endoplasmic reticulum stress in degeneration of cochlear hair cells in type 2 diabetic mice / 吉林大学学报(医学版)

Objective: To investigate the influence of endoplasmic reticulum stress (ERS) in the degeneration of cochlear hair cells in the type 2 diabetic mice∗ and to clarify its mechanism. Methods: Twenty clean Kun Ming male mice aged one month were selected and randomly divided into control group and model group (n= 10). The mice in model group were injected with STZ (40 mg • kg ) to establish the type 2 diabetic models. The fasting blood glucose levels of the mice were measured through collecting the vena caudalis blood of the mice. Auditory brain stem response (ABR) was used to detect the ABR threshold of the mice. Otoacoustic emission (OAE) test was used to detect the OAE threshold of mice. The defect rate of mouse cochlear outer hair cells was calculated by the mouse cochlear spreading technique. The expression levels of GRP78, caspase-12, p-ERK and Nrf2 proteins were detected by Western blotting method. Results: Compared with control group, the fasting blood glucose levels of the mice in model group at the 7th and the 14th days had no significant differences ( P>0. 05) , but the levels were increased significantly at the 21th,28th and 35th days and the level reached the highest value at the 35th day. The ABR thresholds of the mice in model group at 8, 12, and 24 kHZ were increased significantly compared with control group ( P0. 05). The expression levels of p-ERK and Nrf2 in the cochlear hair cells of the mice in model group were lower than those in control group (P<0.05), and the expression levels of GRP78 and caspase-12 were higher than those in control group (P<0. 05). Conclusion: ERS can result in the increase of defect rate of cochlear outer hair cells and ABR brainstem hearing threshold of the diabetic mice and decrease the expression levels of p-ERK and Nrf2 proteins, suggesting that ERS can promote the degenerative lesions of cochlear hair cells in the type 2 diabetic mice.

Atoh1 regulation in the cochlea: more than just transcription / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

More than 80% of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons, and a lack of regeneration of these cells leads to permanent hearing loss. Therefore, the regeneration of lost hair cells is an important goal for the treatment of deafness. Atoh1 is a basic helix-loop-helix (bHLH) transcription factor that is critical in both the development and regeneration of cochlear hair cells. Atoh1 is transcriptionally regulated by several signaling pathways, including Notch and Wnt signalings. At the post-translational level, it is regulated through the ubiquitin-proteasome pathway. In vitro and in vivo studies have revealed that manipulation of these signaling pathways not only controls development, but also leads to the regeneration of cochlear hair cells after damage. Recent progress toward understanding the signaling networks involved in hair cell development and regeneration has led to the development of new strategies to replace lost hair cells. This review focuses on our current understanding of the signaling pathways that regulate Atoh1 in the cochlea.

cVEMP correlated with imbalance in a mouse model of vestibular disorder

BACKGROUND@#Cervical vestibular evoked myogenic potential (cVEMP) testing is a strong tool that enables objective determination of balance functions in humans. However, it remains unknown whether cVEMP correctly expresses vestibular disorder in mice.@*OBJECTIVE@#In this study, correlations of cVEMP with scores for balance-related behavior tests including rotarod, beam, and air-righting reflex tests were determined in ICR mice with vestibular disorder induced by 3,3'-iminodipropiontrile (IDPN) as a mouse model of vestibular disorder.@*METHODS@#Male ICR mice at 4 weeks of age were orally administered IDPN in saline (28 mmol/kg body weight) once. Rotarod, beam crossing, and air-righting reflex tests were performed before and 3-4 days after oral exposure one time to IDPN to determine balance functions. The saccule and utricles were labeled with fluorescein phalloidin. cVEMP measurements were performed for mice in the control and IDPN groups. Finally, the correlations between the scores of behavior tests and the amplitude or latency of cVEMP were determined with Spearman's rank correlation coefficient. Two-tailed Student's t test and Welch's t test were used to determine a significant difference between the two groups. A difference with p < 0.05 was considered to indicate statistical significance.@*RESULTS@#After oral administration of IDPN at 28 mmol/kg, scores of the rotarod, beam, and air-righting reflex tests in the IDPN group were significantly lower than those in the control group. The numbers of hair cells in the saccule, utricle, and cupula were decreased in the IDPN group. cVEMP in the IDPN group was significantly decreased in amplitude and increased in latency compared to those in the control group. cVEMP amplitude had significant correlations with the numbers of hair cells as well as scores for all of the behavior tests in mice.@*CONCLUSIONS@#This study demonstrated impaired cVEMP and correlations of cVEMP with imbalance determined by behavior tests in a mouse model of vestibular disorder.

Influence of endoplasmic reticulum stress in degeneration of cochlear hair cells in type 2 diabetic mice / 吉林大学学报(医学版)

Objective:To investigate the influence of endoplasmic reticulum stress (ERS) in the degeneration of cochlear hair cells in the type 2diabetic mice, and to clarify its mechanism.Methods:Twenty clean Kun Ming male mice aged one month were selected and randomly divided into control group and model group (n=10) .The mice in model group were injected with STZ (40 mg·kg-1) to establish the type 2diabetic models.The fasting blood glucose levels of the mice were measured through collecting the vena caudalis blood of the mice.Auditory brain stem response (ABR) was used to detect the ABR threshold of the mice.Otoacoustic emission (OAE) test was used to detect the OAE threshold of mice.The defect rate of mouse cochlear outer hair cells was calculated by the mouse cochlear spreading technique.The expression levels of GRP78, caspase-12, p-ERK and Nrf2proteins were detected by Western blotting method.Results:Compared with control group, the fasting blood glucose levels of the mice in model group at the 7th and the 14th days had no significant differences (P＞0.05) , but the levels were increased significantly at the 21th, 28th and 35th days and the level reached the highest value at the 35th day.The ABR thresholds of the mice in model group at 8, 12, and 24kHZ were increased significantly compared with control group (P＜0.05) .Under the stimulation of low frequency, there was no significant change in the OAE threshold of the mice in model grouop compared with control group.The OAE thresholds of the mice in model group were increased significantly under the medium frequency and high frequency stimulation compared with control group (P＜0.05) .The defects of the cochlear hair cells were mainly concentrated on the bottom of gyrus of the mice, and the defects in middle temporal gyrus and parietal gyrus were less.Compared with control group, the defect rate in the bottom of gyrus of the mice in model group was increased significantly (P＜0.05) ;the defect rates in the middle temporal gyrus and parietal gyrus were increased, but there was no significant difference (P＞0.05) .The expression levels of p-ERK and Nrf2in the cochlear hair cells of the mice in model group were lower than those in control group (P＜0.05) , and the expression levels of GRP78and caspase-12were higher than those in control group (P＜0.05) .Conclusion:ERS can result in the increase of defect rate of cochlear outer hair cells and ABR brainstem hearing threshold of the diabetic mice and decrease the expression levels of p-ERK and Nrf2proteins, suggesting that ERS can promote the degenerative lesions of cochlear hair cells in the type 2diabetic mice.

Influencia de las zonas cocleares muertas sobre la autopercepción de las habilidades auditivas en adultos con hipoacusia sensorioneural bilateral / Influence of cochlear dead regions on self-perception hearing abilities in adults with bilateral sensorineural hearing loss

RESUMEN Introducción: Las zonas cocleares muertas son áreas de la membrana basilar donde las células ciliadas y/o fibras del nervio auditivo no son funcionales, lo que puede alterar el análisis temporal de una señal acústica. Los efectos funcionales que podrían generar aún no son claros, y establecerlos a través del uso de cuestionarios de autopercepción, proporcionaría información de utilidad para el manejo audiológico de los pacientes. Objetivo: Determinar la relación entre la presencia de zonas cocleares muertas y la autopercepción de las habilidades auditivas en adultos con hipoacusia sensorioneural bilateral. Material y método: Se evaluaron 20 sujetos con hipoacusia bilateral simétrica, entre 51 y 75 años, sin antecedentes de uso de audífonos. Las zonas cocleares muertas fueron evaluadas mediante la prueba TEN en 1, 2, 3 y 4 kHz; y la autopercepción de habilidades auditivas fue medida en los participantes a través del cuestionario de doce preguntas, IROS12. Resultados: La presencia de zonas cocleares muertas se observó en 10 pacientes. No existiendo una diferencia significativa entre grupos, sin embargo, se observaron puntuaciones más bajas de IROS12 en sujetos con zonas cocleares muertas. Conclusiones: La percepción de dificultades auditivas de individuos que presentan una hipoacusia sensorioneural bilateral simétrica moderada, con presencia de zonas cocleares muertas, no difiere significativamente de aquellos individuos que no presentan zonas cocleares muertas.

ABSTRACT Introduction: Cochlear dead regions are areas of the basilar membrane where the hair cells and/or auditory nerve fibers are not functional, which can alter the temporal analysis of an acoustic signal. The functional effects that could generate still are not entirely clear, and set through the use of self-perception questionnaires, provide useful information for audiological management of patients. Aim: To investigate the relationship between the presence of cochlear dead regions and the self-reported of listening difficulties of adults with bilateral sensorineural hearing loss. Material and Method: Twenty adults with symmetric bilateral sensorineural hearing loss, aged 51 to 75 years were tested, without previous story of use of hearing aids. Cochlear dead regions were tested using the TEN test at 1, 2, 3 and 4 kHz. The questionnaire of twelve questions, IROS12, was applied to the participants. Results: Cochlear dead regions were present in 10 participants. No significant differences were observed between groups, however lower IROS12 scores were observed in those who had present of dead regions. Conclusion: The perception of hearing difficulties in individuals with bilateral sensorineural hearing loss with cochlear dead regions did not differ significantly from those individuals without cochlear dead regions.

Screening of microRNAs targeting Notch signaling pathway implicated in inner ear development and the role of microRNA-384-5p / 中华耳鼻咽喉头颈外科杂志

Objective@#To screen the key microRNAs targeting Notch signaling pathway in inner ear and investigate its potential regulating function.@*Methods@#The interaction network and the Core-Notch network, involved with key genes in Notch signal pathway and differential-expressed microRNAs in inner ear, were constructed by bioinformatics methods. The important microRNAs in regulating Notch signaling pathway were screened via topological and GO analysis, followed by in vivo and in vitro investigation.@*Results@#MiRNA-384-5p was identified as a key regulator specifically expressed in mouse brain and inner ear, which could down-regulate Notch1. The Notch1 expression was found significantly down-regulated in miRNA-384-5p-mimic-transfected HeLa cells. The dual-luciferase reporter gene assay further confirmed the effect of miRNA-384-5p on the down-regulation of Notch1 and Dll4 in Notch signaling pathway.@*Conclusions@#The Core-Notch network is constructed to screen microRNAs implicated in inner ear development, and miRNA-384-5p is screened and verified to be target-regulating the Notch signaling pathway, which could be the potential target in the regeneration of impaired hair cells.

The experimental study on endoplasmic reticulum stress-participated outer hair cell apoptosis in cadherin 23 gene mutant mice / 中华耳鼻咽喉头颈外科杂志

Objective@#To test the mechanism and upstream pathway of outer hair cell apoptosis in Cadherin 23 (Cdh23) gene mutant mice.@*Method@#The mutant Cdh23erl/erl(erl) mice were collected as the study group, while the C57BL/6J (B6) mice were chosen as the control group. A total of 70 mice per group were used in this study. The study group and control group underwent auditory-evoked brainstem response (ABR) tests at the same age. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect outer hair cell(OHC) apoptosis. The qRT-PCR was conducted to test the expression of ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP) mRNA. The expression and location of BiP and CHOP protein in OHC were detected by immunostaining. The expression of BiP protein in cochleae was identified by Western blot. The expression and location of CDH23 protein in OHC were discovered by immunostaining.@*Results@#The ABR thresholds in erl mice were significantly higher than those in B6 mice at the age of 1 and 3 months (both P<0.05). The surface preparation with TUNEL staining confirmed OHC apoptosis in erl mouse cochleae which showed a higher TUNEL positive cell ratio than B6 mouse(t=11.291, P<0.01). The ER stress marker Bip and Chop mRNA were upregulated in the erl mouse inner ear, when compared with those in the B6 mouse(both P<0.05). The BiP protein extracted from the erl mouse cochleae was significantly higher than that of B6 mouse measured by Western blot (t=3.66, P=0.02). Immunostaining showed that BiP and CHOP were highly detected in the OHC in erl mouse cochleae, and was mainly detected in the perinuclear region of OHC. However, a bare BiP and CHOP signal were shown in B6 mouse cochleae. The CDH23 protein was specifically localized at the top of the OHC in B6 mice, indicating the localization of the tip links in hair bundle stereocilia. On the contrary, the CDH23erl protein was found to be localized from the top to the nuclei of the OHC in erl mice. Portions of the CDH23erl proteins failed to reach the top of the hair bundles and remained in the OHC cytoplasm.@*Conclusion@#As the downstream response of the Cdh23 gene mutation, portions of the mutant CDH23erl protein was accumulated in ER lumen resulting in the increase of ER loading and ultimately triggered ER stress and hair cell apoptosis in erl mouse cochleae.

Identification and analysis of the proteins interacted with Prestin in cochlear outer hair cells of guinea pig / 中华耳鼻咽喉头颈外科杂志

Objective@#To explore the regulation and mechanism of Prestin protein by identifying the proteins interacted with Prestin in cochlear outer hair cell(OHC) and analyzing their biological function.@*Methods@#Co-immunoprecipitation combined mass spectrometry technology was used to isolate and identify the proteins interacted with Prestin protein of OHC, bioinformatics was used to construct Prestin protein interaction network. The proteins interacted with Prestin in OHC of guinea pig were determined by matching primary interaction mass spectrometry with protein interaction network, and annotated their functions.@*Results@#The results of co-immunoprecipitation combined with mass spectrometry showed that 116 kinds of credible proteins could interact with Prestin. By constructing Prestin protein interaction network, matching the results of mass spectrometry and analyzing of sub-cellular localization, eight kinds of proteins were confirmed that they interacted with Prestin directly, namely EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1, and ERH, respectively, which were mainly involved in the synthesis and transportation, transmembrane folding and localization, structural stability and signal transduction of Prestin protein.@*Conclusion@#EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1 and ERH provide molecular basis for sensory amplification function of OHCs by participating in biotransformation, transmembrane folding and localization, signal transduction and other biological processes of Prestin protein.

Impact of Nicotine Exposure on Hair Cell Toxicity and Embryotoxicity During Zebrafish Development

OBJECTIVES: Nicotine has various adverse effects including negative impacts associated with maternal exposure. In the current study, we examined nicotine-induced damage of hair cells and embryotoxicity during zebrafish development. METHODS: Zebrafish embryos were exposed to nicotine at several concentrations (5, 10, 20, and 40 μM) and embryotoxicity were evaluated at 72 hours, including hatching rate, mortality, teratogenicity rate, and heart rate. Hair cells within the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) neuromasts were identified at 120 hours. Apoptosis and mitochondrial damage of hair cells were analyzed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) and DASPEI (2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide) assays, respectively, and changes of ultrastructure were observed by scanning electron microscopy. RESULTS: The control group without nicotine appeared normal with overall mortality and teratogenicity rate < 5%. The hatching rate and mortality rate was not significantly different according to nicotine concentration (n=400 each). The abnormal morphology rate (n=400) increased and heart rate (n=150) decreased with increasing nicotine concentration (P < 0.05). Nicotine-induced hair cell damage significantly increased as nicotine concentration increased. A significantly greater number of TUNEL-positive cells (P < 0.01) and markedly smaller DASPEI area (P < 0.01) were shown as nicotine concentration increased. CONCLUSION: The current results suggest that nicotine induces dose-dependent hair cell toxicity in embryos by promoting apoptosis and mitochondrial and structural damage.