RESUMO
Hearing loss has become increasingly prevalent and causes considerable disability, thus gravely burdening the global economy. Irreversible loss of hair cells is a main cause of sensorineural hearing loss, and currently, the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids, but these are of limited benefit in patients. It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies. At present, how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research. Multiple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells, and in this article, we first review the principal mechanisms underlying hair cell reproduction. We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration, and we summarize current achievements in hair cell regeneration. Lastly, we discuss potential future approaches, such as small molecule drugs and gene therapy, which might be applied for regenerating functional hair cells in the clinic.
Assuntos
Recém-Nascido , Humanos , Células Ciliadas Auditivas Internas/fisiologia , Orelha Interna/fisiologia , Células Ciliadas Auditivas/fisiologia , Regeneração/genética , Células-TroncoRESUMO
The cochlear auditory epithelium contains two types of sound receptors, inner hair cells (IHCs) and outer hair cells (OHCs). Mouse models for labelling juvenile and adult IHCs or OHCs exist; however, labelling for embryonic and perinatal IHCs or OHCs are lacking. Here, we generated a new knock-in Fgf8P2A-3×GFP/+ (Fgf8GFP/+) strain, in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements. After confirming that GFP expression accurately reflects the expression of Fgf8, we successfully obtained both embryonic and neonatal IHCs with high purity, highlighting the power of Fgf8GFP/+. Furthermore, our fate-mapping analysis revealed, unexpectedly, that IHCs are also derived from inner ear progenitors expressing Insm1, which is currently regarded as an OHC marker. Thus, besides serving as a highly favorable tool for sorting early IHCs, Fgf8GFP/+ will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.
Assuntos
Animais , Camundongos , Células Ciliadas Auditivas Internas , Cóclea/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Modelos Animais de Doenças , Fator 8 de Crescimento de Fibroblasto/metabolismoRESUMO
Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus. Here, we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells (IHCs). We found that IHCs showed significant damage after exposure to a high concentration of salicylate. Whole-cell patch clamp recordings showed that 1-5 mmol/L salicylate did not affect the exocytosis of IHCs, indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input. Instead, salicylate induced a larger peak amplitude, a more negative half-activation voltage, and a steeper slope factor of Ca2+ current. Using noise analysis of Ca2+ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca2+ channels along with CaV1.3 expression. All these changes could act synergistically to enhance the Ca2+ influx into IHCs. Inhibition of intracellular Ca2+ overload significantly attenuated IHC death after 10 mmol/L salicylate treatment. These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.
Assuntos
Animais , Camundongos , Cálcio , Exocitose , Células Ciliadas Auditivas Internas , Salicilato de Sódio/farmacologia , Zumbido/induzido quimicamenteRESUMO
Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell (IHC). This feature is believed to be critical for audition over a wide dynamic range, but whether the spatial gradient of ribbon morphology is fine-tuned in each IHC and how the mitochondrial network is organized to meet local energy demands of synaptic transmission remain unclear. By means of three-dimensional electron microscopy and artificial intelligence-based algorithms, we demonstrated the cell-wide structural quantification of ribbons and mitochondria in mature mid-cochlear IHCs of mice. We found that adjacent IHCs in staggered pairs differ substantially in cell body shape and ribbon morphology gradient as well as mitochondrial organization. Moreover, our analysis argues for a location-specific arrangement of correlated ribbon and mitochondrial function at the basolateral IHC pole.
Assuntos
Animais , Camundongos , Inteligência Artificial , Cóclea/metabolismo , Células Ciliadas Auditivas Internas , Mitocôndrias , Sinapses/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Cochlear dead region (CDR) is a region in the cochlear where hearing loss has occurred due to damage to the inner hair cells and/or neurons. Recently, a subjective test involving a pure-tone test in the presence of threshold-equalizing noise (TEN) was introduced to identify CDR. However, for uncooperative patients, such a subjective method would be unsuitable and objective methods would be needed instead to detect CDR. The acoustic change complex (ACC) is an evoked potential elicited by changes in the ongoing sound. In this study, we developed an objective method of identifying CDR by combining ACC response with a TEN test, namely the TEN-ACC test, and investigated its feasibility in normal-hearing listeners. SUBJECTS AND METHOD: Ten normal-hearing subjects participated in this study. All subjects underwent both behavioral TEN test and electrophysiological TEN-ACC test. The stimuli for the TEN-ACC test consisted of TEN and embedded pure tones with different frequencies/signals to noise ratios (SNRs). To identify the thresholds, the range SNR of stimulation was varied from 0 to 20 dB, in stages of 4 dB. RESULTS: The ACC responses of all subjects who participated in this study were well elicited by stimuli developed for the TEN-ACC test. We confirm that the pure-tones embedded in TEN elicited the objective ACC response. CONCLUSION: The results of this study suggest that the novel TEN-ACC test can be applied to evoke ACC in normal-hearing listeners. Future research should incorporate hearing-impaired listeners to determine the feasibility of the TEN-ACC test as an objective method to identify CDR.
Assuntos
Humanos , Acústica , Potenciais Evocados , Células Ciliadas Auditivas Internas , Perda Auditiva , Métodos , Neurônios , RuídoRESUMO
The synaptic contacts of cochlear afferent fibers (CAFs) with inner hair cells (IHCs) are spatially segregated according to their firing properties. CAFs also exhibit spatially segregated vulnerabilities to noise. The CAF fibers contacting the modiolar side of IHCs tend to be more vulnerable. Noise vulnerability is thought to be due to the absence of neuroprotective mechanisms in the modiolar side contacting CAFs. In this study, we investigated whether the expression of neuroprotective Ca²⁺-buffering proteins is spatially segregated in CAFs. The expression patterns of calretinin, parvalbumin, and calbindin were examined in rat CAFs using immunolabeling. Calretinin-rich fibers, which made up ~50% of the neurofilament (NF)-positive fibers, took the pillar side course and contacted all IHC sides. NF-positive and calretinin-poor fibers took the modiolar side pathway and contacted the modiolar side of IHCs. Both fiber categories juxtaposed the C-terminal binding protein 2 (CtBP2) puncta and were contacted by synaptophysin puncta. These results indicated that the calretinin-poor fibers, like the calretinin-rich ones, were afferent fibers and probably formed functional efferent synapses. However, the other Ca²⁺-buffering proteins did not exhibit CAF subgroup specificity. Most CAFs near IHCs were parvalbumin-positive. Only the pillar-side half of parvalbumin-positive fibers coexpressed calretinin. Calbindin was not detected in any nerve fibers near IHCs. Taken together, of the Ca²⁺-buffering proteins examined, only calretinin exhibited spatial segregation at IHC-CAF synapses. The absence of calretinin in modiolar-side CAFs might be related to the noise vulnerability of the fibers.
Assuntos
Animais , Ratos , Calbindina 2 , Calbindinas , Proteínas de Transporte , Incêndios , Células Ciliadas Auditivas Internas , Filamentos Intermediários , Fibras Nervosas , Ruído , Sensibilidade e Especificidade , Sinapses , SinaptofisinaRESUMO
BACKGROUND AND OBJECTIVES: Locacorten Vioform (Novartis UK) is frequently prescribed for otomycosis. Its component, Clioquinol, also has anti-bacterial properties. Up to this point, its ototoxic potential has not been evaluated. Our objective aims to evaluate Locacorten Vioform’s potential ototoxicity when applied directly to the middle ear cavity. MATERIALS AND METHODS: We performed an experimental prospective animal study in our animal research center with 20 Hartley guinea pigs divided into 2 groups. The first group (experimental) was treated with Locacorten Vioform in one ear and with a physiologic saline solution in the other. The second group (positive control) was treated with concentrated gentamycin in one ear and physiologic saline in the other. Auditory brainstem response measurements were obtained before and after three sets of injections. Statistics were analyzed using a variance analysis with repeated measures. The histological state of cochlear outer hair cells was compared between the two groups using scanning electron microscopy. RESULTS: Average hearing loss in ears treated with Locacorten Vioform was 32.1 dB, compared with a 2.5 dB average loss in the saline-treated ears. Ears treated with gentamycin lost an average of 33.0 dB. There were clinically and statistically significant differences between the two ears of the guinea pigs in both groups (p < 0.001). Scanning electron microscopy revealed severe pericochlear and cochlear inflammation and ossification in the Locacorten Vioform-treated ears. Gentamycin caused significant destruction of outer hair cell architecture. CONCLUSIONS: Locacorten Vioform induces a hearing loss similar to that caused by gentamycin when applied directly to the middle ear of a guinea pig model. Electron microscopy indicates a pericochlear and cochlear inflammatory reaction with ossification.
Assuntos
Animais , Experimentação Animal , Clioquinol , Orelha , Orelha Média , Potenciais Evocados Auditivos do Tronco Encefálico , Gentamicinas , Cobaias , Guiné , Cabelo , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , Perda Auditiva , Inflamação , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Otomicose , Estudos Prospectivos , Cloreto de SódioRESUMO
In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.
Assuntos
Animais , Feminino , Órgão Espiral/cirurgia , Células-Tronco , Transplante de Células-Tronco/métodos , Células Ciliadas Auditivas Internas/transplante , Perda Auditiva Neurossensorial/cirurgia , Limiar Auditivo , Imuno-Histoquímica , Inibidores da Síntese de Proteínas , Neomicina , Sobrevivência Celular , Células Cultivadas , Reprodutibilidade dos Testes , Potenciais Evocados Auditivos do Tronco Encefálico , Resultado do Tratamento , Cobaias , Camundongos Endogâmicos BALB CRESUMO
<p><b>OBJECTIVE</b>To investigate the expression of Dynamin subtypes in inner hair cell (IHC) of mice, and to discuss their possible roles in age-related hearing loss.</p><p><b>METHODS</b>Auditory brainstem response (ABR) was recorded from the Kunming mice on postnatal 3 weeks (young), 10 weeks (adult), and 16 months (aged), 10 mice in each group. The expression of each Dynamin isoforms in the hair cells of the cochlea was observed by immunofluorescence staining and confocal microscope, and the transcription level of Dynamin subtypes mRNA was detected in qRT-PCR. Data analysis using SPSS 18 software.</p><p><b>RESULTS</b>ABR threshold showed no significant difference between the group of young and adult (t = -5.273, P = 0.076), but the threshold of the aged group increased comparing with young group (t = -8.365, P = 0.000), and adult group (t = -6.191, P = 0.000). All subtypes expressed in the inner hair cell of mice, of which Dynamin-1 and 2 expressed in the whole inner hair cell in the group of young and adult. In the aged group, Dynamin-1 was lost beneath the nucleus, and Dynamin-2 only be found near the nucleus. In addition, Dynamin-3 was scattered in the region of the basal part of the cells beneath the nucleus and near the spiral ganglion. The qRT-PCR revealed that mRNA of Dynamin-1 reduced with age (F = 10.410, P = 0.011), mRNA of Dynamin-2 increased to a peak in the adult group and then reduced with age (F = 24.575, P = 0.000). Meanwhile, mRNA of Dynamin-3 was not be detected.</p><p><b>CONCLUSIONS</b>All subtypes of Dynamin express in IHC. The expression of Dynamin-1 and 2 is up-regulated during maturity, which might alter the endocytosis of IHC; and the disorder of endocytosis might modulate the synaptic transmission of IHC. Whether Dynamin-3 plays a role in inner hair cells remains unclear because of the low expression.</p>
Assuntos
Animais , Camundongos , Envelhecimento , Metabolismo , Cóclea , Metabolismo , Dinaminas , Metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas Internas , MetabolismoRESUMO
OBJECTIVE@#To investigate the expression of adaptin-2(AP-2) in mice cochlea and to discuss the probable role in the endocytosis of hair cells.@*METHOD@#Laser scanning confocal microscopy and immune-fluroscence histochemistry were performed in this study.@*RESULT@#In mature mice cochlea, the immunoreactivity for AP-2 was found in the inner hair cells cytoplasm. This protein mainly expressed in the hair cells basal part and nearby the ribbon synapse.@*CONCLUSION@#AP-2 protein mainly expressed in the hair cells synaptic activity zone , which suggested that AP-2 could play an important role in the synaptic vesicle endocytosis. This finding built the foundation for the further research involved in the physiological and pathological role of AP-2.
Assuntos
Animais , Camundongos , Subunidades alfa do Complexo de Proteínas Adaptadoras , Metabolismo , Cóclea , Células Ciliadas Auditivas , Células Ciliadas Auditivas Internas , Metabolismo , Microscopia Confocal , SinapsesRESUMO
OBJECTIVE@#To investigate the expression of Myosin VI and Disabled-2 (Dab2) in the cochlea of mice at different ages.@*METHOD@#Forty KM mice were divided into four groups according to age, named as postnatal 2 week (P2w), P5w, P9w, P16month. The localization of protein in the basilar membrane of mice cochlea was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). The mRNA expression level of protein in cochlear at different ages was evaluated by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Statistical analysis was performed by the SPSS18.0 software.@*RESULT@#Myosin VI and Disabled-2 protein mainly expressed at the apical cytoplasm of hair cells. As for the inner hair cell, Dab2 labeling was abundant especially at the cuticular plate and nearby. Comparing four immunofluorescence staining images of Myosin VI, we found the fluorescence intensity of P2w and P16m were weaker than that of P5w and P9w. After setting P9w as the control group, qRT-PCR revealed that the mRNA expression of MyosinVI and Dab2 in P2w was less than that in the control group (P 0.05).@*CONCLUSION@#Myosin VI and Dab2, two proteins which regulated the clathrin-mediated endocytosis, expressed at hair cells of mice cochlea. In the inner hair cell, this process of endocytosis may be more efficient at the cuticular plate and nearby. The expression level of protein may change in different ages, and this probably leads to a difference of CME, it also may cause a defect of inner hair cells function.
Assuntos
Animais , Camundongos , Proteínas Adaptadoras de Transporte Vesicular , Metabolismo , Envelhecimento , Cóclea , Metabolismo , Endocitose , Células Ciliadas Auditivas , Metabolismo , Células Ciliadas Auditivas Internas , Metabolismo , Microscopia Confocal , Cadeias Pesadas de Miosina , MetabolismoRESUMO
<p><b>BACKGROUND</b>Previous studies have suggested that primary degeneration of hair cells causes secondary degeneration of spiral ganglion neurons (SGNs), but the effect of SGN degeneration on hair cells has not been studied. In the adult mouse inner ear ouabain can selectively and permanently induce the degeneration of type 1 SGNs while leaving type 2 SGNs, efferent fibers, and sensory hair cells relatively intact. This study aimed to investigate the dynamic changes in hair cell ribbon synapse induced by loss of SGNs using ouabain application to the round window niche of adult mice.</p><p><b>METHODS</b>In the analysis, 24 CBA/CAJ mice aged 8-10 weeks, were used, of which 6 normal mice were used as the control group. After ouabain application in the round window niche 6 times in an hour, ABR threshold shifts at least 30 dB in the three experimental groups which had six mice for 1-week group, six for 1-month group, and six for 3-month group. All 24 animals underwent function test at 1 week and then immunostaining at 1 week, 1 month, and 3 months.</p><p><b>RESULTS</b>The loss of neurons was followed by degeneration of postsynaptic specializations at the afferent synapse with hair cells. One week after ouabain treatment, the nerve endings of type 1 SGNs and postsynaptic densities, as measured by Na/K ATPase and PSD-95, were affected but not entirely missing, but their partial loss had consequences for synaptic ribbons that form the presynaptic specialization at the synapse between hair cells and primary afferent neurons. Ribbon numbers in inner hair cells decreased (some of them broken and the ribbon number much decreased), and the arrangement of the synaptic ribbons had undergone a dynamic reorganization: ribbons with or without associated postsynaptic densities moved from their normal location in the basal membrane of the cell to a more apical location and the neural endings alone were also found at more apical locations without associated ribbons. After 1 month, when the neural postsynaptic densities had completed their degeneration, most ribbons were lost and the remaining ribbons had no contact with postsynaptic densities; after 3 months, the ribbon synapses were gone except for an occasional remnant of a CtBP2-positive vesicle. Hair cells were intact other than the loss of ribbons (based on immunohistochemistry and DPOAE).</p><p><b>CONCLUSION</b>These findings define the effect of SGN loss on the precise spatiotemporal size and location of ribbons and the time course of synaptic degeneration and provide a model for studying plasticity and regeneration.</p>
Assuntos
Animais , Feminino , Camundongos , Células Ciliadas Auditivas , Biologia Celular , Fisiologia , Células Ciliadas Auditivas Internas , Biologia Celular , Fisiologia , Camundongos Endogâmicos CBA , Sinapses , FisiologiaRESUMO
OBJECTIVE@#To establish the stable and efficient hearing damage model by using low dosage of cispla tin, and investigate the mechanism.@*METHOD@#C57 mice were divided into 7 groups (every group, n = 8), the first group was the control group, the others were separately intraperitoneally injected with different dosages of cispla tin for different time. We measured the auditory brainstem response (ABR) of the mice, and obtained the basal coil of organ Corti. We observed the shape of inner hair cells (IHC) by staining AgNO3 and marked otoferlin in the IHC by immunofluorescence,successively sliced by laser confocal microscopy. The RNA fragments were amplified by RT-PCR.@*RESULT@#After cisplatin administration for four days, the thresholds of the ABR improved in 1.5 mg/kg and 3.0 mg/kg group, and compared with the control group, the ABR thresholds improved in each group with ciplatin administration for seven days. With the same dosage, the ABR threshold of the 0.75 mg/kg x 7 d group was higher than 0.75 mg/kg x 4 d group, and there was no time-effect relationship existing in other groups with different dosage. The otoferlin was less expressed 3.0 mg/kg groups than the control group. However, the oto ferlin expressed in the 1.5 mg/kg groups were almost the same as the control group. The alteration of the IHC was observed most remarkablely in 3.0 mg/kg x 7 d group.@*CONCLUSION@#Low dosage of cisplatin can impair the hearing, and the expression of otoferlin may involve in the underlying mechanism.
Assuntos
Animais , Camundongos , Cisplatino , Toxicidade , Cóclea , Metabolismo , Patologia , Células Ciliadas Auditivas Internas , Patologia , Proteínas de Membrana , Metabolismo , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE@#To learn the influence the gentamycin on C57BL/6J mice hear and cochlear hair cell ribbon synapses CaV1.3 calcium protein amount. To explore the relationship between hear loss and its dosage correlation change and significance.@*METHOD@#The fixed amino glucoside to C57BL/6J mice was used to make abdominal cavity injection mold every day. The auditory brain-stem response ABR was used to measure the hear of mice in 7th, 14th, 28th after the injection. Immunofluorescence method was used to observe cochlear basement membrane of hair ribbon synapse CaV1.3 calcium channel proteins in the distribution and expression. Inner hair cells synaptic membrane was immune fluorescent tags with CtbP2 and CaV1. 3.@*RESULT@#With the growth of the injected drugs, ABR threshold increased,but all the hair cells and shape had no obvious change. However the amount of hair rib bon synapse CaV1.3 calcium ion channel proteins in the expression had significant differences (P < 0.01). CaV1.3 calcium ion channel proteins increased slightly lower than normal at 7th day, significantly decreased at 14th day, had increased, increased quantity compare with 14th day, but at 28th day after intraperitoneal injection of gentamicin.@*CONCLUSION@#The increasing,decreasing and increasing trend of cochlear hair cells CaV1.3 proteins in the environment of amino glucoside drug toxicity showed that the increase of hair ribbon synapse CaV1.3 proteins may have a compensatory effect on the drug toxicity. With the increase of the drug toxicity effect, this kind of decompensated function could be the listening decline, which may be one of the mechanism of damage to hearing.
Assuntos
Animais , Camundongos , Canais de Cálcio Tipo L , Metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico , Gentamicinas , Farmacologia , Células Ciliadas Auditivas Internas , Metabolismo , Camundongos Endogâmicos C57BL , ProteômicaRESUMO
OBJECTIVE@#To detect the expression variation of microRNA-183 family in cochlea of animal model characterized by noise-induced deafness at various time points, and to explore the mechanisms responsible for noise-induced deafness.@*METHOD@#Fifty mice were randomly divided into 5 groups. In the experimental group, 40 mice were exposed to 2-4 kHz narrow band noise at 100 dB SPL 6h per day for 3 consecutive days. The rest 10 mice served as the control group without receiving any noise. Auditory brainsterm response (ABR) were examined at the 1st, 7th, 14th and 28th day compaired with the ABR before the experiment,to confirm noise lead to the permanent threshold shift. The pathological damage processes of hair cell were detected by the basilar membrane stretched techniques. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was apply to quantify the expression of microRNA183 family members. Statistical analysis was performed by the SPSS 17.0 software.@*RESULT@#The hearing of mice in the experimental group was significantly less than that in the control group. In the experimental group, the hearing of mice exposed to noise were markedly less when compared with the one exposure to null-noise. The hearing in the 1st day group was least among experimental groups, and the followed one was mice in the 7th day group. No statistical difference were observed between the 14th and 28th day groups (P > 0.05). The results of surface preparation showed that the outer hair cells were chaotic, deformational, and their number decreased is time-dependent. The missing of the outer hair cells occurred mainly in the first and second rows, while the inner hair cells were not pronouncedly missing. The qRT-PCR showed that the expressions of the three genes (miR-183/96/182)in the 1st day and 7th day group with exposure to noise were less than in the control group (P 0.05). The expressions rised in the 14th day experimental groups, whereas the 28th day group's expressions of the three genes decreased markedly which were more than that in the 1st day and 7th day group (P < 0.01).@*CONCLUSION@#After noise exposure for some time, the expressions of miRNA-183 family members have significant changes in animal model with noise-induced deafness, which indicated that the miRNA183 family members may play important roles in the pathogenesis and development of noise-induced deafness.
Assuntos
Animais , Feminino , Masculino , Camundongos , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas Internas , Patologia , Células Ciliadas Auditivas Externas , Patologia , Perda Auditiva Provocada por Ruído , Metabolismo , Patologia , MicroRNAs , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To investigate the effects on ribbon synapse of inner hair cells after superpulsed CO2 laser-assisted cochleostomy in SD rats.</p><p><b>METHODS</b>Eighteen SD rats were randomly divided into laser-assisted surgery groups (2 W group and 5 W group), sham-operated group and control group. Ten of those were performed a cochleostomy using superpulsed CO2 laser with a corresponding power. Auditory brainstem responses (ABR) were measured pre-and postoperatively. The ribbon synapses at apical and middle cochlear turns were observed under laser scanning confocal microscope and then were quantified with 3ds Max software.</p><p><b>RESULTS</b>The postoperative ABR thresholds of the 2 W and 5 W groups were larger than the preoperative case (t = -5.65, P < 0.01; t = -4.97, P < 0.01). The synapse number at the middle turn decreased significantly in 5 W group (F = 17.15, P < 0.01), while no significant changes were noted at the apical turn (P > 0.05). There was no statistical difference in 2 W group (P > 0.05).</p><p><b>CONCLUSIONS</b>The superpulsed CO2 laser-assisted cochleostomy with high-power is accompanied by a synaptic injury, while no obvious effects after the low-power laser surgery, which might be a safe strategy to preform cochleostomy.</p>
Assuntos
Animais , Ratos , Cóclea , Cirurgia Geral , Células Ciliadas Auditivas Internas , Efeitos da Radiação , Terapia a Laser , Lasers de Gás , Distribuição Aleatória , Ratos Sprague-Dawley , Sinapses , Efeitos da RadiaçãoRESUMO
<p><b>OBJECTIVE</b>To investigate the function of myosin light chain kinase (MLCK) in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing.</p><p><b>METHODS</b>Cross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response (ABR) to evaluate mice hearing function at different frequencies.</p><p><b>RESULTS</b>Mlck knockout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones (8 kHz, 16 kHz, 32 kHz), and the rise was over 20 dB at high-frequency(32 kHz). Further analyses of waveforms showed that wave-I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone (16 kHz) were less than control group(P < 0.05) on average, but the ratio of wave I/II and I/III were not difference (P > 0.05).</p><p><b>CONCLUSIONS</b>Mlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high-frequencies.</p>
Assuntos
Animais , Camundongos , Audiometria de Tons Puros , Limiar Auditivo , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas Internas , Metabolismo , Perda Auditiva , Genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinase de Cadeia Leve de Miosina , GenéticaRESUMO
Hair cells at the base of the cochlea appear to be more susceptible to damage by the aminoglycoside gentamicin than those at the apex. However, the mechanism of base-to-apex gradient ototoxicity by gentamicin remains to be elucidated. We report here that gentamicin caused rodent cochlear hair cell damages in a time- and dose-dependent manner. Hair cells at the basal turn were more vulnerable to gentamicin than those at the apical turn. Gentamicin-conjugated Texas Red (GTTR) uptake was predominant in basal turn hair cells in neonatal rats. Transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4) expression was confirmed in the cuticular plate, stereocilia and hair cell body of inner hair cells and outer hair cells. The involvement of TRPV1 and TRPV4 in gentamicin trafficking of hair cells was confirmed by exogenous calcium treatment and TRPV inhibitors, including gadolinium and ruthenium red, which resulted in markedly inhibited GTTR uptake and gentamicin-induced hair cell damage in rodent and zebrafish ototoxic model systems. These results indicate that the cytotoxic vulnerability of cochlear hair cells in the basal turn to gentamicin may depend on effective uptake of the drug, which was, in part, mediated by the TRPV1 and TRPV4 proteins.
Assuntos
Animais , Ratos , Morte Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gadolínio/metabolismo , Gentamicinas/metabolismo , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Ratos Sprague-Dawley , Rutênio Vermelho/metabolismo , Canais de Cátion TRPV/metabolismo , Fatores de Tempo , Xantenos/metabolismo , Peixe-ZebraRESUMO
<p><b>OBJECTIVE</b>To investigate the feasibility of orientation-induced differentiation of rat bone marrow-derived mesenchymal stem cells (MSC) into cochlear hair cell-like cells in vitro.</p><p><b>METHODS</b>MSC were extracted, cultured and identified. The organ of Corti was isolated from post natal day 1-day 3 Sprague-Dawley rats. Then MSC were co-cultured with organ of Corti for 14 days in vitro. The expression of hair cell markers (myosin VIIa, math1 and calretinin) was detected by immunocytochemical analyses and by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The differentiated cells were immunopositive for myosin VIIa, calretinin and math1. And RT-PCR results indicated that these differentiated cells expressed hair cell markers Math1 and myosin VIIa.</p><p><b>CONCLUSIONS</b>MSC cultured in vitro could be successfully induced to differentiate into hair cell-like cells with hair cell molecular markers.</p>
Assuntos
Animais , Ratos , Células da Medula Óssea , Biologia Celular , Diferenciação Celular , Células Cultivadas , Células Ciliadas Auditivas Internas , Biologia Celular , Células-Tronco Mesenquimais , Biologia Celular , Ratos Sprague-DawleyRESUMO
<p><b>OBJECTIVE</b>To study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro.</p><p><b>METHODS</b>After the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software.</p><p><b>RESULTS</b>GM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05).</p><p><b>CONCLUSIONS</b>ELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.</p>