RESUMO
Despite the excellent antimicrobial activity of aminoglycoside antibiotics, permanent inner ear damage associated with the use of these drugs has resulted in the need to develop strategies to address the ototoxic risk given their widespread use. In a previous study, we showed that avocado oil protects ear hair cells from damage caused by neomycin. However, the detailed mechanism by which this protection occurs is still unclear. Here, we investigated the auditory cell-protective mechanism of enhanced functional avocado oil extract (DKB122). RNA sequencing followed by pathway analysis revealed that DKB122 has the potential to enhance the expression of detoxification and antioxidant genes associated with glutathione metabolism (Hmox4, Gsta4, Mgst1, and Abcc3) in HEI-OC1 cells. Additionally, DKB122 effectively decreased ROS levels, resulting in the inhibition of apoptosis in HEI-OC1 cells. The expression of the inflammatory genes that encode chemokines and interleukins was also downregulated by DKB122 treatment. Consistent with these results, DKB122 significantly inhibited p65 nuclear migration induced by TNF-α or LPS in HEI-OC1 cells and THP-1 cells and the expression of inflammatory chemokine and interleukin genes induced by TNF-α was significantly reduced. Moreover, DKB122 treatment increased LC3-II and decreased p62 in HEI-OC1 cells, suggesting that DKB122 increases autophagic flux. These results suggest that DKB122 has otoprotective effects attributable to its antioxidant activity, induction of antioxidant gene expression, anti-inflammatory activity, and autophagy activation.
Assuntos
Aminoglicosídeos/efeitos adversos , Antibacterianos/efeitos adversos , Ototoxicidade/tratamento farmacológico , Ototoxicidade/etiologia , Ototoxicidade/genética , Persea/química , Óleos de Plantas/farmacologia , Óleos de Plantas/uso terapêutico , Autofagia/efeitos dos fármacos , Autofagia/genética , Células Cultivadas , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Humanos , Mediadores da Inflamação/metabolismo , Desintoxicação Metabólica Fase I/genética , Ototoxicidade/patologia , Estresse Oxidativo/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: Nicotinamide riboside (NR) has been proved to protect the hearing. To achieve animal models of temporary threshold shift (TTS) and permanent threshold shift (PTS) respectively, evaluate the dynamic change of ribbon synapse before and after NR administration. METHODS: Mice were divided into control group, noise exposure (NE) group and NR group. The noise was exposed to NE and NR group, and NR was injected before noise exposure. Auditory brainstem response (ABR), ribbon synapse count and cochlear morphology were tested, as well as the concentration of hydrogen peroxide (H2O2) and ATP. RESULTS: Ribbon synapse count decrease with the intensity of noise exposure, and the cochlear morphology remains stable during TTS and was damaged during PTS. NR promotes the oxidation resistance to protect the synapse and the inner ear morphology. CONCLUSION: Our findings suggest that TTS mice are more vulnerable to noise, and NR can promote the recovery of the synapse count to protect the animals' hearing.
Assuntos
Estimulação Acústica/efeitos adversos , Células Ciliadas Auditivas Internas/fisiologia , Perda Auditiva Provocada por Ruído/prevenção & controle , Niacinamida/análogos & derivados , Compostos de Piridínio/uso terapêutico , Recuperação de Função Fisiológica/fisiologia , Sinapses/fisiologia , Animais , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Perda Auditiva Provocada por Ruído/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Compostos de Piridínio/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/patologiaRESUMO
Salicylate is widely used to produce animal models of tinnitus in mice and/or rats. The side effects on auditory function, including hearing loss and tinnitus, are considered the results of the auditory nerve dysfunction. A recent study indicated that chronic treatment with salicylate for several weeks reduces compressed action potential amplitude, which is contradictory to the studies reporting excessive activation of NmethylDaspartate receptors (NMDAR) in tinnitusinduced animals. The specific aims of the experiment were to detect the effect of salicylate on the inner hair cells (IHCs), ribbon synapse, as well as the association between the hearing threshold and the number of mismatched ribbon synapses. In the present study, mice were injected intraperitoneally with a low dose of salicylate (200 mg/kg) for 14 days. The auditory brainstem response and otoacoustic emission were measured to assess auditory function of the mice. The postsynaptic regions of IHC were identified with two types of immunostaining targets: Postsynaptic density protein 95 and Glu2/3. The number of spheres was counted and the synapses were reconstructed in 3dimensional images. Increases in distortion product otoacoustic emissions amplitudes of the salicylate group were detected, however, an elevation in the hearing threshold was also observed. A mismatch between preand postribbon synapses was observed. In addition, the cochlear components, including the numbers of outer hair cells and IHCs, were unlikely to be affected by salicylate. IHC ribbon synapses were more susceptible to salicylate stimuli. Furthermore, mismatch of pre and postribbon synapses may indicate a competitive inhibition between NMDAR and α-amino3hydroxy-5-methyl-4-isoxa-zole-propionate receptors and dysfunction of ribbon synapses.
Assuntos
Células Ciliadas Auditivas Internas/efeitos dos fármacos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Salicílico/farmacologia , Sinapses/metabolismo , Estimulação Acústica/métodos , Animais , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Células Ciliadas Auditivas Internas/metabolismo , Audição/efeitos dos fármacos , Perda Auditiva/tratamento farmacológico , Perda Auditiva/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Emissões Otoacústicas Espontâneas/efeitos dos fármacosRESUMO
The zebrafish has become an established model organism for the study of hearing and balance systems in the past two decades. The classical approach to examine hair cells is to use dye to conduct selective staining, which shows the number and morphology of hair cells but does not reveal their function. Startle response is a behavior closely related to the auditory function of hair cells; therefore it can be used to measure the function of hair cells. In this study, we developed a device to measure the startle response of zebrafish larvae. By applying various levels of stimulus, it showed that the system can discern a 10 dB difference. The hair cell in zebrafish can regenerate after damage due to noise exposure or drug treatment. With this device, we measured the startle response of zebrafish larvae during and after drug treatment. The results show a similar trend to the classical hair cell staining method. The startle response was reduced with drug treatment and recovered after removal of the drug. Together it demonstrated the capability of this behavioral assay in evaluating the hair cell functions of fish larvae and its potential as a high-throughput screening tool for auditory-related gene and drug discovery.
Assuntos
Comportamento Animal , Células Ciliadas Auditivas Internas/fisiologia , Reflexo de Sobressalto , Regeneração , Estimulação Acústica/métodos , Animais , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Larva , Neomicina/administração & dosagem , Reflexo de Sobressalto/efeitos dos fármacos , Peixe-ZebraRESUMO
CONTEXT: Ototoxicity due to medications, such as aminoglycosides, is irreversible, and free radicals in the inner ear are assumed to play a major role. Because melatonin has an antioxidant property, we hypothesize that it might mitigate hair cell injury by aminoglycosides. OBJECTIVE: The objective of this study was to evaluate whether melatonin has an alleviative effect on neomycin-induced hair cell injury in zebrafish (Danio rerio). METHODS: Various concentrations of melatonin were administered to 5-day post-fertilization zebrafish treated with 125 µM neomycin for 1 h. Surviving hair cells within four neuromasts were compared with that of a control group. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The changes of ultrastructure were confirmed using a scanning electron microscope. RESULTS: Melatonin alleviated neomycin-induced hair cell injury in neuromasts (neomycin + melatonin 100 µM: 13.88 ± 0.91 cells, neomycin only: 7.85 ± 0.90 cells; n = 10, p < 0.05) and reduced neomycin-induced apoptosis in the TUNEL assay. In ultrastructural analysis, hair cells within the neuromasts in zebrafish were preserved exposed to 125 µM neomycin and 100 µM melatonin for 1 h in SEM findings. CONCLUSION: Melatonin is effective in alleviating aminoglycoside-induced hair cell injury in zebrafish. The results of this study demonstrated that melatonin has the potential to reduce apoptosis induced by aminoglycosides in zebrafish.
Assuntos
Antibacterianos/efeitos adversos , Sequestradores de Radicais Livres/metabolismo , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Melatonina/metabolismo , Neomicina/efeitos adversos , Inibidores da Síntese de Proteínas/efeitos adversos , Animais , Animais Geneticamente Modificados , Antibacterianos/química , Apoptose/efeitos dos fármacos , Suplementos Nutricionais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Neomicina/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Concentração Osmolar , Inibidores da Síntese de Proteínas/química , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Functional maturation of afferent synaptic connections to inner hair cells (IHCs) involves pruning of excess synapses formed during development, as well as the strengthening and survival of the retained synapses. These events take place during the thyroid hormone (TH)-critical period of cochlear development, which is in the perinatal period for mice and in the third trimester for humans. Here, we used the hypothyroid Snell dwarf mouse (Pit1(dw)) as a model to study the role of TH in afferent type I synaptic refinement and functional maturation. We observed defects in afferent synaptic pruning and delays in calcium channel clustering in the IHCs of Pit1(dw) mice. Nevertheless, calcium currents and capacitance reached near normal levels in Pit1(dw) IHCs by the age of onset of hearing, despite the excess number of retained synapses. We restored normal synaptic pruning in Pit1(dw) IHCs by supplementing with TH from postnatal day (P)3 to P8, establishing this window as being critical for TH action on this process. Afferent terminals of older Pit1(dw) IHCs showed evidence of excitotoxic damage accompanied by a concomitant reduction in the levels of the glial glutamate transporter, GLAST. Our results indicate that a lack of TH during a critical period of inner ear development causes defects in pruning and long-term homeostatic maintenance of afferent synapses.
Assuntos
Cóclea/crescimento & desenvolvimento , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Internas/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Tri-Iodotironina/fisiologia , Oxirredutases do Álcool , Animais , Canais de Cálcio Tipo L/metabolismo , Proteínas Correpressoras , Cóclea/efeitos dos fármacos , Cóclea/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Sinapses/efeitos dos fármacos , Fator de Transcrição Pit-1/genética , Tri-Iodotironina/administração & dosagemRESUMO
Poorer hearing in the presence of background noise is a significant problem for the hearing impaired. Ototoxic drugs, ageing, and noise exposure can damage the sensory hair cells of the inner ear that are essential for normal hearing sensitivity. The relationship between outer hair cell (OHC) loss and progressively poorer hearing sensitivity in quiet or in competing background noise is supported by a number of human and animal studies. In contrast, the effect of moderate inner hair cell (IHC) loss or dysfunction shows almost no impact on behavioral measures of hearing sensitivity in quiet, when OHCs remain intact, but the relationship between selective IHC loss and hearing in noise remains relatively unknown. Here, a moderately high dose of carboplatin (75 mg/kg) that produced IHC loss in chinchillas ranging from 40 to 80 % had little effect on thresholds in quiet. However, when tested in the presence of competing broadband (BBN) or narrowband noise (NBN), thresholds increased significantly. IHC loss >60 % increased signal-to-noise ratios (SNRs) for tones (500-11,300 Hz) in competing BBN by 5-10 dB and broadened the masking function under NBN. These data suggest that IHC loss or dysfunction may play a significant role in listening in noise independent of OHC integrity and that these deficits may be present even when thresholds in quiet are within normal limits.
Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Perda Auditiva Provocada por Ruído/etiologia , Animais , Limiar Auditivo , Carboplatina/toxicidade , Chinchila , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Masculino , Ácido gama-Aminobutírico/fisiologiaRESUMO
The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been known to contribute to the pathogenesis of noise-induced hearing loss. In this study, we discovered that in BALB/c mice pretreatment with methylene blue (MB) for 4 consecutive days significantly protected against cochlear injury by intense broad-band noise for 3 h. It decreased both compound threshold shift and permanent threshold shift and, further, reduced outer hair cell death in the cochlea. MB also reduced ROS and RNS formation after noise exposure. Furthermore, it protected against rotenone- and antimycin A-induced cell death and also reversed ATP generation in the in vitro UB-OC1 cell system. Likewise, MB effectively attenuated the noise-induced impairment of complex IV activity in the cochlea. In addition, it increased the neurotrophin-3 (NT-3) level, which could affect the synaptic connections between hair cells and spiral ganglion neurons in the noise-exposed cochlea, and also promoted the conservation of both efferent and afferent nerve terminals on the outer and inner hair cells. These findings suggest that the amelioration of impaired mitochondrial electron transport and the potentiation of NT-3 expression by treatment with MB have a significant therapeutic value in preventing ROS-mediated sensorineural hearing loss.
Assuntos
Perda Auditiva Provocada por Ruído/tratamento farmacológico , Azul de Metileno/uso terapêutico , Estimulação Acústica , Animais , Limiar Auditivo/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/patologia , Cóclea/fisiopatologia , Cóclea/ultraestrutura , Proteína 4 Homóloga a Disks-Large , Transporte de Elétrons/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Guanilato Quinases/metabolismo , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patologia , Perda Auditiva Provocada por Ruído/patologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Imuno-Histoquímica , Masculino , Proteínas de Membrana/metabolismo , Azul de Metileno/administração & dosagem , Azul de Metileno/farmacologia , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurotrofina 3/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Noise trauma, aging, and ototoxicity preferentially damage the outer hair cells of the inner ear, leading to increased hearing thresholds and poorer frequency resolution. Whereas outer hair cells make synaptic connections with less than 10% of afferent auditory nerve fibers (type-II), inner hair cells make connections with over 90% of afferents (type-I). Despite these extensive connections, little is known about how selective inner hair cell loss impacts hearing. In chinchillas, moderate to high doses of the anticancer compound carboplatin produce selective inner hair cell and type-I afferent loss with little to no effect on outer hair cells. To determine the effects of carboplatin-induced inner hair cell loss on the most widely used clinical measure of hearing, the audiogram, pure-tone thresholds were determined behaviorally before and after 75 mg/kg carboplatin. Following carboplatin treatment, small effects on audiometric thresholds were observed even with extensive inner hair cell losses that exceed 80%. These results suggest that conventional audiometry is insensitive to inner hair cell loss and that only small populations of inner hair cells appear to be necessary for detecting tonal stimuli in a quiet background.
Assuntos
Audiometria de Tons Puros , Carboplatina/efeitos adversos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Estimulação Acústica , Animais , Antineoplásicos/efeitos adversos , Limiar Auditivo/efeitos dos fármacos , Limiar Auditivo/fisiologia , Chinchila , Cóclea/efeitos dos fármacos , Cóclea/patologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Audição/fisiologia , Masculino , Psicofísica , Células Receptoras Sensoriais/patologiaRESUMO
Although salicylate is one of the most widely used nonsteroidal anti-inflammatory drugs, it causes moderate hearing loss and tinnitus at high-dose levels. In the present study, salicylate effects on the K currents in inner hair cells were examined. Salicylate reversibly reduced the outward K currents (I(K,f)), but did not affect the inward current (I(K,n)). Salicylate blocked the outward K currents in a concentration-dependent manner according to Hill equation with a half-blocking concentration of 1.66mM, and the Hill coefficient of 1.86.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cóclea/efeitos dos fármacos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/fisiologia , Canais de Potássio/efeitos dos fármacos , Salicilato de Sódio/farmacologia , Animais , Cóclea/fisiologia , Cobaias , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/fisiologiaRESUMO
OBJECTIVE: To study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Gentamicinas/toxicidade , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/patologia , Prescrições , Animais , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/patologia , Camundongos , ComprimidosRESUMO
CONCLUSION: Excess glutamate (Glu) exposure (20 mM) in the cochlear perilymph affects the physiological function of outer hair cells (OHCs) within a 2 h period and induces apoptosis in the modiolus spiral ganglion neurons (SGNs) in an apoptosis-inducing factor (AIF)-dependent manner. OBJECTIVES: To determine whether high-dose Glu affects the function of OHCs and whether it induces AIF- and caspase-3-dependent apoptosis in the cochlear SGNs. METHODS: Perilymphatic perfusions of Glu (20 mM) and artificial perilymph (AP) solutions were performed in adult guinea pig cochleae. Both cochlear microphonics (CM) and electrical auditory brainstem response (eABR) were measured before and 2 h after perfusions. The hair cell morphologies were examined using transmission electron microscopy. The expression of two apoptotic indicators, AIF and caspase-3, was examined 8 h after perfusions. RESULTS: In contrast to AP perfusions, the perfusion of 20 mM Glu caused significant reduction in the CM and eABR amplitudes. Inner hair cells (IHCs) after Glu perfusion were deformed and exhibited vacuolization in the postsynaptic region, whereas the OHC system appeared unaffected. AIF expression was detected in the nuclei of SGNs 8 h after Glu exposure, but the expression of caspase-3 was not shown in any cochlear tissues.
Assuntos
Ácido Glutâmico/toxicidade , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Gânglio Espiral da Cóclea/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Caspase 3/metabolismo , Potenciais Microfônicos da Cóclea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Cobaias , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/patologia , Microscopia Eletrônica de Transmissão , Neurônios/efeitos dos fármacos , Neurônios/patologia , Perfusão , Perilinfa , Gânglio Espiral da Cóclea/patologiaRESUMO
BACKGROUND: Heat shock protein 70 (HSP70) protects inner ear cells from damage and death induced by e.g. heat or toxins. Benzoquinone ansamycin antibiotic geldanamycin (GA) was demonstrated to induce the expression of HSP70 in various animal cell types. The aim of our study was to investigate whether GA induces HSP70 in the organ of Corti (OC), which contains the auditory sensory cells, and whether GA can protect these cells from toxicity caused by a common aminoglycoside antibiotic gentamicin. METHODS: To address these questions, we used the OC explants isolated from p3-p5 rats. As a read-out, we used RT-PCR, ELISA and immunofluorescence. RESULTS: We found that GA at the concentration of 2 microM efficiently induced HSP70 expression on mRNA and protein level in the OC explants. Confocal microscopy revealed that HSP70 induced by GA is expressed by hair cells and interdental cells of spiral limbus. Preincubation of explants with 2 muM GA prior to adding gentamicin (500 microM) significantly reduced the loss of outer but not inner hair cells, suggesting different mechanisms of otoprotection needed for these two cell types. CONCLUSION: GA induced HSP70 in the auditory sensory cells and partially protected them from toxicity of gentamicin. Understanding the molecular mechanisms of GA otoprotection may provide insights for preventative therapy of the hearing loss caused by aminoglycoside antibiotics.
Assuntos
Benzoquinonas/uso terapêutico , Gentamicinas/toxicidade , Proteínas de Choque Térmico HSP70/biossíntese , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Perda Auditiva Neurossensorial/prevenção & controle , Lactamas Macrocíclicas/uso terapêutico , Órgão Espiral/efeitos dos fármacos , Animais , Benzoquinonas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Microscopia Confocal , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.
Assuntos
Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genética , Aminoglicosídeos/antagonistas & inibidores , Aminoglicosídeos/toxicidade , Animais , Sequência de Bases , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Cisplatino/toxicidade , Códon de Terminação/genética , Primers do DNA/genética , DNA Complementar/genética , Avaliação Pré-Clínica de Medicamentos , Epistasia Genética , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Perda Auditiva/etiologia , Perda Auditiva/genética , Perda Auditiva/prevenção & controle , Humanos , Camundongos , Neomicina/antagonistas & inibidores , Neomicina/toxicidade , Mutação Puntual , Sáculo e Utrículo/efeitos dos fármacos , Sáculo e Utrículo/patologia , Tiofenos/química , Tiofenos/farmacologia , Peixe-Zebra/fisiologiaRESUMO
A flavanoid fraction (FF) from Drynaria fortunei, was investigated to see if it has the protective and ameliorative effects against gentamicin (GM) ototoxicity in guinea pigs (n = 36). Eleven (GM-group) animals received GM 100 mg/kg/day. Eleven (GMFF-group) animals received the same dose of GM but 2 days prior were dosed with FF (10 mg/kg/day) for 2 weeks. Seven (S-group) animals received saline and seven (FF-group) animals received the same dose of FF as the GMFF-group. The thresholds of tone-burst auditory evoked response (ABR) at 2 k, 8 k, and 32 k Hz were determined to be as follows: GM-group: 90 dB, 92 dB and 72 dB, GMFF-group: 30 dB, 37 dB and 38 dB, FF-group: 28 dB, 25 dB and 29 dB, S-group: 30 dB, 28 dB and 39 dB. The GM-group had a significantly higher hearing threshold than the other groups (p < 0.05). The GMFF- and FF-groups had hearing thresholds similar to the S-groups (p > 0.1). Repair of damaged hair cells was observed histologically. The percentage of the damaged outer hair cells (OHC) and inner hair cells (IHC) were determined to be as follows: GM-group: 43% and 20%, GMFF-group: 20% and 2%, FF-group: 9% and 2% and S-group: 4% and 1%. The GMFF-group showed less damage to the OHC (p > 0.05) and significantly less damage to the IHC (p < 0.05) than the GM-group. FF did not change the antimicrobial activity of GM and it did not show any intrinsic antibacterial effect. FF did not affect the kinetics of GM during the course of the experiment.
Assuntos
Antibacterianos/farmacologia , Perda Auditiva/prevenção & controle , Fitoterapia , Extratos Vegetais/farmacologia , Polypodiaceae , Animais , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Escherichia coli/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Flavonas/administração & dosagem , Flavonas/farmacologia , Flavonas/uso terapêutico , Gentamicinas , Cobaias , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/ultraestrutura , Perda Auditiva/induzido quimicamente , Testes de Sensibilidade Microbiana , Mycobacterium avium/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêuticoRESUMO
We investigated the effect of glutamate receptor antagonists on progressive inner hair cell (IHC) loss following transient cochlear ischemia in gerbils. Transient cochlear ischemia was induced by 15-min bilateral vertebral artery occlusion. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-type glutamate receptor antagonist, 6-7-dinitroquinoxaline-2,3-dione (DNQX), or an N-methyl-D-aspartate (NMDA)-type receptor antagonist, MK-801, was administered 10 min before the ischemic insult. Hearing was assessed by sequentially recording compound action potentials (CAPs) before, during, and after the ischemia. The degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day after ischemia, the increases in the CAP threshold and the progressive IHC loss were significantly reduced in cochleae treated with DNQX, while MK-801 was ineffective. These results suggest that the AMPA receptor plays a critical role in the development of the progressive IHC loss induced by ischemia/reperfusion injury in the cochlea.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Cóclea/patologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Receptores de AMPA/antagonistas & inibidores , Estimulação Acústica , Animais , Cóclea/irrigação sanguínea , Cóclea/ultraestrutura , Nervo Coclear/efeitos dos fármacos , Nervo Coclear/ultraestrutura , Maleato de Dizocilpina/farmacologia , Gerbillinae , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Internas/ultraestrutura , Audição/efeitos dos fármacos , Imuno-Histoquímica , Isquemia/tratamento farmacológico , Masculino , Microscopia Eletrônica , Quinoxalinas/farmacologia , Receptores de AMPA/ultraestrutura , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/ultraestrutura , Sinapses/efeitos dos fármacos , Sinapses/ultraestruturaRESUMO
In the brain, fast wxcitatory synaptic transmission is mostly mediated by the alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) subtype of the glutamate receptors. Molecular cloning has revealed that four subunits, GluR1, GluR2, GluR3, and GluR4 form heteromeric receptors with high affinity for AMPA. Because antagonists and agonists do not discriminate between individual AMPA receptor subunits, we decided to use antisense oligonucleotides to block the expression of the GluR2 subunit within the receptor complex in adult animals. In the present study, we exploited several advantages afforded by the guinea pig cochlea to determine whether an antisense oligonucleotide directed to the mRNA of the GluR2 subunit could modify primary auditory neurotransmission. While a random probe with the same base composition had no effect, a GluR2 antisense oligonucleotide, continuously delivered into the cochlea, transiently reduced the compound action potential and diminished spontaneous activity of single auditory nerve fibers. Although antisense oligonucleotides penetrated a variety of cells, their effect could be physiologically localized to a single site of GluR2 antisense probe action, the primary auditory neuron. Subunit specificity of this effect was confirmed by a significant reduction in GluR2/3, but not GluR4 immunoreactivity in primary auditory neurons. Besides being the first demonstration that transient knockout of GluR2 subunit in adult animal modifies excitatory synaptic transmission in vivo, these results support the use of the antisense strategy as a powerful tool for blocking expression of any gene in the cochlea.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Receptores de AMPA/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Estimulação Acústica , Potenciais de Ação/efeitos dos fármacos , Animais , Audiometria de Tons Puros , Clonagem Molecular , Nervo Coclear/fisiologia , Difusão , Sistemas de Liberação de Medicamentos , Cobaias , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacocinética , Perilinfa , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genéticaRESUMO
Transient evoked otoacoustic emissions (TEOAE) produced by a 2 kHz tone burst could be detected in 30 out of 37 ears (81% detectability) in 21 cats. The amplitude of tone burst-evoked TEOAE was saturated at a stimulus level between 45 and 50 dB SPL and the latency time of peak amplitude was 6.23 ms on average (5.53-7.28 ms). The effects of pure tone overstimulation and short-term anoxia on the tone burst-evoked TEOAE in cats were evaluated. A permanent detection threshold shift of the TEOAE was confirmed at 24 h and 1 week after the overstimulation at 125 dB SPL. In these cases, damaged first row outer hair cells and inner hair cells were observed over an average length of 3.3 mm (16% of the entire cochlear length) by scanning electron microscopy. These findings suggested that the TEOAE can detect localized cochlear hair cell damage. A temporary detection threshold shift of the TEOAE was observed after the overstimulation at 105 dB SPL, and the threshold shift recovered in 107.5 min on average. In the short-term anoxia trial, the TEOAE amplitude started to decrease 45-90 s after the anoxia and recovered completely when the duration of anoxia was under 1 min. However, the TEOAE amplitude did not recover pre-anoxia values (it remained below 80% of its initial value) after 5 min when the anoxia was over 2 min. These findings demonstrated that the detection threshold and amplitude of the TEOAE were also affected by metabolic changes of the cochlear hair cells. Tone burst-evoked TEOAE are useful for the evaluation of localized histological and functional damage of the cochlear hair cells.
Assuntos
Estimulação Acústica , Hipóxia/fisiopatologia , Órgão Espiral/patologia , Emissões Otoacústicas Espontâneas/fisiologia , Animais , Limiar Auditivo/efeitos dos fármacos , Limiar Auditivo/fisiologia , Gatos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/ultraestrutura , Microscopia Eletrônica de Varredura , Fármacos Neuromusculares não Despolarizantes/toxicidade , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/ultraestrutura , Emissões Otoacústicas Espontâneas/efeitos dos fármacos , Pancurônio/toxicidadeRESUMO
In this study the effect of the neurotrophic ACTH(4-9) analogue, ORG 2766, on cisplatin cochleotoxicity was investigated with both light- and transmission electron microscopy. Guinea pigs were treated with either cisplatin+ORG 2766 (n = 11) or cisplatin + physiological saline (n = 9). All animals treated with cisplatin + physiological saline showed complete loss of outer hair cells (OHC) and degeneration of the organ of Corti in the basal cochlear turns, while partial OHC loss was found in the middle and apical turns. The inner hair cells (IHC) and other cochlear tissues were not affected. Eight animals from the group treated with cisplatin + ORG 2766 demonstrated similar pathological changes, but to a lesser degree, especially in the middle turns. The three remaining animals demonstrated no cochlear alterations at all, light-microscopically, and only minor subcellular changes in the OHCs at the ultrastructural level. Electrophysiologically, these three animals showed normals compound action potential (CAP) amplitudes at stimulus frequencies from 0.5 to 16 kHz and normal cochlear microphonics (CM) in the frequency range from 0.5 to 8 kHz. The other animals treated with cisplatin + ORG 2766 showed a severe loss in their CAPs and CM, except for one showing intermediate loss. All animals from the group treated with cisplatin alone showed a severe loss in their CAPs and CM. Endolymphatic hydrops was present in all animals from the cisplatin- and the cisplatin + ORG 2766-treated groups. These data indicate that daily, concomitant administration of ORG 2766 may reduce OHC loss and subsequent degeneration of the organ of Corti in cisplatin-treated guinea pig cochleas.
Assuntos
Hormônio Adrenocorticotrópico/análogos & derivados , Anticonvulsivantes/farmacologia , Antineoplásicos/toxicidade , Cisplatino/toxicidade , Cóclea/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Estimulação Acústica , Potenciais de Ação/efeitos dos fármacos , Hormônio Adrenocorticotrópico/administração & dosagem , Hormônio Adrenocorticotrópico/farmacologia , Animais , Anticonvulsivantes/administração & dosagem , Antineoplásicos/administração & dosagem , Contagem de Células , Cisplatino/administração & dosagem , Cóclea/patologia , Cóclea/ultraestrutura , Interações Medicamentosas , Eletrofisiologia , Retículo Endoplasmático Rugoso/efeitos dos fármacos , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/patologia , Feminino , Complexo de Golgi/efeitos dos fármacos , Cobaias , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/ultraestrutura , Fragmentos de Peptídeos/administração & dosagem , Solução Salina Hipertônica/administração & dosagem , Inclusão do Tecido , Núcleo Vestibular Lateral/citologia , Núcleo Vestibular Lateral/efeitos dos fármacosRESUMO
Carboplatin produces a selective loss of inner hair cells in chinchilla, substantially reducing the amplitude of the compound action potential. A key question that arises from these experiments is: What effect does a reduction in IHC-eighth-nerve fiber input have on the central auditory nervous system? This investigation evaluated the inferior colliculus potential (ICP) in chinchillas treated with carboplatin. The left ear was surgically destroyed and a recording electrode was placed in the left inferior colliculus. Following thirteen days of recovery time, the ICP was recorded in the awake animal. Click level was varied from 10-20 to 80 dB pSPL. Click rate was varied from 10 to 1000 Hz using both conventional averaging and a cross-correlation procedure. Broadband masking noise was varied from 30 to 70 dB SPL with click level held constant at 80 dB pSPL. The dependent variables were the positive peak latency and peak-to-following trough amplitude of the evoked potential. Following baseline studies, the animals were administered carboplatin (50 mg/kg IP) and retested two weeks later. Prior to carboplatin administration, there was an increase in ICP latency and a decrease in ICP amplitude with decreasing stimulus level, increasing rate and increasing noise level. Mean ICP threshold was 30 dB pSPL. Following carboplatin administration, there was little change in threshold or peak latencies. In contrast, the amplitude of the ICP was reduced on average by one-third, although this effect varied considerably across animals. The magnitude of this amplitude decrement was not strongly dependent on click level, click rate, or the level of background noise.