In order to elucidate the mechanism underlying the attenuation of streptomycin ototoxicity by tetramethylpyrazine (TMP), the present study investigated the effect of TMP on the outward K(+) current in the outer hair cells of guinea pig cochlea. Sixty guinea pigs were divided into 6 groups randomly. Auditory brainstem response (ABR) was used to observe the change in thresholds and to evaluate ototoxicity induced by streptomycin. Whole-cell patch-clamp technique was used to observe the effect of TMP on outward K(+) current in isolated outer hair cells. The results showed that TMP attenuated the threshold shift caused by streptomycin and increased the amplitudes of Ca(2+)-sensitive K(+) current [I(K(Ca))] in the outer hair cells. The present data suggest that TMP displays anti-ototoxicity induced by streptomycin. The augmented amplitudes of I(K(Ca)) of the outer hair cells induced by TMP may be one of the mechanisms underlying its ototoxicity-attenuating effect.
Animals ; Auditory Threshold ; Cochlea ; cytology ; Evoked Potentials, Auditory, Brain Stem ; Guinea Pigs ; Hair Cells, Auditory, Outer ; drug effects ; Patch-Clamp Techniques ; Potassium Channels ; metabolism ; Pyrazines ; Streptomycin ; toxicity

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.
Animals ; Calcium Channels, L-Type ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; pharmacology ; Hair Cells, Auditory, Inner ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Proteomics

OBJECTIVETo explore the feature of the ACh-sensitive potassium current in guinea pig cochlear outer hair cells.

METHODSCochlear outer hair cells of guinea pigs (n=38) were isolated by collagenase type IV. Under the whole-cell patch mode, the ions nature and the pharmacological properties of the ACh-sensitive potassium current were investigated by applying the inhibitors of calcium-dependent potassium currents and the inhibitors of nicotinic ACh receptor.

RESULTSFollowing application of ACh, cochlear outer hair cells displayed a rapidly activating outward potassium current with a fast desensitized kinetic and a reversal (x +/- s) potential of (-67.3 +/- 8.2) mV (n=10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micronmol/L ACh was (506.6 +/- 186.3) pA (n=9). ACh-sensitive potassium current was sensitive to TEA (tetraethylammonium chloride, 10 mmol/L) and potently inhibited by the small conductance calcium-dependent potassium current (SK) blocker, apamin (1 micromol/L). Iberiotoxin (IBTX), the well-known blocker of big conductance calcium-dependent potassium current (BK), failed to inhibit the amplitude of the ACh-sensitive potassium current at the dose of 200 nmol/L. The dose for half-maximal response (EC50) of the ACh-sensitive potassium current was (33.5 +/- 5.7) micromol/L (n=7). The ACh-sensitive potassium current was sensitive to the GABA (gamma-aminobutyric acid)-A receptor blocker, bicuculline, and strongly inhibited by the selective blocker of the alpha 9-nicotinic ACh receptor, strychnine. Strychnine and bicuculline showed the dose-dependent blocking effect with a half inhibition-maximal response (IC50) of (22.3 +/- 2.6) nmol/L (n=7) and (1.2 +/- 0.4) micromol/L (n=6), respectively.

CONCLUSIONSThis work provides direct evidences that the ACh-sensitive SK current was present on guinea pig cochlear outer hair cells. The activation of the ACh-sensitive SK current was most possibly mediated by a alpha 9-nicotinic ACh receptor.

Animals ; Guinea Pigs ; Hair Cells, Auditory, Outer ; drug effects ; metabolism ; Membrane Potentials ; Patch-Clamp Techniques ; Potassium ; metabolism ; pharmacology ; Potassium Channels ; physiology ; Receptors, Cholinergic ; drug effects ; metabolism

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.
Animals ; Cell Death/drug effects ; Cell Polarity/drug effects ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Gadolinium/metabolism ; Gentamicins/*metabolism/pharmacology ; Hair Cells, Auditory/drug effects/*metabolism ; Hair Cells, Auditory, Inner/drug effects/metabolism ; Rats ; Rats, Sprague-Dawley ; Ruthenium Red/metabolism ; TRPV Cation Channels/*metabolism ; Time Factors ; Xanthenes/metabolism ; Zebrafish

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.
Animals ; Cisplatin ; toxicity ; Cochlea ; drug effects ; metabolism ; pathology ; Hair Cells, Auditory, Inner ; drug effects ; pathology ; Membrane Proteins ; metabolism ; Mice ; Mice, Inbred C57BL

AIMTo study the antagonistic action of acanthopanax senticosus injection (ASS) on gentamicin ototoxicity.

METHODSGuinea pigs were randomly divided into control group, GM group, ASS group, and ASS + GM group. The changes of hearing threshold, cochlear morphology, expression of caspases-3 were determined by ABR, TEM, and Western blot, respectively.

RESULTSThe ABR threshold in GM group increased markedly. There was no significant difference in ABR threshold between ASS group and control group, but the ABR threshold in ASS group was much lower than that both in GM group and ASS + GM group. Severely injured hair cells with morphological characteristics of apoptosis were found under TEM in GM group, and the hair cells were less injured in ASS + GM group. The results of Western blot showed that the expression of caspase-3 increased markedly in GM group, but it increased slightly in ASS + GM group.

CONCLUSIONASS may antagonize the GM ototoxicity by inhibiting the expression of caspases-3.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Eleutherococcus ; Gentamicins ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; cytology ; drug effects ; Plant Extracts ; pharmacology

PURPOSE: microRNAs (miRNAs) are non-coding RNAs composed of 20 to 22 nucleotides that regulate development and differentiation in various organs by silencing specific RNAs and regulating gene expression. In the present study, we show that the microRNA (miR)-183 cluster is upregulated during hair cell regeneration and that its inhibition reduces hair cell regeneration following neomycin-induced ototoxicity in zebrafish. MATERIALS AND METHODS: miRNA expression patterns after neomycin exposure were analyzed using microarray chips. Quantitative polymerase chain reaction was performed to validate miR-183 cluster expression patterns following neomycin exposure (500 µM for 2 h). After injection of an antisense morpholino (MO) to miR-183 (MO-183) immediately after fertilization, hair cell regeneration after neomycin exposure in neuromast cells was evaluated by fluorescent staining (YO-PRO1). The MO-183 effect also was assessed in transgenic zebrafish larvae expressing green fluorescent protein (GFP) in inner ear hair cells. RESULTS: Microarray analysis clearly showed that the miR-183 cluster (miR-96, miR-182, and miR-183) was upregulated after neomycin treatment. We also confirmed upregulated expression of the miR-183 cluster during hair cell regeneration after neomycin-induced ototoxicity. miR-183 inhibition using MO-183 reduced hair cell regeneration in both wild-type and GFP transgenic zebrafish larvae. CONCLUSION: Our work demonstrates that the miR-183 cluster is essential for the regeneration of hair cells following ototoxic injury in zebrafish larvae. Therefore, regulation of the miR-183 cluster can be a novel target for stimulation of hair cell regeneration.
Animals ; Animals, Genetically Modified ; Cell Count ; Gene Expression Profiling ; Gene Expression Regulation/drug effects ; Gene Knockdown Techniques ; Green Fluorescent Proteins/metabolism ; Hair Cells, Auditory/drug effects ; Hair Cells, Auditory/physiology ; Larva/drug effects ; Larva/genetics ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Morpholinos/pharmacology ; Neomycin/toxicity ; Regeneration/drug effects ; Regeneration/genetics ; Zebrafish/genetics

Objective: The aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae. Methods: Zebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity. Results: The results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79% and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml Conclusion This study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.
Animals ; Ear, Inner/growth & development* ; Embryo, Nonmammalian/drug effects* ; Gene Expression Regulation, Developmental/drug effects* ; Hair Cells, Auditory/metabolism* ; Lateral Line System/growth & development* ; Locomotion/drug effects* ; Ototoxicity/physiopathology* ; Toluene/toxicity* ; Zebrafish

OBJECTIVE: Effects of ATP and acetylcholine (ACh) on intracellular Ca2+ concentrations ([Ca2+]i) and possible mechanism of Ca2+-induced Ca2+ release (CICR) of the isolated outer hair cells (OHCs) in the guinea pig cochlea were studied with confocal microscopy. METHOD: OHCs were isolated from guinea pig cochlea by enzymatic and mechanical methods. The effects of ATP, ACh, Ryanodine + ATP (or ACh) and Thapsigargin + ATP (or ACh) in the presence or absence of extracellular Ca2+ on [Ca2+]i in OHCs were examined by confocal microscopy. RESULT: In the presence of ATP, Ryanodine + ATP, Thapsigargin + ATP, ACh, Ryanodine + ACh and Thapsigargin + ACh increased [Ca2+]i and evoked an evident wave, respectively, the relative magnitude of fluorescence were 1.60 +/- 0.01(ATP), 1.644 +/- 0.005 (Ryanodine + ATP), 1.491 +/- 0.005 (Thapsigargin + ATP), 1.43 +/- 0.01 (ACh), 1.58 +/- 0.02 (Ryanodine + ACh), 1.398 +/- 0.003 (Thapsigargin + ACh) in OHCs in the presence of extracellular Ca2+ respectively. In the absence of extracellular Ca2+, ATP and Ryanodine + ATP induced a gradual and small [Ca2+]i wave, the relative magnitude of fluorescence were 1.341 +/- 0.006 and 1.386 +/- 0.008, however, ACh, Ryanodine + ACh, Thapsigargin + ACh and Thapsigargin + ATP can not induce wave but a gradual [Ca2+]i elevation. ACh can not increase [Ca2+]i. CONCLUSION In the presence of extracellular Ca2+, ATP and ACh increased [Ca2+]i in OHCs not only by Ca2+ influx through ion channel on cell membrane but also a release of Ca2+ from IP3-sensitive calcium reservoir and CICR. In the absence of extracellular Ca2+, ATP activated IP3 sensitive calcium reservoir and Ca2+ release through IP3 sensitive calcium reservoir, in turn CICR was induced. ACh can not activate IP3 sensitive calcium reservoir and CICR in the absence of extracellular Ca2+, therefore, the effect of ACh was dependent of extracellular Ca2+.
Acetylcholine ; pharmacology ; Adenosine Triphosphate ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; metabolism ; Cells, Cultured ; Cochlea ; cytology ; metabolism ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; Microscopy, Confocal ; Ryanodine ; pharmacology ; Thapsigargin ; pharmacology

OBJECTIVETo investigate the regulatory effect of miR-181a with abnormal expression on the expression of c-fos in cochlear hair cells undergoing oxidative damage.

METHODSHouse Ear Institute-Organ of Corti1 (HEI-CO1) cells were assigned to 50, 100, and 200 µmol/L tert-butyl hydroperoxide (t-BHP) exposure groups and control group. The HEI-CO1 cells in the exposure groups were exposed to 50, 100, or 200 µmol/L t-BHP for 12 h. Then, total RNA and total protein were extracted from the HEI-CO1 cells, and the expression of miR-181a/-181d was measured by qPCR. The miR-181a with abnormal expression was selected as the subject of study. The putative miR-181a target sequence in the 3' untranslated region (3'-UTR) of c-fos was predicted by searching on a bioinformatics website. The HEI-CO1 cells were transfected with miR-181a mimics by lipofection, and the transfection efficiency was measured by qPCR. The mRNA and protein expression of c-fos was measured by qPCR and Western blot. The pGL3-c-fos-3'UTR-WT plasmid was constructed, and the luciferase activity of the plasmid in the case of high miR-181a expression was measured using the Dual-Luciferase Reporter Assay System.

RESULTSCompared with those in the control group, the expression of miR-181a in 100 and 200 µmol/L t-BHP exposure groups was significantly decreased, with expression ratios of 0.744 and 0.766 (P < 0.01), while the expression of miR-181d in 50 µmol/L t-BHP exposure group was significantly increased, with an expression ratio of 1.29 (P < 0.01). There was no significant difference in miR-181a expression between the 100 and 200 µmol/L t-BHP exposure groups (P > 0.05). The predication results revealed that c-fos was regulated by miR-181a in humans and mice, with complete complementarity to the seed region of miR-181a, and there was high degree of target sequence conservation across species. The expression of miR-181a in the HEI-OC1 cells transfected with miR-181a mimics was elevated 892.979 times at 24 hours after transfection. As compared with those of controls, the mRNA and protein expression levels of c-fos in the transfected HEI-OC1 cells were significantly increased (P < 0.05 and P < 0.01). The luciferase activity of pGL3-c-fos-3'UTR-WT plasmid was not suppressed but increased in the case of high miR-181a expression.

CONCLUSIONmiR-181a has no direct inhibitory effect on the mRNA and protein expression of c-fos, which may not be the target gene of miR-181a. Bioinformatic prediction might produce false-positive results.

Animals ; Apoptosis ; drug effects ; genetics ; Cell Line ; Hair Cells, Auditory ; cytology ; drug effects ; metabolism ; Mice ; MicroRNAs ; genetics ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; RNA, Messenger ; genetics ; Transfection ; tert-Butylhydroperoxide ; toxicity