Novel synthetic protective compound,KR‐22335, against cisplatin‐induced auditory cell death

Cisplatin [cis‐diammine‐dichloroplatinum (II)] is a widely used chemotherapeutic agent, and one of its most severe side effects is ototoxicity. In the course of developing a new protective agent against cisplatin‐induced ototoxicity, we have been interested in a novel synthetic compound, 3‐Amino‐3‐(4‐fluoro‐phenyl)‐1H‐quinoline‐2,4‐dione (KR‐22335). We evaluated the effectiveness of KR‐22335 as an otoprotective agent against cisplatin‐induced toxicity. The otoprotective effect of KR‐22335 against cisplatin was tested in vitro in cochlear organs of Corti‐derived cell lines, HEI‐OC1, and in vivo in a zebrafish (Danio rerio) model. Cisplatin‐induced apoptosis, cell cycle arrest and an increase in intracellular reactive oxygen species (ROS) generation were demonstrated in HEI‐OC1 cells. KR‐22335 inhibited cisplatin‐induced apoptosis and mitochondrial injury in HEI‐OC1 cells. KR‐22335 inhibited cisplatin‐induced activation of JNK, p‐38, caspase‐3 and PARP in HEI‐OC1 cells. Scanning and transmission electron micrographs showed that KR‐22335 prevented cisplatin‐induced destruction of kinocilium and stereocilia in zebrafish neuromasts. Tissue TUNEL of neuromasts in zebrafish demonstrated that KR‐22335 blocked cisplatin‐induced TUNEL positive hair cells in neuromasts. The results of this study suggest that KR‐22335 may prevent ototoxicity caused by the administration of cisplatin through the inhibition of mitochondrial dysfunction and suppression of ROS generation. KR‐22335 may be considered as a potential candidate for protective agents against cisplatin‐induced ototoxicity. Copyright © 2013 John Wiley & Sons, Ltd.     

Epicatechin protects the auditory organ by attenuating cisplatin-induced ototoxicity through inhibition of ERK

Cisplatin, a widely used chemotherapeutic drug, causes ototoxicity in a large percentage of patients. The purpose of this study was to determine the efficacy of epicatechin (EC) as an otoprotective agent to prevent cisplatin toxicity and to investigate the molecular mechanism of EC. The effects of EC on cisplatin-induced ototoxicity were investigated in a cochlear organ of Corti-derived cell line, HEI-OC1 and in a rat model. In addition, signaling mechanisms were investigated, specifically those involving MAP kinase. Cisplatin induced apoptosis and demonstrated, conjugation of annexin V/PI in FACS, and an increase of subG1 in HEI-OC1. EC protected HEI-OC1 against cisplatin and showed inhibition of cisplatin-induced apoptosis of the HEI-OC1 by transmission electron microscopy. Intratympanic administration of EC protected against cisplatin-induced ototoxicity in the rat model, as determined by auditory brainstem responses. EC inhibited activation of JNK, ERK, cytochrome-c and caspase-3 by cisplatin. An ERK Inhibitor, cisplatin-induced ototoxicity in a dose dependent manner but a JNK inhibitor did not. The results of this study suggest that EC may provide a mechanism by which ototoxicity caused by the administration of cisplatin can be reduced through the inhibition of ERK. EC may have clinical use as a chemopreventive agent that prevents cisplatin ototoxicity.     

Ototoxicity: mechanisms of cochlear impairment and its prevention

Aminoglycosides, cisplatin, and non-steroidal anti-inflammatory drugs (NSAIDs) are widely used pharmacological agents. There is a possibility, however, that the use of these agents may induce transient or permanent hearing loss and tinnitus as side effects. Recent animal studies have clarified mechanisms leading to the ototoxicity induced by these agents, at least in part. The permanent hearing loss caused by aminoglycosides and cisplatin is suggested to be predominantly associated with the apoptotic death of outer hair cells. Both drugs generate reactive oxygen species (ROS) in the inner ear. ROS can activate cell-death pathways such as the c-Jun Nterminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, which in turn, induce hair cell apoptosis. On the other hand, the abuse of NSAIDs may transiently cause tinnitus and mild to moderate hearing loss. NSAIDs impair the active process of the outer hair cells and affect peripheral and central auditory neurons. Conversely, recent reports clarified that NSAIDs are potential therapeutic agents against cochlear injuries. In this review, recent findings from animal studies regarding the ototoxicity induced by aminoglycosides, cisplatin, and NSAIDs are summarized. Their ototoxic mechanisms are focused on.     

M40403, a superoxide dismutase mimetic,protects cochlear hair cells from gentamicin,but not cisplatin toxicity

Gentamicin, an aminoglycoside antibiotic, and cisplatin, a platinum-based anticancer drug, are two commonly used clinical drugs with ototoxic side effects. The ototoxicity of gentamicin and cisplatin has been linked to the production of reactive oxygen species (ROS), although the specific ROS pathways have not been identified. One ROS that might play a role in ototoxicity is the superoxide radical, which is enzymatically dismutated to molecular oxygen and hydrogen peroxide by endogenous superoxide dismutase (SOD) enzymes. M40403, a manganese-based nonpeptidyl molecule that mimics the activity of SOD, was tested for its ability to protect against gentamicin and cisplatin toxicity in cochlear organotypic cultures from neonatal C57BL/10J mice. Cultures were treated with gentamicin or cisplatin alone or in combination with M40403. M40403 alone had no effect on outer hair cell (OHC) or inner hair cell (IHC) survival at doses of 1, 5, and 10 microM, but a high dose of 30 microM reduced hair cell numbers by approximately 30%. Gentamicin alone and cisplatin alone killed OHCs and IHCs in a dose-dependent manner. The addition of M40403 to gentamicin-treated cultures significantly increased OHC and IHC survival in a dose-dependent manner, whereas M40403 failed to protect hair cells in cisplatin-treated cultures at any dose. The results suggest that the toxicity of gentamicin and cisplatin to cochlear hair cells are mediated by different pathways. Clinically, increased levels of SOD or SOD mimetics might provide significant protection against aminoglycoside ototoxicity.     

Use of the biodegradable polymer chitosan as a vehicle for applying drugs to the inner ear

Development of efficient local delivery systems for the auditory organ has an important role in clinical practice for the management of inner ear disorders using pharmacological means. Chitosan, a biodegradable polymer, is a good drug carrier with bioadhesive properties. The aim of this study was to investigate the feasibility of using chitosan to deliver drugs to the inner ear across the round window membrane (RWM).Three structurally different chitosans loaded with a tracer drug, neomycin, were injected into the middle ear cavity of albino guinea pigs (n = 35). After 7 days the effect of chitosans and neomycin was compared among the treatment groups. The hearing organ was analysed for hair cell loss and the RWM evaluated in term of thickness.All tested chitosan formulations successfully released the loaded neomycin which then diffused across the RWM, and exerted ototoxic effect on the cochlear hair cells in a degree depending on the concentrations used. Chitosans per se had no noxious effect on the cochlear hair cells. It is concluded that the chitosans, and especially glycosylated derivative, are safe and effective carriers for inner ear therapy.     

Aminoglycoside induced ototoxicity

Aminoglycosides are bactericidal aminoglycosidic aminocyclitols. They are cost effective and therefore widely used, however ototoxicity is a prominent dose-limiting side effect. Aminoglycoside induced ototoxicity leads to permanent bilaterally severe, high-frequency sensorineural hearing loss and temporary vestibular hypofunction. The permanent hearing loss is accompanied by degeneration of hair cells and neurons in the cochlea. An iron-aminoglycoside complex is believed to potentiate ROS-induced cellular degeneration in the cochlea. The development of aminoglycoside otoprotective strategies is a primary goal in ototoxicity research. Animal experiments have provided encouraging evidence for the protection of cochlear hair cells and neurons from aminoglycoside toxicity. However, the extent to which such protection, generalize to human ototoxicity remains unresolved.     

A mitochondrial targeting tetrapeptide Bendavia protects lateral line hair cells from gentamicin exposure

The hearing loss induced by aminoglycosides is caused by the permanent loss of mechanosensory hair cells of the inner ear. The aim of the present study is therefore to evaluate the protective effect of Bendavia, a novel antioxidant, on gentamicin‐induced hair cell damage in zebrafish lateral lines. The results demonstrated the pretreatment of Bendavia exhibited dose‐dependent protection against gentamicin in both acute and chronic exposure. We found that Bendavia at 150 μm conferred optimal protection from either acute or chronic exposure with ototoxin. Bendavia reduced uptake of fluorescent‐tagged gentamicin via mechanoelectrical transduction channels, suggesting its protective effects may be partially due to decreasing ototoxic molecule uptake. The intracellular death pathways inhibition triggered by gentamicin might be also included as no blockage of gentamicin was observed. Our data suggest that Bendavia represents a novel otoprotective drug that might provide a therapeutic alternative for patients receiving aminoglycoside treatment.     

Cisplatin ototoxicity: involvement of iron and enhanced formation of superoxide anion radicals.

Since there are indications that iron influences cisplatin nephrotoxicity, we studied the role of iron in cisplatin ototoxicity in an in vitro model of the neurosensory epithelium of the guinea pig cochlea. Viability tests showed that Deiters and Hensen cells were not damaged and inner hair cells were only slightly damaged by cisplatin (50 microM). The outer hair cells were most sensitive to cisplatin toxicity. The iron chelator 2,2'-dipyridyl provided partial protection against cisplatin-induced cell death. In addition, we studied the influence of the iron chelators 2,2'-dipyridyl and deferoxamine on the chelatable iron pool in the various cells of the neurosensory epithelium using the fluorescent iron indicator Phen Green SK. Both chelators decreased the chelatable iron accessible to Phen Green SK, although the effect of deferoxamine was weaker because it entered the cells more slowly. The cellular concentration of the chelatable iron was measured using Phen Green SK and quantitative laser scanning microscopy. The concentration of chelatable iron in the inner ear cells ranged from 1.3 +/- 0.4 microM iron in inner hair cells to 3.7 +/- 1.7 microM iron in Hensen cells and did not correlate with the various cell types' susceptibility to cisplatin. Furthermore, cisplatin did not raise the intracellular chelatable iron concentration but enhanced the production of superoxide anions inside the neurosensory epithelium, especially inside the hair cells, as detected by the nitrotetrazolium blue reduction assay. Our conclusion is that cisplatin ototoxicity is partially mediated by an iron-dependent pathway and is associated with an enhanced formation of superoxide anions.     

Ototoxicity of cisplatin administered to guinea pigs via the round window membrane

Animal models of ototoxicity represent an elementary tool in otolaryngologic research. Such models are usually created via the consecutive injection of ototoxic drugs or the co-administration of ethacrynic acid and low-dose ototoxic drugs. Injection via the round window membrane (RWM) is one approach that allows for local drug delivery into the inner ear. In this study, 47 guinea pigs received an injection of varying doses of cisplatin via the RWM, and data concerning the animals' auditory brainstem responses, hair cells, and spiral ganglion neurons were analyzed. Our results indicate the high efficiency and generally small reaction of the subjects, suggesting that the application of cisplatin via the RWM is an effective animal model for ototoxicity research.     

Functional and morphological effects of styrene on the auditory system of the rat

The effect of inhaled styrene on the structure and function of the auditory organ of the male Wistar rat was studied. The animals were exposed either to 600, 300 or 100 ppm styrene (12 h/day, 5 days/week, for 4 weeks). Auditory sensitivity was tested prior to and after the exposure by auditory brain stem audiometry (ABR) at frequencies of 1.0, 2.0, 4.0 and 8.0 kHz. Inner ear morphological changes were studied by light- and electron-microscopy. Exposure to 600 ppm styrene caused a 3 dB hearing loss only at the highest test frequency (8 kHz). Quantitative morphological analysis of cochlear hair cells (cytocochleograms) showed that 600 ppm styrene caused a severe outer hair cell (OHC) loss particularly in the third OHC row of the upper basal and lower middle coil. The inner hair cells were usually intact. Exposure to lower styrene concentrations (100 and 300 ppm) caused no unequivocal functional deficit or hair cell damage. We conclude that there appears to be a concentration threshold for styrene ototoxicity in rats (between 300 and 600 ppm).     

Protective effects of apocynin on cisplatin‐induced ototoxicity in an auditory cell line and in zebrafish

Cisplatin is a very effective anticancer drug and generates reactive oxygen species (ROS) such as superoxide anions that can deplete antioxidant protective molecules in the cochlea. These processes result in the death of cochlear hair cells by induction of apoptosis. Apocynin, which is used as a specific nicotinamide adenine dinucleotide phosphate oxidase inhibitor, has a preventive effect for intracellular ROS generation. In this study, the effect of apocynin was investigated in a cochlear organ of Corti‐derived cell line, HEI‐OC1 cells, and in transgenic zebrafish (Brn3C: EGFP). To investigate the protective effects of apocynin, HEI‐OC1 cells were treated with various concentrations of apocynin and a 20 µm concentration of cisplatin, simultaneously. An in vivo study of transgenic zebrafish (Brn3C: EGFP) was used to investigate the protective effects of apocynin on cisplatin‐induced hair cell death. In an in vitro study, apocynin appeared to protect against cisplatin‐induced apoptotic features on Hoechst 33258 staining in the HEI‐OC1 cells. Treatment of the HEI‐OC1 cells with 100 µm of apocynin, significantly decreased caspase‐3 activity. Treatment of the cells with a 100 µm concentration of apocynin and a 20 µm concentration of cisplatin significantly decreased the intracellular ROS production. In the in vivo study, apocynin significantly decreased the TUNEL reaction and prevented cisplatin‐induced hair cell loss of the neuromasts in the transgenic zebrafish at low concentrations (125 and 250 µm ). These findings suggest that apocynin has antioxidative effects and prevents cisplatin‐induced apoptotic cell death in HEI‐OC1 cells as well as in zebrafish. Copyright © 2011 John Wiley & Sons, Ltd.     

In Vitro Ototoxicity of aminoglycosides and platin derivatives. A semi-automatic assay for sensory hair cell damage in explanted rat organ of corti

The ototoxic damage that drugs such as neomycin, kanamycin, colistin, cisplatin, transplatin and carboplatin cause on outer and inner hair cells in postnatal day 3 rat cochlear explants was investigated. Phalloidin–fluorescein conjugate-stained stereocilia bundles of sensory hair cells were quantified by video image analysis as a measurement of ototoxic effect. The video image quantification system established dose–response curves for ototoxic drugs (e.g. calculation of an IC₅₀) and allowed comparisons between several ototoxins from the same family. This methodology provided the means to assess the efficacy of otoprotectant agents in preventing ototoxicity. Poly-l-aspartate (10⁻⁵ ) and poly-l-glutamate (10⁻⁵ ) protected auditory hair cells from neomycin (10⁻³ ) toxicity while reduced glutathione (10⁻³ ) provided protection against cisplatin (10⁻⁴ )-induced hair cell damage.     

Adenosine and the Auditory System

Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A₁, A_2A, A_2B, and A₃. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A₁ adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A₁ and A_2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea.Key Words: Adenosine, adenosine receptors, cochlea, hearing, deafness, oxidative stress, noise, ototoxicity.     

In utero and lactational β-carotene supplementation attenuates d-galactose-induced hearing loss in newborn rats

d-Galactose could give rise to free radical damage by disturbing the some maternal antioxidants. The oxidative stress induced by d-galactose is a potent inducer of apoptosis, which is accompanied by the activation of protein-splitting enzymes called caspases. Apoptosis is a crucial physiological determinant of embryonic and neonatal development, and play an essential role in the development of the inner ear structures. Recently the increasing of d-galactose exposure is due to high consumption of dairy foods or reduced galactose metabolism. An overwhelming presence of d-galactose is known to become highly ototoxicity to humans. The purpose of this study was to investigate whether supplementation of pregnant and lactational mothers with β-carotene could attenuate cochlear function damage and hair cells apoptosis induced by d-galactose in newborn rats. Pregnant rats were supplemented with d-galactose, or d-galactose and β-carotene from gestational day (GD) 7 until postnatal day (PND) 21. On PND 22, offspring were examined in the distortion product otoacoustic emission (DPOAE) task, cochleae were then harvested for assessment of apoptosis by immunohistochemical stain for cysteine-aspartic acid proteases 3 (caspase-3) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Maternal and offspring blood samples were then collected by direct cardiac puncture in heparin tubes, blood levels of d-galactose and β-carotene were measured, plasma was separated for malondialdehyde (MDA) analysis, erythrocytes were left for superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH). d-Galactose could significantly disturb the balance between maternal antioxidants and free radicals, and induce hearing loss in the offspring and cochlear hair cell apoptosis. In contrast, β-carotene supplementation, coincidentally with d-galactose exposure, ameliorated these changes. Our data offer a conceptual framework for designing clinical trials using a safe micronutrient, β-carotene, as a simple preventive strategy for d-galactose-induced ototoxicity.     

Expression of myeloperoxidase in the inner ear of cisplatin-treated guinea pigs

Cisplatin is known to cause inner ear damage (ototoxicity). The role of myeloperoxidase (MPO) in the inner ear of the guinea pigs after injections of cisplatin i.p. was examined immunohistochemically. Three days after the injection, the animals were sacrificed with intracardiac perfusion of fixative, and temporal bones were removed and processed for immunohistochemistry with the anti-MPO antibody. MPO could be detected after 3 days in the lateral wall, the organ of Corti, supporting cells of the sensory epithelium and dark cells. These results suggest that MPO and reactive oxygen species are involved in the inner ear dysfunction after the application of cisplatin.     

Styrene induced alterations in biomarkers of exposure and effects in the cochlea: mechanisms of hearing loss.

It is known that styrene is ototoxic and causes cochlear damage starting from the middle turn. However, the cellular mechanism underlying styrene ototoxicity is still unclear. In this study, rats were exposed to styrene by gavage at different doses once a day for varying periods. Styrene levels in the cochlear tissues, styrene-induced permanent hearing loss, cochlear disruptions, and cell death pathways were determined. Styrene concentration in the cochlea varied along with the basilar membrane with the lowest level in the basal turn being consistent with the lowest styrene-induced threshold shift and hair cell loss in this region. After 3 weeks of exposure (5 days per week), a dose-dependent permanent hearing loss and a hair cell loss, especially in the midfrequency region, were observed. The styrene exposure at a dose of 200 mg/kg, which induced a blood level of 6.0 +/- 1.0 microg/g, caused an average of 4.4 +/- 0.5% OHC (outer hair cell) loss and 2-5 dB threshold shift in the cochlear region of 20-70% from the apex. A significant OHC loss was not observed until 7 days of exposure at a dose of 800 mg/kg. Deiters cells appeared to be the most vulnerable target of styrene. When condensed nuclei were observed in Deiters cells after a few days of styrene exposure (800 mg/kg), other cells were still intact. Apoptotic cell death appeared to be the main cell death pathway in the cochlea after styrene exposure. In the styrene-induced apoptotic OHCs, histochemical staining detected activated caspases-9 and 8, indicating that both mitochondrial-dependent pathway and death receptor-dependent pathway were involved in the styrene-induced cell death.     

High accumulation of platinum-DNA adducts in strial marginal cells of the cochlea is an early event in cisplatin but not carboplatin ototoxicity

Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of drug-induced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3- to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.     

Ototoxicity of cis-dichlorodiammine platinum (II) in the guinea pig.

cis-Dichlorodiammine platinum (II) (NSC-119 875), an agent with potent antineoplastic activity which also induces renal, intestinal, and bone marrow toxicity, was tested for ototoxic effects in guinea pigs. Ototoxicity was evaluated by the disappearance of Preyer's reflex in response to pure tones of 5, 7, and 10 kHz and by histopathological evaluation of the inner ear with surface preparations or midmodiolar sections. Groups of five guinea pigs treated with 8–40 ip injections of cis-dichlorodiammine platinum (II) 1 mg/kg (5 doses/wk) or with 10–15 doses of 1.5 mg/kg developed permanent deafness and histopathological lesions with pronounced loss of outer hair cells in the lower turns of the organ of Corti. Frequently, entire rows of outer hair cells disappeared and only a few isolated hair cells remained intact. Moderate numbers of outer pillar cells were missing and occasional inner hair cells and pillar cells were also damaged. Hair cell lesions included degeneration, complete cytolysis, and replacement by phalangeal scars. Single doses of 6, 9, 12, or 18 mg/kg of cis-dichlorodiammine platinum (II) produced permanent hearing loss as early as Day 3 and a scattered pattern of outer hair cell loss on Day 4 with cytological changes similar to but less severe than those observed for multiple doses. Neomycin sulfate-treated guinea pigs used as positive controls (eight consecutive daily doses of 150–250 mg/kg) showed a similar pattern of hair cell loss as produced by cis-dichlorodiammine platinum (II). Positive controls treated with a single dose of 6-aminonicotinamide from 2.5 to 20 mg/kg, developed severe damage to both inner and outer hair cells, pillar cells, and spiral ganglia. Occasional strial atrophy was also observed.     

Regeneration and replacement in the vertebrate inner ear

Deafness affects more than 40 million people in the UK and the USA, and many more world-wide. The primary cause of hearing loss is damage to or death of the sensory receptor cells in the inner ear, the hair cells. Birds can readily regenerate their cochlear hair cells but the mammalian cochlea has shown no ability to regenerate after damage. Current research efforts are focusing on gene manipulation, gene therapy and stem cell transplantation for repairing or replacing damaged mammalian cochlear hair cells, which could lead to therapies for treating deafness in humans.     

Protective role of edaravone against neomycin‐induced ototoxicity in zebrafish

Aminoglycosides such as neomycin are one of the most commonly prescribed types of antibiotics worldwide. However, these drugs appear to generate free radicals within the inner ear, which can result in permanent hearing loss. We evaluated the effects of edaravone, a neuroprotective agent, on neomycin‐induced ototoxicity in transgenic zebrafish. The 5‐day post fertilization (dpf) zebrafish larvae were exposed to 125 μM neomycin and various concentrations of edaravone for 1 h. Hair cell survival was calculated as average numbers of the hair cells in the control group, which was not exposed to neomycin. Ultrastructural changes were evaluated using a scanning electron microscope (SEM) and transmission electron microscope (TEM). Edaravone protected against neomycin‐induced hair cell loss in the neuromasts (1000 μM: 11.6 ± 1.1 cells, neomycin only: 5.5 ± 0.5 cells; n = 10, P < 0.05) and decreased the TUNEL reaction for detecting apoptosis. In ultrastructural analysis, structures of mitochondria and hair cells within neuromasts were preserved in zebrafish exposed to 125 μM neomycin and 1000 μM edaravone for 1 h. Edaravone protected against neomycin‐induced hair cell loss by preventing apoptosis. Copyright © 2013 John Wiley & Sons, Ltd.