Number Of Hair Cells Research Articles

N-acetylcysteine attenuates noise-induced permanent hearing loss in diabetic rats

The purpose of this study is to investigate whether repeated noise exposure aggravates the level of permanent noise-induced hearing loss (NIHL) in diabetic rats and whether N-acetylcysteine (NAC), a precursor of glutathione, attenuates the level of noise-induced permanent hearing loss in diabetic rats. Fifty male Wistar rats were divided into four groups: 12 non-diabetic control rats with saline injection (Control-Saline), 11 non-diabetic control rats with NAC injection (Control-NAC), 13 streptozotocin-induced diabetic rats with saline injection (Diabetes-Saline) and 14 streptozotocin-induced diabetic rats with NAC injection (Diabetes-NAC). NAC (325mg/kg) was given by intraperitoneal injection twice per day (b.i.d.) for 14 days starting 2 days before noise exposure. All rats were exposed to noise for 8hours per day for 10 consecutive days to develop noise-induced permanent hearing loss. The hearing status of all animals was evaluated with auditory brainstem responses (ABR) evoked by clicks and tone bursts. ABRs were measured before and at 1hour, 1 week, 2 weeks and 4 weeks after noise exposure. After a recovery time of 4 weeks, animals were decapitated, and the loss of hair cells was assessed microscopically. In all groups, ABR thresholds failed to return to pre-exposure values throughout the experimental period. The ABR threshold to clicks was markedly elevated in the Diabetes-Saline group (36.9±2.3dB SPL), less elevated in the Control-Saline and Diabetes-NAC groups and least in the Control-NAC group (19.5±2.0dB SPL) at 4 weeks after noise exposure. Diabetes caused increased susceptibility to noise-induced hearing loss, and NAC treatment reduced the loss in both control and diabetic rats. Cochleograms revealed no gross destruction of hair cells in the non-diabetic groups or the Diabetes-NAC group; however, a significant number of outer hair cells (OHCs) were lost in the Diabetes-Saline group. This study demonstrated that diabetics were prone to developing more severe NIHL than non-diabetics and that NAC could preserve most OHCs and attenuate the permanent noise-induced hearing loss in both groups.

Anti-apoptotic factor z-Val-Ala-Asp-fluoromethylketone promotes the survival of cochlear hair cells in a mouse model for human deafness

A major challenge in the inner ear research field is to restore hearing loss of both non-genetic and genetic origin. A large effort is being made to protect hair cells from cell death after exposure to noise or drugs that can cause hearing loss. Our research focused on protecting hair cells from cell death occurring in a genetic model for human deafness. POU4F3 is a transcription factor associated with human hearing impairment. Pou4f3 knockout mice ( Pou4f3 −/−) have no cochlear hair cells, resulting in complete deafness. Although the hair cells appear to form properly, they progressively degenerate via apoptosis. In order to rescue the hair cells in the knockout mice, we produced explant cultures from mouse cochleae at an early embryonic stage and treated the cells with z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a general caspase inhibitor. Hair cell numbers in the knockout mice treated with z-VAD-fmk were significantly higher than in the untreated mice. We found that the time window that z-VAD-fmk has a protective effect is between E14.5 ( P=0.001) to E16.5 ( P=0.03), but not after E18.5. The source of the surviving hair cells is not due to proliferation, as measured by 5-bromo-2-deoxyuridine (BrdU) labeling, or to supporting cell transdifferentiation to hair cells, since there was no change in supporting cell numbers. Instead, the survival appears to be a direct effect of the anti-apoptotic agent on the dying hair cells with an early developmental window. These results help towards providing a comprehensive understanding of the molecular mechanisms of hair cell death, which might lead to the development of new therapeutic anti-apoptotic agents to alleviate hereditary hearing loss (HL).

Development and Regeneration of the Inner Ear: Coordinating cell cycle and differentiation – Constraining Notch signaling.

Generating the correct number and type of cells, in the correct ratio and spatial relationship is required for normal functional development of complex tissues. However, the mechanisms coordinating cell proliferation with growth and differentiation to achieve correct organ size and function remain a poorly understood aspect of embryonic development and postnatal regeneration. In this presentation, I will discuss our work on the molecular basis for the timing of cell cycle exit in the embryonic inner ear, and how this is coordinated with differentiation to produce the correct number of hair cell and supporting cell precursors to build a functional organ of Corti. In the second part of my talk I will discuss the mechanisms that govern cell fate decisions between sensory hair cells and the various supporting cell types in the developing organ of Corti. Specifically, I will focus on the role of Notch‐dependent and Notch‐independent mechanisms governing Hes and Hey gene expression, and their potential role in the maintenance of cell fate during subsequent maturation. Finally, I will discuss the potential role of these developmental processes in the failure of regeneration in the mammalian inner ear.

MicroRNA-183 Family Members Regulate Sensorineural Fates in the Inner Ear

Members of the microRNA (miRNA) 183 family (miR-183, miR-96, and miR-182) are expressed abundantly in specific sensory cell types in the eye, nose, and inner ear. In the inner ear, expression is robust in the mechanosensory hair cells and weak in the associated statoacoustic ganglion (SAG) neurons; both cell types can share a common lineage during development. Recently, dominant-progressive hearing loss in humans and mice was linked to mutations in the seed region of miR-96, with associated defects in both development and maintenance of hair cells in the mutant mice. To understand how the entire triplet functions in the development of mechanosensory hair cells and neurons of the inner ear, we manipulated the levels of these miRNAs in zebrafish embryos using synthesized miRNAs and antisense morpholino oligonucleotides (MOs). Overexpression of miR-96 or miR-182 induces duplicated otocysts, ectopic or expanded sensory patches, and extra hair cells, whereas morphogenesis of the SAG is adversely affected to different degrees. In contrast, knockdown of miR-183, miR-96, and miR-182 causes reduced numbers of hair cells in the inner ear, smaller SAGs, defects in semicircular canals, and abnormal neuromasts on the posterior lateral line. However, the prosensory region of the posterior macula, where the number of hair cells is reduced by approximately 50%, is not significantly impaired. Our findings suggest both distinct and common roles for the three miRNAs in cell-fate determination in the inner ear, and these principles might apply to development of other sensory organs.

Stereological estimation of total cell numbers in the young human utricular macula

Conclusion: There is no change in the total cell population and hair cell:supporting cell ratio in the human utricular macula from gestational week 16 and onwards, whereas the lower hair cell:supporting cell ratio and lower total number of cells in the youngest specimens indicate that the utricle is still differentiating and adding new cells at the 10th to 12th gestational week. Objectives: Archival temporal bones were investigated to quantify cell numbers in the utricular macula in fetuses and children. Methods: The age of the subjects ranged from gestational week 10 to 15 years. The optical fractionator was used to estimate the total number of cells in the utricular macula. Results: The total cell number was found to be 143 000 in subjects older than gestational week 16. The number of hair cells and supporting cells did not change between the 16th gestational week and 15 years and was 36 000 and 107 000, respectively. In the youngest specimen (10th and 12th gestational week) there was a statistically significant lower total number of cells (62 000) and a lower hair cell:supporting cell ratio, resulting in a mean number of 13 000 hair cells and 49 000 supporting cells.

The Effect of Memantine on Experimentally Gentamicin Induced Vestibulotoxicity in Guinea Pigs

Background and ObjectivesZZIn general, aminoglycosides are known to cause ototoxicity through the glutamate induced nitric oxide production. The N-methyl-D-aspartate (NMDA) related glutamate receptors have a pivotal role in aminoglycoside induced ototoxicity. Memantine is known as a safe NMDA antagonist and is also used in some neurologic insults, such as the Alzheimer disease. In this study, we observed the effect of memantine on gentamicin induced vestibulotoxicity in an animal model. Materials and MethodZZVestibulotoxicity was induced with intratympanic administration of gentamicin and memantine was injected intraperitoneally to a study group. Histomorphological studies for vestibule were performed via light and electron microscopy. Immunohistochemical studies were performed for iNOS, nitrotyrosine and apoptosis via TUNEL staining. ResultsZZThe numbers of hair cells were decreased significantly in the gentamicin group than in the gentamicin-memantine group. Increased immunoreactivities for iNOS and nitrotyrosine were observed in the gentamicin group than in the memantine-pretreated gentamicin group. TUNEL positive cells were more frequently observed in the gentamicin group than in the memantinepretreated gentamicin group. ConclusionZZThis result shows that memantine has a protection effect on gentamicin-induced vestibulotoxicity in an animal model. Korean J Otorhinolaryngol-Head Neck Surg 2010;53:77-83 Key WordsZZGentamicin·Memantine·Vestibulotoxicity.

The Small GTPase Rac1 Regulates Auditory Hair Cell Morphogenesis

Morphogenesis of sensory hair cells, in particular their mechanotransduction organelle, the stereociliary bundle, requires highly organized remodeling of the actin cytoskeleton. The roles of Rho family small GTPases during this process remain unknown. Here we show that deletion of Rac1 in the otic epithelium resulted in severe defects in cochlear epithelial morphogenesis. The mutant cochlea was severely shortened with a reduced number of auditory hair cells and cellular organization of the auditory sensory epithelium was abnormal. Rac1 mutant hair cells also displayed defects in planar cell polarity and morphogenesis of the stereociliary bundle, including bundle fragmentation or deformation, and mispositioning or absence of the kinocilium. We further demonstrate that a Rac-PAK (p21-activated kinase) signaling pathway mediates kinocilium-stereocilia interactions and is required for cohesion of the stereociliary bundle. Together, these results reveal a critical function of Rac1 in morphogenesis of the auditory sensory epithelium and stereociliary bundle.

Na +/K +-ATPase and vacuolar-type H +-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation

The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na +/K +-ATPase (NKA) and vacuolar-type H +-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na + concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model.

Distribution of high‐conductance calcium‐activated potassium channels in rat vestibular epithelia

Voltage- and calcium-activated potassium channels (BK) are important regulators of neuronal excitability. BK channels seem to be crucial for frequency tuning in nonmammalian vestibular and auditory hair cells. However, there are a paucity of data concerning BK expression in mammalian vestibular hair cells. We therefore investigated the localization of BK channels in mammalian vestibular hair cells, specifically in rat vestibular neuroepithelia. We find that only a subset of hair cells in the utricle and the crista ampullaris express BK channels. BK-positive hair cells are located mainly in the medial striolar region of the utricle, where they constitute at most 12% of hair cells, and in the central zone of the horizontal crista. A majority of BK-positive hair cells are encapsulated by a calretinin-positive calyx defining them as type I cells. The remainder are either type I cells encapsulated by a calretinin-negative calyx or type II hair cells. Surprisingly, the number of BK-positive hair cells in the utricle peaks in juvenile rats and declines in early adulthood. BK channels were not found in vestibular afferent dendrites or somata. Our data indicate that BK channel expression in the mammalian vestibular system differs from the expression pattern in the mammalian auditory and the nonmammalian vestibular system. The molecular diversity of vestibular hair cells indicates a functional diversity that has not yet been fully characterized. The predominance of BK-positive hair cells within the medial striola of juvenile animals suggests that they contribute to a scheme of highly lateralized coding of linear head movements during late development.

Quantitative analysis of the ribbon synapse number of cochlear inner hair cells in C57BL/6J mice using the three-dimensional modeling method

In mammals, the ribbon synapses of cochlear inner hair cells are a synaptic structure of the first sensory nerve in the pathway of acoustical signal transmission to the acoustic center, and it is directly involved in voice coding and neurotransmitter release. It is difficult to quantitatively analyze the ribbon synaptic number only using an electron microscope, because the ribbon synaptic number is relatively limited and their location is deep. In this study, the specific presynaptic structure-RIBEYE, and non-specific postsynaptic structure-GluR 2 & 3 in C57BL/6J mouse basilar membrane samples were treated by immunofluorescent labeling. Serial section was performed on the samples using a laser scanning confocal microscope, and then the serial sections were used to build three-dimensional models using 3DS MAX software. Each fluorescein color pair indicates one synapse, so the number of ribbon synapses of inner hair cells is obtained. The spatial distribution and the number of ribbon synapses of cochlear inner hair cells were clearly shown in this experiment, and the mean number of ribbon synapses per inner hair cell was 16.10+/-1.03. Our results have demonstrated the number of ribbon synapses is accurately calculated by double immunofluorescent labeling to presynaptic and postsynaptic structures, serial sections obtained using a laser scanning confocal microscope, and three-dimensional modeling obtained using 3DS MAX software. The method above is feasible and has important significance for further exploring the mechanism of sensorineural deafness.

The influence of viscous hydrodynamics on the fish lateral-line system

Fish exhibit many behaviors that involve sensing water flows with their lateral-line system. In many situations, viscosity affects how the flow interacts with the body of the fish and the neuromasts of the lateral line. Here we discuss how viscosity influences the stimulus to the fish lateral-line system. The movement of a fish's body creates flows that can interfere with the detection of external signals, but these flows can also serve as a source of information about nearby obstacles and the fish's own hydrodynamic performance. The viscous boundary layer on the surface of the skin alters external signals by attenuating the low-frequency components of stimuli. The stimulus to each neuromast depends on the interaction of the fluid surrounding the neuromast and the structural properties of that neuromast, including the number of mechanosensory hair cells it contains. A consideration of the influences of viscosity on flow, at both the whole-body and receptor levels, offers the promise of a more comprehensive understanding of the signals involved in behaviors mediated by the lateral-line system.

Hair Cell Regeneration

Hair Cell Regeneration

Effects of Aging on Inner Ear Morphology in Dogs in Relation to Brainstem Responses to Toneburst Auditory Stimuli

Age-related hearing loss (ARHL) is the most common form of hearing loss in humans and is increasingly recognized in dogs. Cochlear lesions in dogs with ARHL are similar to those in humans and the severity of the histological changes is reflected in tone audiograms. Ten geriatric dogs (mean age: 12.7 years) and three 9-month-old dogs serving as controls for histological analysis. Observational study. Auditory thresholds were determined by recording brainstem responses (BERA) to toneburst auditory stimuli (1, 2, 4, 8, 12, 16, 24, and 32 kHz). After euthanasia and perfusion fixation, the temporal bones were harvested and processed for histological examination of the cochleas. The numbers of outer hair cells (OHCs) and inner hair cells (IHCs) were counted and the spiral ganglion cell (SGC) packing density and stria vascularis cross-sectional area (SVCA) were determined. A combination of cochlear lesions was found in all geriatric dogs. There were significant reductions (P .001) in OHC (42%, 95% confidence interval [CI]; 24-64%) and IHC counts (21%, 95% CI; 62-90%) and SGC packing densities (323, 95% CI; 216-290) in the basal turn, SVCA was smaller in all turns. The greatest reduction in auditory sensitivity was at 8-32 kHz. ARHL in this specific population of geriatric dogs was comparable histologically to the mixed type of ARHL in humans. The predominance of histological changes in the basal cochlear turn was consistent with the large threshold shifts observed in the middle- to high-frequency region.

Ultrastructure and distribution of superficial neuromasts of blind cavefish, Phreatichthys andruzzii, juveniles

Transmission and scanning electron microscopy (TEM, SEM) were used to study the ultrastructure of superficial neuromasts in 15 six-month old blind cavefish juveniles, Phreatichthys andruzzii (Cyprinidae). In five specimens examined with SEM, the number of superficial neuromasts over the fish body (480-538) was recorded. They were localized mainly on the head (362-410), including the dorsal surface, the mentomandibular region, and laterally from the mouth to the posterior edge of the operculum. Neuromasts were also present laterally on the trunk and near the caudal fin (116-140). A significantly higher number of neuromasts were present on the head compared to the trunk (t-test, P < 0.05). Superficial neuromasts of the head and those along the trunk were similar in ultrastructure. Each neuromast comprised sensory hair cells surrounded by nonsensory support cells (mantle cells and supporting basal cells) with the whole covered by a cupula. Each hair cell was pear-shaped, 15-21 microm high and 4-6 microm in diameter, with a single long kinocilium and several short stereocilia. Most support cells were elongated, with nuclei occupying a large portion of the cytoplasm. In the margin of the neuromast, mantle cells were particularly narrow. Both types of support cells had well-developed Golgi apparatus and rough endoplasmic reticulum. The number of hair cells and nonsensory support cells of the anterior lateral line (head) did not differ significantly from those of the posterior lateral line (trunk) (t-test, P > 0.05).

Anatomie fonctionnelle du nerf cochléaire et du système auditif central

The auditory pathways are a system of afferent fibers (through the cochlear nerve) and efferent fibers (through the vestibular nerve), which are not limited to a simple information transmitting system but create a veritable integration of the sound stimulus at the different levels, by analyzing its three fundamental elements: frequency (pitch), intensity, and spatial localization of the sound source. From the cochlea to the primary auditory cortex, the auditory fibers are organized anatomically in relation to the characteristic frequency of the sound signal that they transmit (tonotopy). Coding the intensity of the sound signal is based on temporal recruitment (the number of action potentials) and spatial recruitment (the number of inner hair cells recruited near the cell of the frequency that is characteristic of the stimulus). Because of binaural hearing, commissural pathways at each level of the auditory system and integration of the phase shift and the difference in intensity between signals coming from both ears, spatial localization of the sound source is possible. Finally, through the efferent fibers in the vestibular nerve, higher centers exercise control over the activity of the cochlea and adjust the peripheral hearing organ to external sound conditions, thus protecting the auditory system or increasing sensitivity by the attention given to the signal.

Plasma membrane calcium ATPase required for semicircular canal formation and otolith growth in the zebrafish inner ear

Fish otoliths consist of >90% calcium carbonate, the accretion of which depends on acellular endolymph. This study confirms the presence of plasma membrane calcium ATPase 1a isoform (Atp2b1a) in the auditory and vestibular system of a teleost fish. As shown by in situ hybridization, zebrafish atp2b1a is expressed mainly in larval otic placode and lateral-line neuromast as well as in the hair cells within the adult zebrafish inner ear chamber. Zebrafish atp2b1a knockdown by antisense morpholinos reduced the number of hair cells and produced malformation of semicircular canals and smaller otoliths. These defects coincide with unbalanced body orientation. The formation of smaller otoliths in atp2b1a morphants may stem from an impairment of calcium supply in the endolymph. However, otolith formation persists in most morphants, suggesting that other zebrafish Atp2b isoforms or paracellular pathways may also transport calcium into the endolymph. These results suggest that Atp2b1a plays an important role for normal development of the auditory and vestibular system as well as calcium transport in the inner ear of zebrafish.

Cooperative functions of Hes/Hey genes in auditory hair cell and supporting cell development

Notch-mediated lateral inhibition has been reported to regulate auditory hair cell and supporting cell development from common precursors. While the Notch effector genes Hes1, Hes5 and Hey1 are expressed in the developing cochlea, inactivation of either of them causes only mild abnormality, suggesting their functional redundancy. To explore the roles of Hes/Hey genes in cochlear development, we examined compound heterozygous or homozygous mutant mice that lacked Hes1, Hes5 and Hey1 alleles. We found that a reduction in Hes/Hey gene dosage led to graded increase of hair cell formation. However, if at least one allele of Hes1, Hes5 or Hey1 was intact, excessive hair cells were accompanied by overproduction of supporting cells, suggesting that the hair cell increase does not occur at the expense of supporting cells, and that each Hes/Hey gene functions to induce supporting cells. By contrast, when all alleles of Hes1, Hes5 and Hey1 were inactivated, the number of hair cells increased more drastically, whereas that of supporting cells was unchanged compared with control, suggesting that supporting cell formation was balanced by their overproduction and fate conversion into hair cells. The increase of the cell numbers seemed to occur after the prosensory domain formation in the mutants because the proliferation state and the size of the prosensory domain were not affected. Thus, Hes1, Hes5 and Hey1 cooperatively inhibit hair cell formation, and one allele of Hes1, Hes5 or Hey1 is sufficient for supporting cell production probably by lateral inhibition in the sensory epithelium. Strikingly, Hes/Hey mutations lead to disorganized cell alignment and polarity and to hearing loss despite hair cell overproduction. These results suggest that Hes/Hey gene dosage is essential not only for generation of appropriate numbers of hair cells and supporting cells by controlling cell proliferation and lateral inhibition but also for the hearing ability by regulating the cell alignment and polarity.

Prednisolone prevents transient ischemia-induced cochlear damage in gerbils

Conclusions. Prednisolone protects against inner ear damage, even when administered after ischemic injury in Mongolian gerbils. Objective. The effect of prednisolone on ischemia-induced cochlear damage was investigated in Mongolian gerbils. Materials and methods. The bilateral vertebral arteries were occluded for 15 min to transiently induce cochlear ischemia, followed by an intraperitoneal injection of prednisolone (1 mg/kg) or physiological saline (control). Sequential changes in hearing were evaluated by recording the auditory brainstem response (ABR) before and at 1, 4, and 7 days after treatment. In our histologic analysis, the numbers of dead and intact inner hair cells (IHCs) were counted in specimens stained with rhodamine-phalloidin. Results. In control animals, transient ischemia increased the ABR threshold (24.2±8.6 dB) within 7 days of treatment, whereas prednisolone-treated animals exhibited a threshold of 14.2±9.2 dB. Furthermore, the percent IHC loss at the basal turn of the cochlea was 26.5±11.4% in control animals compared with 5.3±3.0% in the prednisolone-treated group.

Preventive and Therapeutic Effects of Low Level Laser Irradiation on Gentamicin-Induced Vestibulotoxicity in Rat Utricles

Background and Objectives：The purpose of this study was to investigate the effects of low level laser for the prevention and treatment of aminoglycoside-induced vestibular ototoxicity. Materials and Method：An organotypic culture of 2 to 4 days old rat utricular maculae was established. Rats were divided into 6 groups according to the treadtment of the utricles：G (gentamicin-treated), L (laser-irradiated), LG (laser-irradiated and gentamicin-treated), GL (gentamicin-treated and laser-irradiated), LGL (gentamicin-treated during laser-irradiated) and C (control). After organotypic culture, the utricles of 6 groups were examined by confocal laser scanning electron microscope and scanning electron microscope. The results of each group were compared with each other by statistical methods. Results：The number of vestibular hair cells of the group G was smaller compared to that of the group C. The group L had no difference compared with the group C. The groups LG and GL showed more vestibular hair cells compared with the group G. The group LG showed more vestibular hair cells than the group GL. The group LGL showed most vestibular hair cells compared to that of the groups G, LG, and GL. Conclusion：The most effective treatment of aminoglycoside-induced vestibular otoxicity is the irradiation of low level laser before and after the insult of the aminoglycoside. Further clinical studies using low level laser were needed to prevent aminoglycoside-induced ototoxicity and to promote the regeneration of vestibular hair cells.

Antioxidant and Anti-Apoptotic Effect of Melatonin on the Vestibular Hair Cells of Rat Utricles

ObjectivesAminoglycosides are commonly used antibiotic agents, and they are known to generate free oxygen radicals within the inner ear and to cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Melatonin, a pineal secretory product, has the properties of being a powerful direct and indirect antioxidant. The aim of the present study was to prove the antioxidant effect of melatonin against gentamicin-induced ototoxicty.MethodsThe utricular maculae of Sprague-Dawley rats were prepared from postnatal day 2-4, and these maculae were were divided into 6 groups as follows: 1) control, 2) melatonin only, 3) gentamicin only, and 4), 5), and 6) gentamicin plus melatonin (10, 50, and 100 µM, respectively). To count the number of hair cells, 5 utricles from each group were stained with phalloidin-FITC on the 1st, 4th, and 7th days after drug administration. Reactive oxygen species (ROS) was assessed by using the fluorescent probe hydrofluorescent diacetate acetyl ester. The caspase-3 activity was also examined with using the fluorescent caspase-3 substrate and performing Western blotting.ResultsThe result of this study showed that gentamicin induced the loss of utricular hair cells, and this loss of hair cells was significantly attenuated by co-administration of melatonin. Melatonin reduced ROS production and caspase-3 activation in the gentamicin treated utricular hair cells.ConclusionOur findings conclusively reveal that melatonin has protective effects against gentamicin-induced hair cell loss in the utricles of rat by inhibiting both ROS production and caspase-3 activity.