Candidate Key Proteins in Tinnitus: A Bioinformatic Study of Synaptic Transmission in Spiral Ganglion Neurons.

To study key proteins associated with changes in synaptic transmission in the spiral ganglion in tinnitus, we build three gene lists from the GeneCard database: 1. Perception of sound (PoS), 2. Acoustic stimulation (AcouStim), and 3. Tinnitus (Tin). Enrichment analysis by the DAVID database resulted in similar Gene Ontology (GO) terms for cellular components in all gene lists, reflecting synaptic structures known to be involved in auditory processing. The STRING protein-protein interaction (PPI) network and the Cytoscape data analyzer were used to identify the top two high-degree proteins (HDPs) and their high-score interaction proteins (HSIPs) identified by the combined score (CS) of the corresponding edges. The top two protein pairs (key proteins) for the PoS are BDNF-GDNF and OTOF-CACNA1D and for the AcouStim process BDNF-NTRK2 and TH-CALB1. The Tin process showed BDNF and NGF as HDPs, with high-score interactions with NTRK1 and NGFR at a comparable level. Compared to the PoS and AcouStim process, the number of HSIPs of key proteins (CS > 90. percentile) increases strongly in Tin. In the PoS and AcouStim networks, BDNF receptor signaling is the dominant pathway, and in the Tin network, the NGF-signaling pathway is of similar importance. Key proteins and their HSIPs are good indicators of biological processes and of signaling pathways characteristic for the normal hearing on the one hand and tinnitus on the other.

Differential expression of transcription factors and inflammation-, ROS-, and cell death-related genes in organotypic cultures in the modiolus, the organ of Corti and the stria vascularis of newborn rats.

Cells respond to injury and hypoxia by changing gene expression. To study how the main compartments of the cochlea, the stria vascularis (SV), the organ of Corti (OC), and the modiolus (MOD), respond to such stress, we analyzed the expression of selected genes using microarray analysis. Organotypic cultures of SV, OC, and MOD from newborn rats were used as an experimental model. In the present study, we compare the expression of a total of 50 genes involved in apoptosis and necrosis, reactive oxygen species (ROS) metabolism, inflammation as well as selected transcription factors (TF) and analyze their role for the different cell death patterns observed in the three regions. MOD, OC, and SV differ not only in their basal gene profiles but also in their ability to respond to injury and hypoxia. The results provide two coexpression clusters across the three regions, a Hif-1a coexpression cluster and a cluster around the cell death-associated transcripts Casp3, Capn1, Capn2, and Capns1. These clusters include the TF Jun, Bmyc, Nfyc, Foxd3, Hes1, the ROS-associated molecules Sod3, and Nos2, and the inflammatory chemokine Ccl20. The evidence of both clusters indicates the complex and regulated character of gene expression following injury and hypoxia across the three regions SV, OC, and MOD. The high vulnerability of spiral ganglion neurons in the MOD region, previously explained on the basis of the availability of neuro-trophic factors, is associated with the increased endogenous production of ROS and nitric oxide and inadequate activation of protective acting genes.

Potential Key Proteins, Molecular Networks, and Pathways in Perinatal Hypoxia.

Perinatal hypoxia is a common risk factor for CNS development. Using bioinformatics databases, a list of 129 genes involved in perinatal hypoxia was selected from the literature and analyzed with respect to proteins important for biological processes influencing the brain development. Functional enrichment analysis using the DAVID database was performed to identify relevant Gene Ontology (GO) biological processes like response to hypoxia, inflammatory response, positive and negative regulation of apoptosis, and positive and negative regulation of cell proliferation. The selected GO processes contain 17-30 proteins and show an enrichment of 6.3-14.3-fold. The STRING protein-protein interaction network and the Cytoscape data analyzer were used to identify interacting proteins playing a significant role in these processes. The two top protein pairs referring to the proteins with highest degrees and the corresponding proteins connected by high score edges exert opposite or regulatory functions and are essential for the balance between damaging, repairing, protective, or epigenetic processes. The GO response to hypoxia is characterized by the high score protein-protein interaction pairs CASP3/FAS promoting apoptosis and by the protective acting BDNF/MECP2 protein pair. Core components of the GO processes positive and negative regulation of apoptosis are the proteins CASP3/FAS/AKT/eNOS/RPS6KB1 involved in several signal pathways. The GO processes cell proliferation are characterized by the high-score protein-protein interaction pairs MYC/ MAPK1, JUN/MAPK1, IL6/IL1B, and JUN/HDAC1. The study provides new insights into the pathophysiology of perinatal hypoxia and is of importance for future investigations, diagnostics, and therapy of perinatal hypoxia.

Role of mitochondrial uncoupling protein 4 in rat inner ear.

The uncoupling protein 4 (UCP4) belongs to the mitochondrial anion transporter family. Protein tissue distribution and functions are still a matter of debate. Using an antibody we have previously shown that UCP4 appears in neurons and to a lesser extent in astrocytes of murine neuronal tissue as early as days 12-14 of embryonic development (Smorodchenko et al., 2009). Here we demonstrated for the first time that neurosensory cells such as hair cells of the inner ear and mechanosensitive Merkel cells in skin also express a significant amount of UCP4. We tested the hypothesis about whether UCP4 contributes to the regulation of oxidative stress using the model of oxygen deprivation. For this we compared the protein expression level in freshly isolated explants of organ of Corti, modiolus and stria vascularis from neonatal rats with explants cultured under hypoxia. Western blot analysis revealed that the UCP4 level was not increased under hypoxic conditions, when compared to the mitochondrial outer membrane protein VDAC or to the anti-oxidative enzyme SOD2. We moreover demonstrated that UCP4 expression is differently regulated during postnatal stages and is region-specific. We hypothesized that UCP4 may play an important role in functional maturation of the rat inner ear.

Expression analysis of prestin and selected transcription factors in newborn rats.

Transcription factors (TFs) have a central role to play in regulating gene expression. To analyze the co-expression patterns of selected TFs with the motor protein prestin of the outer hair cells, we applied an real-time PCR approach combining several kinds of information: (i) expression changes during postnatal development, (ii) expression changes by exposure of organotypic cultures of the organ of Corti to factors which significantly affect prestin expression [thyroid hormone (T4), retinoic acid (RA), butyric acid (BA), increased KCl concentration] and (iii) changes along the apical-basal gradient. We found that the mRNA levels of the TF Brn-3c (Pou4f3), a member of the POU family, are significantly associated with the regulation of prestin during postnatal development and in cultures supplemented with T4 (0.5 µM), BA (0.5-2.0 mM), and high KCl (50 mM) concentration. The mRNA level of the constitutively active TF C/ebpb (CCAAT/enhancer binding protein beta) correlates positively with the prestin expression during postnatal development and in cultures exposed to T4 and RA (50-100 µM). The mRNA levels of the calcium-dependent TF CaRF correlates significantly with the prestin expression in cultures exposed to T4 and high KCl concentration. The observed coexpression patterns may suggest that the TFs Brn-3c, C/ebpb, and Carf contribute to regulating the expression of prestin under the investigated conditions.

Evaluation of vardenafil for the treatment of subjective tinnitus: a controlled pilot study.

BACKGROUND: Vardenafil (Levitra(R)) represents a potent and highly selective phosphodiesterase type 5 (PDE5) inhibitor, which is established for treatment of various diseases. There are several unpublished reports from patients stating that vardenafil has a considerable therapeutic effect on their concomitant tinnitus. This pilot study was conducted to specifically assess the effect of vardenafil in patients with chronic tinnitus. METHODS: This trial was based on a prospective, randomized, double-blind, placebo-controlled, parallel group design. Fourty-two consecutive subjects with mon- or binaural chronic tinnitus received 10 mg vardenafil (N = 21) or matching placebo tablets (N = 21) administered orally twice a day over a period of 12 weeks. Clinical examination and data acquisition took place at each visit: at baseline, after 4 weeks, after 12 weeks (end of treatment with study medication), and at non-medicated follow-up after 16 weeks. Assessment of clinical effectiveness was based on a standardized tinnitus questionnaire (TQ), the Short Form 36 health survey (SF-36), audiometric measurements (mode, pitch and loudness of tinnitus; auditory thresholds) and biomarkers of oxidative stress in patients' blood (malondialdehyde, protein carbonyl, homocysteine and total antioxidative status). Therapeutic efficacy was evaluated by comparison of subjective and objective parameters with baseline data between both treatment groups (ANCOVA). RESULTS: Vardenafil had no superior efficacy over placebo in the treatment of chronic tinnitus during this study. The primary efficacy criterion 'TQ total score' failed to demonstrate significant improvement compared to placebo. Subjective reports of TQ subscales and general quality of life areas (SF-36), objective audiometric examinations as well as investigated biomarkers for oxidative stress did not reveal any significant treatment effects. The safety profile was favorable and consistent with that in other vardenafil studies. CONCLUSION: Although hypoxia and ischemia play a special role in the pathogenesis of tinnitus, the PDE5-inhibitor-induced increase of nitric oxide-mediated vasodilatation exerted no specific influence on tinnitus symptomatology. Considering the unclear risk of rarely associated hearing impairment, systemic application of vardenafil or other PDE5 inhibitors prove to be not appropriate for therapy of chronic tinnitus.

Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia.

The aim of the present study was to analyze the relations between the serum anti-erythropoietin antibody (AEAb) levels and the antibodies' neutralizing activity in 20 patients with renal anemia and rhEPO-induced antibodies. AEAb levels were determined by the enzyme-linked immunosorbent assay (ELISA, double antigen-bridging) and by radioimmunoprecipitation assay (RIPA). The bone marrow neutralization test was used to determine the neutralizing activity of the antibodies. RIPA and ELISA data resulted in closely correlated measurements. The relations between AEAb levels and the neutralizing activity of the antibodies are variable as shown by follow-up and cross-sectional evaluations of the data. Serum samples with a high antibody level (>1000 ng/ml) are associated with 100% neutralizing activity, whereas serum samples with lower AEAb levels show partial neutralizing activities or have no effect. Determining the neutralizing activity might be helpful when it comes to deciding of whether or not rhEPO therapy should be continued, specifically in patients who have low antibody levels. The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC(50)) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activities.

Expression of apoptosis-related genes in the organ of Corti, modiolus and stria vascularis of newborn rats.

Cell death in the inner ear tissues is an important mechanism leading to hearing impairment. Here, using microarrays and real-time RT-PCR we analyzed expression of selected apoptosis-related genes in rat's inner ear. We determined the gene expression in tissues freshly isolated from neonatal rats (3-5 days old) and compared it to that of explants cultured for 24 h under normoxic or hypoxic conditions. For the analyses, we used pooled samples of the organ of Corti (OC), modiolus (MOD) and stria vascularis (SV), respectively. We observed region-specific changes in gene expression between the fresh tissues and the normoxic culture. In the OC, expression of the proapoptotic genes caspase-2, caspase-3, caspase-6 and calpain-1 was downregulated. In the MOD, the antioxidative defense SOD-2 and SOD-3 were upregulated. In the SV, caspase-2, caspase-6, calpain-1 and SOD-3 were downregulated and SOD-2 upregulated. We speculate that these changes could reflect survival shift in transcriptome of inner ear explants tissues under in vitro conditions. With the exception of SOD-2, hypoxic culture conditions induced the same changes in gene expression as the normoxic conditions indicating that culture preparation is likely the dominating factor, which modifies the gene expression pattern. We conclude that various culture conditions induce different expression pattern of apoptosis-related genes in the organotypic cochlear cultures, as compared to fresh tissues. This transcriptional pattern may reflect the survival ability of specific tissues and could become a tempting target for a pharmacological intervention in inner ear diseases.

Up-regulation of prestin mRNA expression in the organs of Corti of guinea pigs and rats following unilateral impulse noise exposure.

Prestin is the motor protein of the outer hair cells (OHCs) and is required for both their electromotility and for cochlear amplification. We investigated the prestin mRNA expression in guinea pigs and rats in relation to the degree of noise-induced hearing loss (NIHL) induced by unilateral impulse noise exposure (167dB peak SPL) for 2.5-5 min. Distortion product otoacoustic emissions (DPOAE) and auditory brainstem responses were recorded before and one week post exposure. Prestin mRNA was examined by quantitative reverse transcription-polymerase chain reaction. Either the whole organs of Corti or the apical, middle and basal parts were examined separately. The specimens were pooled and grouped according to the degree of NIHL measured in the exposed ears. In rats, the number of hair cells was counted. A clear base-to-apex gradient in the prestin mRNA expression was found to exist in guinea pig and rat controls. In both species, there was an increase in the number of prestin RNA transcripts at a mean NIHL of about 15-25 dB indicating an up-regulation in the remaining intact cells. In rats, this degree of NIHL corresponded to an OHC loss of about 40%. Interestingly, the contralateral ears also revealed an up-regulation of prestin mRNA accompanied by significant DPOAE improvements.

Coenzyme Q10 does not protect cochlear hair cells from death in the ischemic organotypic culture.

OBJECTIVE: To evaluate in vitro the effect of coenzyme Q10 (CoQ(10)) on ischemia-induced hair cell death. STUDY DESIGN: Organotypic cochlear cultures of newborn rats were subjected to ischemia with and without CoQ(10). RESULTS: Addition of CoQ(10) has not prevented HC loss. CONCLUSION: CoQ(10) seems to protect against only certain modes of cell death.

A pilot clinical trial of the effects of coenzyme Q10 on chronic tinnitus aurium.

OBJECTIVE: To determine the short-term effects of coenzyme Q10 (CoQ10) on the antioxidative status and tinnitus expression in patients with chronic tinnitus aurium. STUDY DESIGN: A 16-week prospective nonrandomized clinical trial (n = 20). Tinnitus and Short Form-36 Questionnaires (TQ/SF-36) were evaluated together with the plasma concentrations of CoQ10, malondialdehyde, and the total antioxidant status. RESULTS: The mean plasma CoQ10 concentration rose under external CoQ10 supply and remained elevated after medication stopped without overall effects on the tinnitus score. However, in a subgroup of 7 patients with low initial plasma CoQ10 concentration and significant increase in the plasma CoQ10 level, a clear decrease in the TQ score was observed. CONCLUSION: In patients with a low plasma CoQ10 concentration, CoQ10 supply may decrease the tinnitus expression. SIGNIFICANCE: This is the first study to examine the effect of CoQ10 in chronic tinnitus aurium.

Detection of anti-EPO antibodies in human sera by a bridging ELISA is much more sensitive when coating biotinylated rhEPO to streptavidin rather than using direct coating of rhEPO.

Sensitive and efficient methods for detecting anti-erythropoietin (anti-EPO) antibodies are needed for analysis and, above all, for large scale screening of human serum samples. ELISA is an attractive alternative to labor-intensive radioimmunoprecipitation assays but apparently conflicting reports question its sensitivity. We sought to resolve this issue by directly comparing different reported ELISA approaches to determine whether rhEPO-coating methods affect detection of anti-EPO antibodies. Investigators reporting low sensitivity had used ELISAs in which rhEPO was directly coated to microtiter plates while the high sensitivity ELISA used plate-bound streptavidin to bind biotinylated rhEPO. Using anti-EPO positive human sera, our results confirmed a large (100- to 300-fold) difference in sensitivity between the ELISAs and suggested that the inferiority of the low sensitivity ELISA was caused by the direct coating of rhEPO which may disrupt epitopes by masking recognition sites or introducing conformational changes. Thus, a bridging ELISA can be an appropriate and effective system for antibody analysis and screening of human sera with high sensitivity and specificity but only if performed with streptavidin binding of biotinylated antigen. This finding may also be more generally applicable to the detection of antibodies against other protein antigens.

Recombinant human erythropoietin prevents ischemia-induced apoptosis and necrosis in explant cultures of the rat organ of Corti.

This study was designed to evaluate the effect of recombinant human erythropoietin (rhEPO), insulin-like growth factor-1 (rhIGF-1) and epidermal growth factor (rhEGF) on ischemia-induced hair cell loss in an organotypic cochlea culture. The apical, middle and basal parts of the organs of Corti (newborn rat, postnatal days 3-5) were exposed to ischemia (3.5 h) in glucose-free artificial perilymph (pO2 10-20 mmHg) with or without growth factors. Controls were exposed to normoxia. Twenty-four hours after the onset of ischemia, the cultures were stained using tetramethyl rhodamine isothiocyanate (TRITC) phalloidin (hair cells), propidium iodide (membrane integrity) and apoptosis detection kit (DNA-fragmentation). Ischemia (3.5 h) induced a hair cell loss of 20 and 40% in the middle and basal cochlear parts, respectively, and an increase of the numbers of PI-stained and DNA-fragmented nuclei (controls 0-1, ischemia 4-7 nuclei/100 microm). The basal part was more affected than the apical one. rhEPO and rhIGF-1 significantly attenuated the ischemia-induced hair cell loss by reducing processes involved in apoptosis and necrosis. rhEPO has been in clinical use for more than a decade and found to be well tolerated. Therefore, rhEPO could be an effective drug for the prevention of hearing loss via a hair cell protective mechanism.

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes.

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.

High potassium concentrations protect inner and outer hair cells in the newborn rat culture from ischemia-induced damage.

Several studies indicate that an increase in the extracellular potassium (K+) concentration is a factor exerting a damaging effect on cochlear hair cells (HCs). The present study was designed to examine the effects of high extracellular K+ concentrations on the HCs under normoxic and ischemic conditions. Organotypic cultures of the organ of Corti of newborn rats were exposed to normoxia and ischemia at K+ concentrations of 5-70 mM in artificial perilymph for 3-4h. The number of IHCs and OHCs in the apical, medial and basal parts of the cochlea were counted 24h later. The work resulted in two main findings: (1) extracellular K+ concentrations of 30-70 mM had no effect on the HCs under normoxic conditions; (2) under ischemic conditions, a clear HC loss, mainly in the medial and basal cochlear parts, was observed at 5 mM K+ as previously reported. In contrast, a high extracellular K+ concentration strongly attenuated the HC loss. This effect nearly completely disappeared by the addition of both eosin, an inhibitor of the plasma membrane calcium ATPase (PMCA), and linopirdine, an inhibitor of the KCNQ4 channel, indicating that a normal activity of the PMCA and the KCNQ4 channels are key factors for HC survival under ischemia and depolarizing conditions.

Pure red cell aplasia due to treatment with epoietin beta: first case report of PRCA from Poland.

Pure red cell aplasia (PRCA) emerged recently as the potentially life-threatening condition secondary to anti-Epo antibodies. Vast majority of cases were reported from Western European countries and were secondary to subcutaneous injections of Epoietin alpha. Here we describe possibly for the first time in the literature the case of PRCA from Poland or even Central and Eastern Europe in patient treated exclusively with Epoietin beta from multi-dose vial. In the last section of the paper the current epidemiological, diagnostic and therapeutic aspect of PRCA were discussed.

Apoptosis, necrosis and hypoxia inducible factor-1 in human head and neck squamous cell carcinoma cultures.

The objective of this study was to examine the mode of cell death and the hypoxia inducible factor-1 (HIF-1) expression of human head and neck squamous cell carcinoma (HNSCC) exposed to hypoxia in vitro. Apoptosis and necrosis rates were examined using flow cytometry. The findings suggest that HNSCC cells show a considerable heterogeneity in cell size and in response to hypoxia. A small-cell population showed a high spontaneous apoptosis and necrosis rate which was in-sensitive to hypoxia. A large-cell population responded to hypoxia by increase of apoptosis rate in parallel to recruitment of HIF-1. Hypoxia led to increased HIF-1alpha protein levels in nuclear extract using ELISA-binding activity. In all cells, accumulation of HIF-1 in the nuclei during hypoxia and a rapid degradation of HIF-1 in the post-hypoxic period were observed immunocytochemically. The HIF-1alpha mRNA level showed an expression of 10-40 pg/microg total RNA and remained unchanged in one cell line, while slightly decreasing in the other. Remarkably, no increased luciferase activity response was found on the reporter gene level using pGL3 reporter gene with three erythropoietin hypoxia responsive elements, either by hypoxia or by application of lactacystin, desferrioxamine or CoCl2. These findings suggest that, in HNSCC cells, hypoxia induces HIF-1alpha to stabilize and accumulate in the cell nuclei but have a cell-specific transcriptional complex.

Effect of perinatal asphyxia on tyrosine hydroxylase and D2 and D1 dopamine receptor mRNA levels expressed during early postnatal development in rat brain.

This study was designed to investigate the postnatal developmental plasticity of the mesostriatal and mesolimbic dopamine systems that occurs following perinatal asphyxia. The time course and patterning of the changes in levels of tyrosine hydroxylase (TH), and D1 and D2 dopamine receptor (R) mRNA in the cell body region, substantia nigra and ventral tegmental area (SN/VTA), and projection fields, striatum and limbic regions at the age of 6 and 24 h, and 1 week after asphyxia were studied with a quantitative reverse transcription polymerase chain reaction method with appropriate internal cRNA standard. In Caesarean-delivered control rats (Sprague-Dawley), TH, D2R and D1R mRNA levels showed regional and temporal specificity in both absolute levels and developmental kinetics during the first week of life. TH mRNA levels were >10-fold higher in SN/VTA than in striatum and limbic regions. Compared to Caesarean delivered controls, severe asphyxia (15-20 min) induced an increase of TH and D2R mRNA in SN/VTA 6 h and 1 week after birth. In addition, asphyxia induced an increase of TH mRNA in the projection fields, striatum and limbic regions, at 1 week. Perinatal asphyxia did not appear to exert any effect on D1R mRNA levels. No differences in any of the parameters were observed between spontaneous- and Caesarean-delivered animals. The present results indicate that perinatal asphyxia triggers coordinated changes in the expression of TH, and dopamine receptor mRNA in SN/VTA, striatum and limbic regions. These changes may affect differently dopamine D2R and D1R expression along development, contributing to long-term neurocircuitry imbalances.

Expression of prestin mRNA in the organotypic culture of rat cochlea.

To quantitate in absolute terms the prestin mRNA levels in the explant culture of rat cochlea, we used competitive RT-PCR with a synthetic internal cRNA standard. Prestin gene expression was found at levels of 100 fg specific mRNA/microg total RNA on postnatal day 3, which corresponds to about 300 copies per outer hair cell (OHC) and is indicative of an intermediate level of expression. Two days of culturing resulted in an increase of prestin mRNA levels and in the formation of an apical-basal gradient (p<0.001). To elucidate the variations the prestin mRNA levels undergo as a result of damage to the organ of Corti, we exposed the explant cultures to ischemia and hypoxia. While total RNA was observed to remain unchanged, the numbers of OHCs and the prestin mRNA levels were found to decrease by about 20% and 35%, respectively, compared to normoxia. In conclusion, we showed that the prestin mRNA levels during in vitro development increase and form an apical-basal gradient within 2 days in culture, similar to the postnatal in vivo development. Hypoxia and ischemia result in a decrease of the prestin mRNA level in parallel with OHC loss. The prestin mRNA level can therefore be used as marker of damage to or loss of OHCs.

Argon protects hypoxia-, cisplatin- and gentamycin-exposed hair cells in the newborn rat's organ of Corti.

During the last few years, an important protective effect of the noble gas xenon against neuronal hypoxic damage was observed. However, argon (Ar), a gas from the same chemical group, but less expensive and without anesthetic effect at normobaric pressure, has not been studied in terms of possible biological effects on cell protection. Ar was tested for its ability to protect organotypic cultures of the organ of Corti from 3-5 day old rats against hypoxia, cisplatin, and gentamycin toxicity. Cultures were exposed to nitrogen hypoxia (5% CO2, 95% N2), Ar hypoxia (5% CO2, 95% Ar) or normoxia for 30 h. Ar protected the hair cells from hypoxia-induced damage by about 25%. Ar-oxygen (O2) mixtures (21% O2, 5% CO2, 74% Ar) had no effect on the hair cell survival. Cisplatin (7.5-25 microM) and gentamycin (5-40 microM) exposed in medium under air damaged the hair cells in a dose-dependent manner. The exposure of cisplatin- and gentamycin-treated cultures to the Ar-O2 atmosphere significantly reduced the hair cell damage by up to 25%. This protective effect of Ar might provide a new protective approach against ototoxic processes.