Dimensions of Tinnitus-Related Distress.

OBJECTIVES: (1) To determine which psychosocial aspects predict tinnitus-related distress in a large self-reported dataset of patients with chronic tinnitus, and (2) to identify underlying constructs by means of factor analysis. METHODS: A cohort of 1958 patients of the Charité Tinnitus Center, Berlin completed a large questionnaire battery that comprised sociodemographic data, tinnitus-related distress, general psychological stress experience, emotional symptoms, and somatic complaints. To identify a construct of "tinnitus-related distress", significant predictive items were grouped using factor analysis. RESULTS: For the prediction of tinnitus-related distress (linear regression model with R2 = 0.7), depressive fatigue symptoms (concentration, sleep, rumination, joy decreased), the experience of emotional strain, somatization tendencies (pain experience, doctor contacts), and age appeared to play a role. The factor analysis revealed five factors: "stress", "pain experience", "fatigue", "autonomy", and low "educational level". CONCLUSIONS: Tinnitus-related distress is predicted by psychological and sociodemographic indices. Relevant factors seem to be depressive exhaustion with somatic expressions such as sleep and concentration problems, somatization, general psychological stress, and reduced activity, in addition to higher age.

Towards a unification of treatments and interventions for tinnitus patients: The EU research and innovation action UNITI.

Tinnitus is the perception of a phantom sound and the patient's reaction to it. Although much progress has been made, tinnitus remains a scientific and clinical enigma of high prevalence and high economic burden, with an estimated prevalence of 10%-20% among the adult population. The EU is funding a new collaborative project entitled "Unification of Treatments and Interventions for Tinnitus Patients" (UNITI, grant no. 848261) under its Horizon 2020 framework. The main goal of the UNITI project is to set the ground for a predictive computational model based on existing and longitudinal data attempting to address the question of which treatment or combination of treatments is optimal for a specific patient group based on certain parameters. Clinical, epidemiological, genetic and audiological data, including signals reflecting ear-brain communication, as well as patients' medical history, will be analyzed making use of existing databases. Predictive factors for different patient groups will be extracted and their prognostic relevance validated through a Randomized Clinical Trial (RCT) in which different patient groups will undergo a combination of tinnitus therapies targeting both auditory and central nervous systems. From a scientific point of view, the UNITI project can be summarized into the following research goals: (1) Analysis of existing data: Results of existing clinical studies will be analyzed to identify subgroups of patients with specific treatment responses and to identify systematic differences between the patient groups at the participating clinical centers. (2) Genetic and blood biomarker analysis: High throughput Whole Exome Sequencing (WES) will be performed in well-characterized chronic tinnitus cases, together with Proximity Extension Assays (PEA) for the identification of blood biomarkers for tinnitus. (3) RCT: A total of 500 patients will be recruited at five clinical centers across Europe comparing single treatments against combinational treatments. The four main treatments are Cognitive Behavioral Therapy (CBT), hearing aids, sound stimulation, and structured counseling. The consortium will also make use of e/m-health applications for the treatment and assessment of tinnitus. (4) Decision Support System: An innovative Decision Support System will be implemented, integrating all available parameters (epidemiological, clinical, audiometry, genetics, socioeconomic and medical history) to suggest specific examinations and the optimal intervention strategy based on the collected data. (5) Financial estimation analysis: A cost-effectiveness analysis for the respective interventions will be calculated to investigate the economic effects of the interventions based on quality-adjusted life years. In this paper, we will present the UNITI project, the scientific questions that it aims to address, the research consortium, and the organizational structure.

Decrease of prestin expression by increased potassium concentration in organotypic cultures of the organ of Corti of newborn rats.

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high sensitivity level of mammalian hearing. In the present study, we examined the effects of increased extracellular potassium (K(+)) concentration on the expression of prestin mRNA and the transcription factors Gata-3 and Carf in the organotypic culture of the organ of Corti of newborn rats. Mannitol and NaCl were used to analyze possible effects of hyperosmotic stress or ion-specific changes, respectively. An increase in prestin expression by a factor of 1.5-2.0 was seen in cultures grown in the presence of 5mM K(+). Potassium concentration of 35 and 55 mM induced a parallel decrease in prestin and Carf expression, but Gata-3 expression increased. Mannitol had no effect on gene expression whereas increased NaCl concentrations decreased prestin, but not Carf expression. The data suggest that chronic depolarization might decrease the prestin expression and possibly contribute to hearing loss and tinnitus.

Expression of genes implicated in oxidative stress in the cochlea of newborn rats.

Oxidative stress is an important mechanism inducing ototoxicity-, age- and noise-induced hearing loss. To better understand this phenomenon, we examined cochlear tissues for the expression of following genes involved directly or indirectly in the oxidative stress response: glyceraldehyde-3-phosphate dehydrogenase (Gapdh); solute carrier family-2 (facilitated glucose transporter), member-1 (Slc2a1); heme oxygenase-1 (Hmox1); heme oxygenase-2 (Hmox2); inducible nitric oxide synthase-2 (Nos2); transferrin (Tf); transferrin receptor (Tfrc); glutathione S-transferase A3 (Gsta3) and metallothionein-1a (Mt1a). Cochlear tissues were dissected from the p3-p5 Wistar rats, divided into the organ of Corti (OC), modiolus (MOD) and stria vascularis together with spiral ligament (SV + SL) and processed immediately or cultured under normoxic conditions or a short-term, mild hypoxia followed by re-oxygenation. After 24 h, explants were collected and total RNA isolated, transcribed and amplified in the real time RT-PCR. We found all genes listed above expressed in the freshly isolated cochlear tissues. In the OC and MOD, Slc2a1, Tf, and Mt1a were expressed on a lower level than in the SV + SL. In the OC, Hmox1 was expressed on a lower level than in the MOD and SV + SL. Hypoxic and normoxic cultures increased the transcript number of Gapdh, Slc2a1 and Hmox1 in all cochlear tissues. The expression of Nos2, Tf, Gsta3 and Mt1a increased in a tissue-specific manner. In the SV + SL, Mt1a expression decreased after normoxic and hypoxic conditions. Taken together, using real time RT-PCR, our results imply that oxidative stress may be an important component of cochlear injury during the developing period. In spite of the immaturity of the tissue, a differential response of antioxidant enzymes/proteins with respect to the pathway, the expression levels and regions was observed.

Effects of retinoic acid and butyric acid on the expression of prestin and Gata-3 in organotypic cultures of the organ of corti of newborn rats.

Prestin is the motor protein of the outer hair cells of the organ of Corti and a key factor in ensuring a high level of sensitivity of mammalian hearing. The factors that influence prestin expression are still largely unknown. We studied the effects of the application of retinoic acid, a ligand of a nuclear receptor, and of butyric acid, an inhibitor of histone deacetylase activity, on the expression of mRNA of prestin and Gata-3 in the organotypic culture of the organ of Corti of newborn rats using RT-PCR. Application of retinoic acid at concentrations of 1-50 µM results in a dose-dependent expression decrease after two days in culture. Treatment with sodium butyrate (0.5-2 mM) elevated the expression of prestin and Gata-3. Statistically significant correlations between Gata-3 and prestin mRNA levels were observed under all conditions. The data indicate that retinoid nuclear transcription factors, GATA-3 and histone acetylation/deacetylation processes may have a regulatory role to play in prestin expression.

Expression of prestin and Gata-3,-2,-1 mRNA in the rat organ of Corti during the postnatal period and in culture.

Based on observations that mutations of GATA-3 are responsible for the HDR-syndrome (hypoparathyroidism, deafness, renal defects) and that GATA-transcription factors have an important role to play in inner ear development, we hypothesized that these transcription factors may be involved in regulatory changes of prestin transcription. To prove this, we examined in parallel the expression of mRNA of prestin and Gata-3,-2 and Gata-1 in the organ of Corti during early postnatal development of rats and in organotypic cultures. Remarkable relations are observed between prestin and Gata-3,-2 expression in organ of Corti preparations in vivo and in vitro: (i) Gata-3,-2 expression display similar apical-basal gradients as prestin mRNA levels. (ii) The prestin expression increases between postnatal day two and postnatal day eight by a factor of about four in the apical and middle segments and by a factor of two in the basal part. Highly significant Pearson correlation coefficients were observed between Gata-3,-2 mRNA and prestin levels when the data were evaluated by regression analyses. (iii) Parallel changes of prestin mRNA and Gata-3,-2 mRNA levels were observed in response to thyroid hormone and to gemfibrozil application. These observations suggest a regulatory role played by the Gata-3,-2 transcription factors in prestin expression.

MRNA expression of members of the IGF system in the organ of Corti, the modiolus and the stria vascularis of newborn rats.

We analyzed the mRNA expression of the insulin-like growth factor (IGF) family genes and of selected downstream pathway genes using the Affymetrix microarray system and confirmatory RT-PCR in the freshly prepared organ of Corti (OC), modiolus (MOD) and stria vascularis (SV) from neonatal rats (3-5 days old) and after 24h in culture. Among the seven members of the IGF family analyzed in this paper, IGF1, IGF2 and IGF-binding protein (IGFBP2) had the highest basal expression in all regions. Preparatory stress and culture increased the expression of IGF2, IGFBP2, IGFBP3, IGFBP5, glucose transporterl (GLUT1), signal transducer, and activator of transcription3 (STAT3), phosphoinositide-3-kinase regulatory subunit (Pik3r1), Jun oncogene (c-jun) and decreased that of mitogen-activated protein kinases MAPK3 and MAPK14 in all regions. Region-specific changes were observed in OC (GLUT1), MOD (IGFBP3 and c-jun) and SV (IGF2 and IGFBP2).

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.

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.

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.

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.