Screening for Circulating Inflammatory Proteins Does Not Reveal Plasma Biomarkers of Constant Tinnitus.

BACKGROUND AND OBJECTIVE: Tinnitus would benefit from an objective biomarker. The goal of this study is to identify plasma biomarkers of constant and chronic tinnitus among selected circulating inflammatory proteins. METHODS: A case-control retrospective study on 548 cases with constant tinnitus and 548 matched controls from the Swedish Tinnitus Outreach Project (STOP), whose plasma samples were examined using Olink's Inflammatory panel. Replication and meta-analysis were performed using the same method on samples from the TwinsUK cohort. Participants from LifeGene, whose blood was collected in Stockholm and Umeå, were recruited to STOP for a tinnitus subtyping study. An age and sex matching was performed at the individual level. TwinsUK participants (n = 928) were selected based on self-reported tinnitus status over 2 to 10 years. Primary outcomes include normalized levels for 96 circulating proteins, which were used as an index test. No reference standard was available in this study. RESULTS: After adjustment for age, sex, BMI, smoking, hearing loss, and laboratory site, the top proteins identified were FGF-21, MCP4, GDNF, CXCL9, and MCP-1; however, these were no longer statistically significant after correction for multiple testing. Stratification by sex did not yield any significant associations. Similarly, associations with hearing loss or other tinnitus-related comorbidities such as stress, anxiety, depression, hyperacusis, temporomandibular joint disorders, and headache did not yield any significant associations. Analysis in the TwinsUK failed in replicating the top candidates. Meta-analysis of STOP and TwinsUK did not reveal any significant association. Using elastic net regularization, models exhibited poor predictive capacity tinnitus based on inflammatory markers [sensitivity = 0.52 (95% CI 0.47-0.57), specificity = 0.53 (0.48-0.58), positive predictive value = 0.52 (0.47-0.56), negative predictive values = 0.53 (0.49-0.58), and AUC = 0.53 (0.49-0.56)]. DISCUSSION: Our results did not identify significant associations of the selected inflammatory proteins with constant tinnitus. Future studies examining longitudinal relations among those with more severe tinnitus and using more recent expanded proteomics platforms and sampling of cerebrospinal fluid could increase the likelihood of identifying relevant molecular biomarkers.

Delivery of gene therapy through a cerebrospinal fluid conduit to rescue hearing in adult mice.

Inner ear gene therapy has recently effectively restored hearing in neonatal mice, but it is complicated in adulthood by the structural inaccessibility of the cochlea, which is embedded within the temporal bone. Alternative delivery routes may advance auditory research and also prove useful when translated to humans with progressive genetic-mediated hearing loss. Cerebrospinal fluid flow via the glymphatic system is emerging as a new approach for brain-wide drug delivery in rodents as well as humans. The cerebrospinal fluid and the fluid of the inner ear are connected via a bony channel called the cochlear aqueduct, but previous studies have not explored the possibility of delivering gene therapy via the cerebrospinal fluid to restore hearing in adult deaf mice. Here, we showed that the cochlear aqueduct in mice exhibits lymphatic-like characteristics. In vivo time-lapse magnetic resonance imaging, computed tomography, and optical fluorescence microscopy showed that large-particle tracers injected into the cerebrospinal fluid reached the inner ear by dispersive transport via the cochlear aqueduct in adult mice. A single intracisternal injection of adeno-associated virus carrying solute carrier family 17, member 8 (Slc17A8), which encodes vesicular glutamate transporter-3 (VGLUT3), rescued hearing in adult deaf Slc17A8-/- mice by restoring VGLUT3 protein expression in inner hair cells, with minimal ectopic expression in the brain and none in the liver. Our findings demonstrate that cerebrospinal fluid transport comprises an accessible route for gene delivery to the adult inner ear and may represent an important step toward using gene therapy to restore hearing in humans.

Gender-Specific Risk Factors and Comorbidities of Bothersome Tinnitus.

OBJECTIVE: This study aims to identify gender-specific risk factors associated with the presence of bothersome tinnitus (compared with non-bothersome tinnitus), including sociodemographic and lifestyle factors, tinnitus-associated phenomena (hearing loss, traumatic experiences, sleep disturbances), and physical as well as mental comorbidities. METHODS: We conducted a cross-sectional study using survey data from the Swedish LifeGene cohort containing information on self-reported tinnitus (N = 7615). We (1) analyzed risk factor and comorbidity frequencies, (2) computed multivariate logistic regression models to identify predictors of bothersome tinnitus within both genders, and (3) moderated logistic regression models to compare effects between genders. RESULTS: (1) The majority of factors that differed in frequencies between bothersome and non-bothersome tinnitus were equal for both genders. Women with bothersome tinnitus specifically reported higher rates of cardiovascular disease, thyroid disease, epilepsy, fibromyalgia, and burnout, and men with bothersome tinnitus reported higher rates of alcohol consumption, Ménière's disease, anxiety syndrome, and panic (compared with non-bothersome tinnitus, respectively). (2) Across both genders, multivariate logistic regression analyses revealed significant associations between bothersome tinnitus and age, reduced hearing ability, hearing-related difficulties in social situations, and reduced sleep quality. In women, bothersome tinnitus was specifically associated with cardiovascular disease and epilepsy; in men, with lower education levels and anxiety syndrome. (3) Moderated logistic regression analyses revealed that the effects of low education and anxiety syndrome were present in men, but not in women, whereas the effects of age, reduced hearing ability and related difficulties, cardiovascular disease, epilepsy, and burnout were not gender specific. CONCLUSION: Irrespective of gender, bothersome tinnitus is associated with higher age, reduced hearing ability, hearing-related difficulties, cardiovascular disease, epilepsy, and burnout. Gender-specific effects comprise low levels of education and the presence of anxiety syndrome for men. These findings need to be interpreted with caution, yet they suggest the presence of gender-specific biopsychosocial influences in the emergence or maintenance of bothersome tinnitus. Future studies ought to investigate the underlying mechanisms of the observed relationships.

Circadian vulnerability of cisplatin-induced ototoxicity in the cochlea.

The chemotherapeutic agent cisplatin is renowned for its ototoxic effects. While hair cells in the cochlea are established targets of cisplatin, less is known regarding the afferent synapse, which is an essential component in the faithful temporal transmission of sound. The glutamate aspartate transporter (GLAST) shields the auditory synapse from excessive glutamate release, and its loss of function increases the vulnerability to noise, salicylate, and aminoglycosides. Until now, the involvement of GLAST in cisplatin-mediated ototoxicity remains unknown. Here, we test in mice lacking GLAST the effects of a low-dose cisplatin known not to cause any detectable change in hearing thresholds. When administered at nighttime, a mild hearing loss in GLAST KO mice was found but not at daytime, revealing a potential circadian regulation of the vulnerability to cisplatin-mediated ototoxicity. We show that the auditory synapse of GLAST KO mice is more vulnerable to cisplatin administration during the active phase (nighttime) when compared to WT mice and treatment during the inactive phase (daytime). This effect was not related to the abundance of platinum compounds in the cochlea, rather cisplatin had a dose-dependent impact on cochlear clock rhythms only after treatment at nighttime suggesting that cisplatin can modulate the molecular clock. Our findings suggest that the current protocols of cisplatin administration in humans during daytime may cause a yet undetectable damage to the auditory synapse, more so in already damaged ears, and severely impact auditory sensitivity in cancer survivors.

Circadian integration of inflammation and glucocorticoid actions: Implications for the cochlea.

Auditory function has been shown to be influenced by the circadian system. Increasing evidence point towards the regulation of inflammation and glucocorticoid actions by circadian rhythms in the cochlea. Yet, how these three systems (circadian, immune and endocrine) converge to control auditory function remains to be established. Here we review the knowledge on immune and glucocorticoid actions, and how they interact with the circadian and the auditory system, with a particular emphasis on cochlear responses to noise trauma. We propose a multimodal approach to understand the mechanisms of noise-induced hearing loss by integrating the circadian, immune and endocrine systems into the bearings of the cochlea. Considering the well-established positive impact of chronotherapeutic approaches in the treatment of cardiovascular, asthma and cancer, an increased knowledge on the mechanisms where circadian, immune and glucocorticoids meet in the cochlea may improve current treatments against hearing disorders.

The genetic vulnerability to cisplatin ototoxicity: a systematic review.

Ototoxicity is one of the major side-effects of platinum-based chemotherapy, in particular cisplatin (cis-diammine dichloroplatinum II). To our knowledge, no systematic review has previously provided a quantitative summary estimate of the impact of genetics upon the risk of developing hearing loss. We searched Embase, Medline, ASSIA, Pubmed, Scopus, and Web of Science, for studies documenting the genetic risk of ototoxicity in patients with cancer treated with cisplatin. Titles/abstracts and full texts were reviewed for inclusion. Meta-analytic estimates of risk (Odds Ratio) from the pooled data were calculated for studies that have been repeated twice or more. The search identified 3891 papers, of which 30 were included. The majority were retrospective (44%), ranging from n = 39 to n = 317, some including only patients younger than 25 years of age (33%), and some on both genders (80%). The most common cancers involved were osteosarcoma (53%), neuroblastoma (37%), prostate (17%) and reproductive (10%). Most studies performed genotyping, though only 5 studies performed genome-wide association studies. Nineteen single-nucleotide polymorphisms (SNPs) from 15 genes were repeated more than twice. Meta-analysis of group data indicated that rs1872328 on ACYP2, which plays a role in calcium homeostasis, increases the risk of ototoxicity by 4.61 (95% CI: 3.04-7.02; N = 696, p < 0.0001) as well as LRP2 rs4668123 shows a cumulated Odds Ratio of 3.53 (95% CI: 1.48-8.45; N = 118, p = 0.0059), which could not be evidenced in individual studies. Despite the evidence of heterogeneity across studies, these meta-analytic results from 30 studies are consistent with a view of a genetic predisposition to platinum-based chemotherapy mediated ototoxicity. These new findings are informative and encourage the genetic screening of cancer patients in order to identify patients with greater vulnerability of developing hearing loss, a condition having a potentially large impact on quality of life. More studies are needed, with larger sample size, in order to identify additional markers of ototoxic risk associated with platinum-based chemotherapy and investigate polygenic risks, where multiple markers may exacerbate the side-effects.

Association of Genetic vs Environmental Factors in Swedish Adoptees With Clinically Significant Tinnitus.

Importance: No effective treatments are currently available for severe tinnitus, which affects 1% of the population and lowers the quality of life. The factors that contribute to the transition from mild to severe tinnitus are poorly known. Before performing genetic analyses and determining the mechanisms involved in the development of severe tinnitus, its heritability needs to be determined. Objectives: To examine whether clinically significant tinnitus is associated with genetic factors and to evaluate the genetic risk in the transmission of tinnitus using adoptees. Design, Setting, and Participants: Data from adoptees and their biological and adoptive parents from Swedish nationwide registers were collected from January 1, 1964, to December 31, 2015, and used to separate genetic from environmental factors in familial clustering. In all, 11 060 adoptees, 19 015 adoptive parents, and 17 025 biological parents were investigated. The study used a cohort design and a case-control approach to study genetic and nongenetic factors in tinnitus among adoptees. Main Outcomes and Measures: The primary outcome was odds ratio (OR) of tinnitus in adoptees with at least 1 affected biological parent compared with adoptees without any affected biological parent using logistic regression. The secondary outcome was OR in adoptees with at least 1 affected adoptive parent compared with adoptees without any affected adoptive parent. Results: A total of 1029 patients (440 [42.8%] male; mean [SD] age, 62 [14] years) with tinnitus were identified. The prevalence of diagnosed tinnitus was 2.2%. The OR for tinnitus was 2.22 for adoptees (95% CI, 1.03-4.81) of biological parents diagnosed with tinnitus, whereas the OR was 1.00 (95% CI, 0.43-2.32) for adoptees from adoptive parents diagnosed with tinnitus. Mean (SE) heritability determined using tetrachoric correlations was 31% (14%). Conclusions and Relevance: The findings suggest that genetic factors are associated with the familial clustering of clinically significant tinnitus with no shared-environment association, revealing that the transition from negligible to severe tinnitus may be associated with genetic factors. These findings may provide insight for future genetic analyses that focus on severe tinnitus.

Estradiol treatment and hormonal fluctuations during the estrous cycle modulate the expression of estrogen receptors in the auditory system and the prepulse inhibition of acoustic startle response.

Estrogens' effects on hearing are documented across species, but the responsible molecular mechanisms remain unknown. The presence of estrogen receptors (ER) throughout the auditory system offers a potential pathway of direct estrogenic effects on auditory function, but little is known about how each ER's expression is regulated by the overall hormonal status of the body. In the present study, we determined the effects of ovariectomy and chronic 17ß-estradiol treatment on mRNA and protein expression of ERα and ERß in peripheral (cochlea) and central (inferior colliculus) auditory structures of mice, as well as on auditory-related behavior using the acoustic startle response (ASR), prepulse inhibition (PPI), and habituation of the startle response. 17ß-Estradiol treatment down-regulated ERα but not ERß and increased PPI and latency of the ASR. Neither the magnitude nor the habituation of ASR was affected. Furthermore, ER's mRNA and protein expression in the inner ear were analyzed throughout the estrous cycle (proestrus, estrus, metestrus, and diestrus), revealing a negative correlation of circulating estrogens with ERα expression, whereas ERß was stable. Our findings show that ER not only are present in both the peripheral and central auditory system but also that circulating estrogen levels down-regulate ERα expression in the auditory system and affect PPI and the latency of ASR, suggesting a key role of ERα as a hormone-induced modulator of the auditory system and behavior.

Epidermal growth factor receptor expression in human fetal cochlea with Turner syndrome.

HYPOTHESIS: Growth hormones have beneficial effects on increasing height in adults with Turner syndrome (TS) and may also affect auditory function. BACKGROUND: Turner syndrome is the most common sex-linked chromosomal abnormality in female conceptions. Epidermal growth factor and its receptor (EGFR) affect differentiation, proliferation, and migration of epithelial cells and function as survival factors. The expression of EGFR is found in the developing and juvenile inner ear of experimental animals but is absent in adults. METHODS: To determine whether EGFR plays a role in TS, its expression was analyzed in the cochlea of healthy fetus and fetus with TS and in healthy adults. RESULTS: In healthy fetuses, EGFR protein expression was localized to the inner and outer hair cells and the Reissner membrane. The fetuses with TS on the 13th gestational week (GW) showed a similar pattern of immunoreactivity as the normal 16th and 20th GW cochlea. By the 23rd GW, EGFR immunoreactivity was not detectable in the TS hair cells or the Reissner membrane, and less intensive staining was found in the surrounding fibrocytes of the spiral ganglion. CONCLUSION: This is the first demonstration of EGFR immunoreactivity in the human cochlea and illustrates how EGFR expression is altered during development in TS. These findings indicate the importance of growth hormone receptors for inner ear development in humans.

Functional responses of estrogen receptors in the male and female auditory system.

Recently significant progress was made in understanding the mechanisms by which the two estrogen receptors (alpha and beta) are involved in different pathways of estrogen action in a wide variance of tissues. Divergent responses of cells and tissues to estrogens or their ligands have been attributed to various isoforms and signaling pathways of estrogen receptors. Both subtypes of estrogen receptors have been identified in the cochlea and there are indications that they have neuroprotective effects but there is still limited information on the role and specific mechanisms of these two receptors in the auditory system. This review will examine the molecular and functional actions of the two estrogen receptor subtypes, the pivotal role they play in the auditory system and their therapeutic strategies for protecting against hearing loss.

Prenatal dexamethasone impairs behavior and the activation of the BDNF exon IV promoter in the paraventricular nucleus in adult offspring.

Prenatal manipulations to the hypothalamic-pituitary-adrenal axis are shown to affect auditory responses to an acoustic challenge as well as behavior in adult life. To achieve these results, we examined the effect of prenatal dexamethasone (DEX) treatment in male and female adult rat offspring by assessing body and adrenal weight, anxiety using the elevated plus maze (EPM), and acoustic startle responses as well as the effects of acoustic challenge in the paraventricular nucleus (PVN). DEX male offspring had reduced adrenal gland weight in adult life and demonstrated anxiolytic-like behavior when tested on the EPM. The acoustic startle amplitude in naive DEX-treated male offspring was significantly higher compared with saline (SAL)-treated males and females and DEX-treated females. When challenged with either a glucocorticoid agonist or antagonist, the startle response of the SAL-treated males and females significantly increased or decreased in the presence of agonist and antagonist treatment, respectively, whereas DEX males and females were not affected. Acoustic challenge caused an increase in c-fos mRNA and glucocorticoid receptor nuclear translocation in the PVN of all groups. BDNF and TrkB mRNA increased in the PVN after acoustic challenge in the SAL-treated males and females but not in the DEX males or females. These findings exemplify the differential sensitivity of the developing nervous and endocrine systems to prenatal hormonal stress and demonstrate that prenatal DEX treatment elicits long-term behavioral alterations related to anxiety and auditory processing.

Estrogen receptor beta protects against acoustic trauma in mice.

The hormone estradiol affects the auditory system both by itself and by its interaction with neuroprotective factors. In this study, we examined the role of estrogen receptors (ERs) in response to auditory trauma. We found a ligand-dependent protective role for ERbeta in the auditory system by investigating mice deficient in ERalpha (ERKO mice), ERbeta (BERKO mice), and aromatase (ARKO mice). Basal auditory brainstem response (ABR) thresholds were similar in all animals. An acoustic trauma causing a temporary hearing loss raised ABR thresholds in male and female BERKO and ARKO mice compared with WT and ERKO mice. The ERalpha-selective agonist, propyl(1H) pyrazole-1,3,5-triyl-trisphenol (PPT), partially protected ARKO mice from trauma, while the ERbeta-selective agonist, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN), protected WT and ARKO mice. Immunohistochemistry and western blotting confirmed the expression of ERbeta in cochlea of WT males and females. Levels of brain-derived neurotrophic factor (BDNF), a neuroprotective peptide that can be induced by estrogen, was lower in BERKO and ARKO mice compared with WT. DPN treatment increased BDNF expression in ARKO mice. These data indicate ERbeta-mediated neuroprotection involving BDNF in the auditory system of males and females.

Effects of a single high dose of cisplatin on the melanocytes of the stria vascularis in the guinea pig.

The antineoplastic drug cisplatin is known to cause a reduction in endocochlear potential. The hypothesis to be tested was whether a single high dose of cisplatin affects the melanocytes by altering the expression of melanin. Pigmented guinea pigs received a bolus injection of cisplatin (8 mg/kg as a 15-second intravenous infusion). Auditory brainstem response (ABR) thresholds and morphological analysis of the hair cells and the stria vascularis were made 96 h after injection. ABR thresholds were elevated (15-40 dB) at 12-30 kHz and a significant loss of outer hair cells in the more basal regions was found. Cisplatin caused a significantly lower density of melanin in the intermediate cells in the basal region without any signs of apoptosis. Changes in melanin content were not noted in the middle or apical cochlear regions. Significant correlations were found between melanin density, ABR threshold shifts and outer hair cell loss in the region corresponding to 30 kHz. The findings reported here further support the multiple cytotoxic effect of cisplatin on the inner ear.

N-Acetyl L-cysteine does not protect against premature age-related hearing loss in C57BL/6J mice: a pilot study.

A compound capable of preventing age-related hearing loss would be very useful in an aging population. N-acetyl-L-cysteine (L-NAC) has been shown to be protective against noise exposure, a condition that leads to increased oxidative stress. Not withstanding environmental factors, there is evidence that age-related hearing loss (AHL) in the mouse is linked to more than one genetic loci and, by extension, in humans. Our hypothesis is that AHL defect results in increased sensitivity to oxidative stress and L-NAC would be able to protect the hearing of a mouse model of pre-mature AHL, the C57BL/6J (B6) mouse strain. L-NAC was added to the regular water bottle of B6 mice (experimental group) and available ad lib. The other group received normal tap water. Hearing was tested monthly by the ability to generate the auditory brainstem response (ABR). After the final ABR test, mice were sacrificed by an overdose of Avertin, ears were harvested and hair cell loss was quantified. There was no difference in ABR thresholds or in histopathology between the control group and the group receiving L-NAC in their drinking water. In contrast to the protective effects of L-NAC against noise-induced hearing loss, the lack of protective effect in this study may be due to (i) the dosage level; (ii) the duration of treatment; (iii) the biochemical mechanisms underlying age-induced hearing loss; or (iv) how the mouse metabolizes L-NAC.

Activation of tyrosine hydroxylase in the lateral efferent terminals by sound conditioning.

Preconditioning to sound is a well-documented strategy to provide protections against a subsequent acoustic trauma. In the present study, preconditioning (1.0 kHz tone at 81 dB sound pressure level (SPL) for 24 h) protected ABR thresholds by 17-28 dB from an acoustic trauma (2.7 kHz, 103 dB SPL, 30 min) that resulted in a temporary threshold shift. The protection afforded by sound conditioning was shown to be blocked by the administration of 6-hydroxydopamine which disrupts tyrosine hydroxylase in the nerve terminals of the lateral efferent fibers. Furthermore, tyrosine hydroxylase immunoreactivity was up-regulated both by sound conditioning alone, and by the combined treatment of sound conditioning and acoustic trauma. In contrast, acoustic trauma alone resulted in a reduction in tyrosine hydroxylase immunoreactivity compared to unexposed controls. These findings are the first demonstration that tyrosine hydroxylase in the lateral efferents are up-regulated during sound conditioning and suggests a role for the lateral efferent system in protecting against acoustic trauma by sound conditioning.

Protective mechanisms of sound conditioning.

Evidence continues to accumulate demonstrating the importance of reducing the deleterious effects of noise trauma by sound conditioning. Sound conditioning is an active process induced by low-level, nondamaging noise exposure that creates long-term protective effects to subsequent detrimental forms of noise trauma. This phenomenon is now shown to occur in a variety of mammals, including gerbils, chinchillas, guinea pigs, rabbits, rats, mice and human subjects. Different sound-conditioning paradigms have been proven successful in preventing pathological changes to the auditory system. These studies are reviewed in the present chapter and the possible biological mechanisms underlying this phenomenon are discussed.

The Identification and Localization of the Guanine Nucleotide Binding Protein G0 in the Auditory System.

The identification of guanine nucleotide binding proteins (G proteins) in guinea-pig tissues was assessed by the adenosine diphosphate-ribosylation of the alpha subunit by Bordetella pertussis toxin using [alpha32P]nicotinamide adenine dinucleotide as the substrate followed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis and autoradiography. Three tissues (inferior colliculus, neuroblastoma cells, and the organ of Corti) contained G0alpha (39 kD), as well as Gi2alpha (40 kD) and Gi1alpha and/or Gi3alpha (41 kD). The stria vascularis and the VIIIth nerve contained mainly Gi2alpha, Gi1alpha and/or Gi3alpha, but G0alpha was barely detectable. A purified preparation of outer hair cells from the organ of Corti contained all three pertussis toxin substrates including G0alpha, with the Gi2alpha (40 kD) subunit being the most prominent. The immunocytochemical localization of the G0alpha subunit was determined by light microscopy after incubating isolated outer hair cells, Hensen cells and the stria vascularis with affinity-purified anti-G0alpha antibodies. In hair cells a positive reaction was observed along the plasma membrane and around the perimeter of the cuticular plate (zona adherens). Positive reaction was also observed within the infracuticular network extending from the cuticular plate towards the nucleus in outer hair cells. Finally, the base of the outer hair cells also contained G0alpha. However, it is likely that the G0alpha that is present in this cell region is not within the hair cell itself, but rather in nerve terminals which remained attached during dissection.