Noise-induced hearing disorders: Clinical and investigational tools.

A series of articles discussing advanced diagnostics that can be used to assess noise injury and associated noise-induced hearing disorders (NIHD) was developed under the umbrella of the United States Department of Defense Hearing Center of Excellence Pharmaceutical Interventions for Hearing Loss working group. The overarching goals of the current series were to provide insight into (1) well-established and more recently developed metrics that are sensitive for detection of cochlear pathology or diagnosis of NIHD, and (2) the tools that are available for characterizing individual noise hazard as personal exposure will vary based on distance to the sound source and placement of hearing protection devices. In addition to discussing the utility of advanced diagnostics in patient care settings, the current articles discuss the selection of outcomes and end points that can be considered for use in clinical trials investigating hearing loss prevention and hearing rehabilitation.

Integrating pharmacogenomics into clinical trials of hearing disorders.

In 2019, the U.S. Food and Drug Administration issued guidance to increase the efficiency of drug development and support precision medicine, including tailoring treatments to those patients who will benefit based on genetic variation even in the absence of a documented mechanism of action. Although multiple advancements have been made in the field of pharmacogenetics (PGx) for other disease conditions, there are no approved PGx guidelines in the treatment of hearing disorders. In studies of noise-induced hearing loss (NIHL), some progress has been made in the last several years associating genomic loci with susceptibility to noise damage. However, the power of such studies is limited as the underlying physiological responses may vary considerably among the patient populations. Here, we have summarized previous animal studies to argue that NIHL subtyping is a promising strategy to increase the granularity of audiological assessments. By coupling this enhanced phenotyping capability with genetic association studies, we suggest that drug efficacy will be better predicted, increasing the likelihood of success in clinical trials when populations are stratified based on genetic variation or designed with multidrug combinations to reach a broader segment of individuals suffering or at risk from NIHL.

Optimizing non-invasive functional markers for cochlear deafferentation based on electrocochleography and auditory brainstem responses.

Accumulating evidence suggests that cochlear deafferentation may contribute to suprathreshold deficits observed with or without elevated hearing thresholds, and can lead to accelerated age-related hearing loss. Currently there are no clinical diagnostic tools to detect human cochlear deafferentation in vivo. Preclinical studies using a combination of electrophysiological and post-mortem histological methods clearly demonstrate cochlear deafferentation including myelination loss, mitochondrial damages in spiral ganglion neurons (SGNs), and synaptic loss between inner hair cells and SGNs. Since clinical diagnosis of human cochlear deafferentation cannot include post-mortem histological quantification, various attempts based on functional measurements have been made to detect cochlear deafferentation. So far, those efforts have led to inconclusive results. Two major obstacles to the development of in vivo clinical diagnostics include a lack of standardized methods to validate new approaches and characterize the normative range of repeated measurements. In this overview, we examine strategies from previous studies to detect cochlear deafferentation from electrocochleography and auditory brainstem responses. We then summarize possible approaches to improve these non-invasive functional methods for detecting cochlear deafferentation with a focus on cochlear synaptopathy. We identify conceptual approaches that should be tested to associate unique electrophysiological features with cochlear deafferentation.

Detecting Cochlear Synaptopathy Through Curvature Quantification of the Auditory Brainstem Response.

The sound-evoked electrical compound potential known as auditory brainstem response (ABR) represents the firing of a heterogenous population of auditory neurons in response to sound stimuli, and is often used for clinical diagnosis based on wave amplitude and latency. However, recent ABR applications to detect human cochlear synaptopathy have led to inconsistent results, mainly due to the high variability of ABR wave-1 amplitude. Here, rather than focusing on the amplitude of ABR wave 1, we evaluated the use of ABR wave curvature to detect cochlear synaptic loss. We first compared four curvature quantification methods using simulated ABR waves, and identified that the cubic spline method using five data points produced the most accurate quantification. We next evaluated this quantification method with ABR data from an established mouse model with cochlear synaptopathy. The data clearly demonstrated that curvature measurement is more sensitive and consistent in identifying cochlear synaptic loss in mice compared to the amplitude and latency measurements. We further tested this curvature method in a different mouse model presenting with otitis media. The change in curvature profile due to middle ear infection in otitis media is different from the profile of mice with cochlear synaptopathy. Thus, our study suggests that curvature quantification can be used to address the current ABR variability issue, and may lead to additional applications in the clinic diagnosis of hearing disorders.

Early Physiological and Cellular Indicators of Cisplatin-Induced Ototoxicity.

Cisplatin chemotherapy often causes permanent hearing loss, which leads to a multifaceted decrease in quality of life. Identification of early cisplatin-induced cochlear damage would greatly improve clinical diagnosis and provide potential drug targets to prevent cisplatin's ototoxicity. With improved functional and immunocytochemical assays, a recent seminal discovery revealed that synaptic loss between inner hair cells and spiral ganglion neurons is a major form of early cochlear damage induced by noise exposure or aging. This breakthrough discovery prompted the current study to determine early functional, cellular, and molecular changes for cisplatin-induced hearing loss, in part to determine if synapse injury is caused by cisplatin exposure. Cisplatin was delivered in one to three treatment cycles to both male and female mice. After the cisplatin treatment of three cycles, threshold shift was observed across frequencies tested like previous studies. After the treatment of two cycles, beside loss of outer hair cells and an increase in high-frequency hearing thresholds, a significant latency delay of auditory brainstem response wave 1 was observed, including at a frequency region where there were no changes in hearing thresholds. The wave 1 latency delay was detected as early cisplatin-induced ototoxicity after only one cycle of treatment, in which no significant threshold shift was found. In the same mice, mitochondrial loss in the base of the cochlea and declining mitochondrial morphometric health were observed. Thus, we have identified early spiral ganglion-associated functional and cellular changes after cisplatin treatment that precede significant threshold shift.

Evidence for independent peripheral and central age-related hearing impairment.

Deleterious age-related changes in the central auditory nervous system have been referred to as central age-related hearing impairment (ARHI) or central presbycusis. Central ARHI is often assumed to be the consequence of peripheral ARHI. However, it is possible that certain aspects of central ARHI are independent from peripheral ARHI. A confirmation of this possibility could lead to significant improvements in current rehabilitation practices. The major difficulty in addressing this issue arises from confounding factors, such as other age-related changes in both the cochlea and central non-auditory brain structures. Because gap detection is a common measure of central auditory temporal processing, and gap detection thresholds are less influenced by changes in other brain functions such as learning and memory, we investigated the potential relationship between age-related peripheral hearing loss (i.e., audiograms) and age-related changes in gap detection. Consistent with previous studies, a significant difference was found for gap detection thresholds between young and older adults. However, among older adults, no significant associations were observed between gap detection ability and several other independent variables including the pure tone audiogram average, the Wechsler Adult Intelligence Scale-Vocabulary score, gender, and age. Statistical analyses showed little or no contributions from these independent variables to gap detection thresholds. Thus, our data indicate that age-related decline in central temporal processing is largely independent of peripheral ARHI.

Alcohol abuse and disorder of granulopoiesis.

Granulocytes are the major type of phagocytes constituting the front line of innate immune defense against bacterial infection. In adults, granulocytes are derived from hematopoietic stem cells in the bone marrow. Alcohol is the most frequently abused substance in human society. Excessive alcohol consumption injures hematopoietic tissue, impairing bone marrow production of granulocytes through disrupting homeostasis of granulopoiesis and the granulopoietic response. Because of the compromised immune defense function, alcohol abusers are susceptible to infectious diseases, particularly septic infection. Alcoholic patients with septic infection and granulocytopenia have an exceedingly high mortality rate. Treatment of serious infection in alcoholic patients with bone marrow inhibition continues to be a major challenge. Excessive alcohol consumption also causes diseases in other organ systems, particularly severe alcoholic hepatitis which is life threatening. Corticosteroids are the only therapeutic option for improving short-term survival in patients with severe alcoholic hepatitis. The existence of advanced alcoholic liver diseases and administration of corticosteroids make it more difficult to treat serious infection in alcoholic patients with the disorder of granulopoieis. This article reviews the recent development in understanding alcohol-induced disruption of marrow granulopoiesis and the granulopoietic response with the focus on progress in delineating cell signaling mechanisms underlying the alcohol-induced injury to hematopoietic tissue. Efforts in exploring effective therapy to improve patient care in this field will also be discussed.

Otoprotective Effects of Stephania tetrandra S. Moore Herb Isolate against Acoustic Trauma.

Noise is the most common occupational and environmental hazard, and noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit. Although therapeutics that target the free-radical pathway have shown promise, none of these compounds is currently approved against NIHL by the United States Food and Drug Administration. The present study has demonstrated that tetrandrine (TET), a traditional Chinese medicinal alkaloid and the main chemical isolate of the Stephania tetrandra S. Moore herb, significantly attenuated NIHL in CBA/CaJ mice. TET is known to exert antihypertensive and antiarrhythmic effects through the blocking of calcium channels. Whole-cell patch-clamp recording from adult spiral ganglion neurons showed that TET blocked the transient Ca2+ current in a dose-dependent manner and the half-blocking concentration was 0.6 + 0.1 µM. Consistent with previous findings that modulations of calcium-based signaling pathways have both prophylactic and therapeutic effects against neural trauma, NIHL was significantly diminished by TET administration. Importantly, TET has a long-lasting protective effect after noise exposure (48 weeks) in comparison to 2 weeks after noise exposure. The otoprotective effects of TET were achieved mainly by preventing outer hair cell damage and synapse loss between inner hair cells and spiral ganglion neurons. Thus, our data indicate that TET has great potential in the prevention and treatment of NIHL.

Detection of single mRNAs in individual cells of the auditory system.

Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.

An operant-based detection method for inferring tinnitus in mice.

BACKGROUND: Subjective tinnitus is a hearing disorder in which a person perceives sound when no external sound is present. It can be acute or chronic. Because our current understanding of its pathology is incomplete, no effective cures have yet been established. Mouse models are useful for studying the pathophysiology of tinnitus as well as for developing therapeutic treatments. NEW METHOD: We have developed a new method for determining acute and chronic tinnitus in mice, called sound-based avoidance detection (SBAD). The SBAD method utilizes one paradigm to detect tinnitus and another paradigm to monitor possible confounding factors, such as motor impairment, loss of motivation, and deficits in learning and memory. RESULTS: The SBAD method has succeeded in monitoring both acute and chronic tinnitus in mice. Its detection ability is further validated by functional studies demonstrating an abnormal increase in neuronal activity in the inferior colliculus of mice that had previously been identified as having tinnitus by the SBAD method. COMPARISON WITH EXISTING METHODS: The SBAD method provides a new means by which investigators can detect tinnitus in a single mouse accurately and with more control over potential confounding factors than existing methods. CONCLUSION: This work establishes a new behavioral method for detecting tinnitus in mice. The detection outcome is consistent with functional validation. One key advantage of mouse models is they provide researchers the opportunity to utilize an extensive array of genetic tools. This new method could lead to a deeper understanding of the molecular pathways underlying tinnitus pathology.

Noise-induced cochlear synaptopathy: Past findings and future studies.

For decades, we have presumed the death of hair cells and spiral ganglion neurons are the main cause of hearing loss and difficulties understanding speech in noise, but new findings suggest synapse loss may be the key contributor. Specifically, recent preclinical studies suggest that the synapses between inner hair cells and spiral ganglion neurons with low spontaneous rates and high thresholds are the most vulnerable subcellular structures, with respect to insults during aging and noise exposure. This cochlear synaptopathy can be "hidden" because this synaptic loss can occur without permanent hearing threshold shifts. This new discovery of synaptic loss opens doors to new research directions. Here, we review a number of recent studies and make suggestions in two critical future research directions. First, based on solid evidence of cochlear synaptopathy in animal models, it is time to apply molecular approaches to identify the underlying molecular mechanisms; improved understanding is necessary for developing rational, effective therapies against this cochlear synaptopathy. Second, in human studies, the data supporting cochlear synaptopathy are indirect although rapid progress has been made. To fully identify changes in function that are directly related this hidden synaptic damage, we argue that a battery of tests including both electrophysiological and behavior tests should be combined for diagnosis of "hidden hearing loss" in clinical studies. This new approach may provide a direct link between cochlear synaptopathy and perceptual difficulties.

Canertinib induces ototoxicity in three preclinical models.

Neuregulin-1 (NRG1) ligand and its epidermal growth factor receptor (EGFR)/ERBB family regulate normal cellular proliferation and differentiation in many tissues including the cochlea. Aberrant NRG1 and ERBB signaling cause significant hearing impairment in mice. Dysregulation of the same signaling pathway in humans is involved in certain types of cancers such as breast cancer or non-small cell lung cancer (NSCLC). A new irreversible pan-ERBB inhibitor, canertinib, has been tested in clinical trials for the treatment of refractory NSCLC. Its possible ototoxicity was unknown. In this study, a significant dose-dependent canertinib ototoxicity was observed in a zebrafish model. Canertinib ototoxicity was further confirmed in two mouse models with different genetic backgrounds. The data strongly suggested an evolutionally preserved ERBB molecular mechanism underlying canertinib ototoxicity. Thus, these results imply that clinical monitoring of hearing loss should be considered for clinical testing of canertinib or other pan-ERBB inhibitors.

Identification of multiple metabolic enzymes from mice cochleae tissue using a novel functional proteomics technology.

A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.

T-type calcium channel blockers as neuroprotective agents.

T-type calcium channels are expressed in many diverse tissues, including neuronal, cardiovascular, and endocrine. T-type calcium channels are known to play roles in the development, maintenance, and repair of these tissues but have also been implicated in disease when not properly regulated. Calcium channel blockers have been developed to treat various diseases and their use clinically is widespread due to both their efficacy as well as their safety. Aside from their established clinical applications, recent studies have suggested neuroprotective effects of T-type calcium channel blockers. Many of the current T-type calcium channel blockers could act on other molecular targets besides T-type calcium channels making it uncertain whether their neuroprotective effects are solely due to blocking of T-type calcium channels. In this review, we discuss these drugs as well as newly developed chemical compounds that are designed to be more selective for T-type calcium channels. We review in vitro and in vivo evidence of neuroprotective effects by these T-type calcium channel blockers. We conclude by discussing possible molecular mechanisms underlying the neuroprotective effects by T-type calcium channel blockers.

Prophylactic and therapeutic functions of drug combinations against noise-induced hearing loss.

Noise is the most common occupational and environmental hazard. Noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit, after age-related hearing loss (presbycusis). Although promising approaches have been identified for reducing NIHL, currently there are no effective medications to prevent NIHL. Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL. We turned this difficulty into an advantage by asking whether NIHL could be effectively prevented by targeting multiple signaling pathways with a combination of drugs already approved by U.S. Food and Drug Administration (FDA). We previously found that antiepileptic drugs blocking T-type calcium channels had both prophylactic and therapeutic effects for NIHL. NIHL can also be reduced by an up-regulation of glucocorticoid (GC) signaling pathways. Based on these findings, we tested a combination therapy for NIHL that included ethosuximide and zonisamide (anticonvulsants) and dexamethasone and methylprednisolone (synthetic GCs) in mice under exposure conditions typically associated with dramatic permanent threshold shifts (PTS). We first examined possible prophylactic effects for each drug when administered alone 2 h before noise, and calculated the median effective dose (ED50). We then tested for synergistic effects of two-drug combinations (anticonvulsant + GC), and identified combinations with the strongest synergy against NIHL, based on a previously established combination index (CI) metric. We repeated similar tests to determine their therapeutic effects when administered the same drugs 24 h after the noise exposure. Our study shows the feasibility of developing pharmacological intervention in multiple pathways, and discovering drug combinations with optimal synergistic effects in preventing permanent NIHL.

Reprogramming of single-cell-derived mesenchymal stem cells into hair cell-like cells.

HYPOTHESIS: Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. BACKGROUND: Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. METHODS: Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer, and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed using reverse transcriptase-polymerase chain reaction, and cell structures were examined using immunocytochemistry. RESULTS: Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells before overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. CONCLUSION: We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells.

Recent advances in the study of age-related hearing loss: a mini-review.

Hearing loss is a common age-associated affliction that can result from the loss of hair cells and spiral ganglion neurons (SGNs) in the cochlea. Although hair cells and SGNs are typically lost in the same cochlea, recent analysis suggests that they can occur independently, via unique mechanisms. Research has identified both environmental and genetic factors that contribute to degeneration of cochlear cells. Additionally, molecular analysis has identified multiple cell-signaling mechanisms that likely contribute to pathological changes that result in hearing deficiencies. These analyses should serve as useful primers for future work, including genomic and proteomic analysis, to elucidate the mechanisms driving cell loss in the aging cochlea. Significant progress in this field has occurred in the past decade. As our understanding of aging-induced cochlear changes continues to improve, our ability to offer medical intervention will surely benefit the growing elderly population.

MicroRNA-181a regulates local immune balance by inhibiting proliferation and immunosuppressive properties of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) exhibit extensive self-renewal potential and can modulate immunocyte activation. Our previous study reported that miR-181a expression was significantly increased in placenta from women with severe preeclampsia (PE), but the mechanisms by which miR-181a regulates MSCs are unknown. In this study, we asked if and how miR-181a regulates MSCs' proliferation and immunosuppressive properties. We found that the expression of miR-181a in the MSCs derived from the umbilical cord and decidua of PE patients increased relative to MSCs derived from normal patients. Transfection with miR-181a oligos prevented MSCs proliferation but did not affect MSCs apoptosis. Overexpression of miR-181a blocked activation of the TGF-ß signaling pathway and caused downregulation of target gene (TGFBR1 and TGFBRAP1) mRNA and protein expression. Reporter genes with putative miR-181a binding sites from the TGFBR1 and TGFBRAP1 3'-untranslated regions (3'-UTRs) were downregulated in the presence of miR-181a, suggesting that miR-181a binds to TGFBR1 and TGFBRAP1 3'-UTRs. In contrast, transfection of MSCs with miR-181a oligo enhanced expression of IL-6 and indoleamine 2,3-dioxygenase by activating p38 and JNK signaling pathways, respectively. MSCs transfected with miR-181a also enhanced the proliferation of T cells in a short-term culture. Additionally, treatment with control MSCs, but not miR-181a transfected MSCs, improved dextran sulfate sodium-induced experimental colitis, suggesting that miR-181a attenuates the immunosuppressive properties of MSCs in vivo. Together, our data demonstrate that miR-181a is an important endogenous regulator in the proliferation and immunosuppressive properties of MSCs.

Two-phase analysis of molecular pathways underlying induced pluripotent stem cell induction.

Induced pluripotent stem cells (iPSCs) can be reprogrammed from adult somatic cells by transduction with Oct4, Sox2, Klf4, and c-Myc, but the molecular cascades initiated by these factors remain poorly understood. Impeding their elucidation is the stochastic nature of the iPS induction process, which results in heterogeneous cell populations. Here we have synchronized the reprogramming process by a two-phase induction: an initial stable intermediate phase following transduction with Oct4, Klf4, and c-Myc, and a final iPS phase following overexpression of Sox2. This approach has enabled us to examine temporal gene expression profiles, permitting the identification of Sox2 downstream genes critical for induction. Furthermore, we have validated the feasibility of our new approach by using it to confirm that downregulation of transforming growth factor ß signaling by Sox2 proves essential to the reprogramming process. Thus, we present a novel means for dissecting the details underlying the induction of iPSCs, an approach with significant utility in this arena and the potential for wide-ranging implications in the study of other reprogramming mechanisms.

Anti-epileptic drugs delay age-related loss of spiral ganglion neurons via T-type calcium channel.

Loss of spiral ganglion neurons is a major cause of age-related hearing loss (presbycusis). Despite being the third most prevalent condition afflicting elderly persons, there are no known medications to prevent presbycusis. Because calcium signaling has long been implicated in age-related neuronal death, we investigated T-type calcium channels. This family is comprised of three members (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3), based on their respective main pore-forming alpha subunits: α1G, α1H, and α1I. In the present study, we report a significant delay of age-related loss of cochlear function and preservation of spiral ganglion neurons in α1H null and heterozygous mice, clearly demonstrating an important role for Ca(v)3.2 in age-related neuronal loss. Furthermore, we show that anticonvulsant drugs from a family of T-type calcium channel blockers can significantly preserve spiral ganglion neurons during aging. To our knowledge, this is the first report of drugs capable of diminishing age-related loss of spiral ganglion neurons.