Hear and Now: Ongoing Clinical Trials to Prevent Drug-Induced Hearing Loss.

Each year over half a million people experience permanent hearing loss caused by treatment with therapeutic drugs with ototoxic side effects. There is a major unmet clinical need for therapies that protect against this hearing loss without reducing the therapeutic efficacy of these lifesaving drugs. At least 17 clinical trials evaluating 10 therapeutics are currently underway for therapies aimed at preventing aminoglycoside- and/or cisplatin-induced ototoxicity. This review describes the preclinical and clinical development of each of these approaches, provides updates on the status of ongoing trials, and highlights the importance of appropriate outcome measures in trial design and the value of reporting criteria in the dissemination of results.

Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

Hearing Loss after Round Window Surgery in Mice Is due to Middle Ear Effusion.

BACKGROUND: Delivery of therapeutic agents directly through the round window (RW) offers promise for treating sensorineural hearing loss. However, hearing loss can result from the surgical approach itself, and the reasons for this are poorly understood. We examined the hearing loss following the 3 major steps involved with the RW approach to access the mouse cochlea: bullostomy, RW puncture, and RW injection. METHODS: Twenty-one adult CBA/J mice underwent bullostomy alone, 10 underwent RW puncture, and 8 underwent RW injection with PBS with 5% glycerol. Auditory brainstem responses (ABR) and otoscopy were performed preoperatively and up to 6 weeks postoperatively. Hair cells were stained, and survival was assessed using immunofluorescence. RESULTS: One week postoperatively, mice in all groups showed significant threshold shifts. Otoscopy revealed approximately half of all mice had middle ear effusion (MEE), with a higher incidence of effusion in the RW puncture and RW injection groups. Those with MEE had significant ABR threshold shifts, whereas those without MEE had minimal hearing loss. MEE persisted through 6 weeks in a majority of cases, but in those mice with MEE resolution, there was at least partial improvement in hearing. Immunohistochemistry showed minimal loss of hair cells in all animals. CONCLUSION: MEE is highly correlated with hearing loss in mice undergoing RW surgery. Otoscopy is an important adjunct to consider after ear surgery in mice, as MEE may contribute to postsurgical hearing loss.

Gene therapy for sensorineural hearing loss.

Gene therapy is a promising treatment modality that is being explored for several inherited disorders. Multiple human gene therapy clinical trials are currently ongoing, but few are directed at hearing loss. Hearing loss is one of the most prevalent sensory disabilities in the world, and genetics play an important role in the pathophysiology of hearing loss. Gene therapy offers the possibility of restoring hearing by overcoming the functional deficits created by the underlying genetic mutations. In addition, gene therapy could potentially be used to induce hair cell regeneration by delivering genes that are critical to hair cell differentiation into the cochlea. In this review, we examine the promises and challenges of applying gene therapy to the cochlea. We also summarize recent studies that have applied gene therapy to animal models of hearing loss.

Early development of the glucocorticoid stress response in poison frog tadpoles.

In vertebrates, the glucocorticoid response through the hypothalamic-pituitary-adrenal (HPA) axis controls many essential functions, including behavior, metabolism, and ontogenetic transitions. However, there are tradeoffs associated with high levels of glucocorticoids, including reduced growth rate and lowered immunity. These tradeoffs drive variation in the timing of the development of the HPA axis across taxa. In anurans (frogs and toads), corticosterone has critical roles in development and behavior, and concentrations can fluctuate in response to environmental stressors. Given the role of corticosterone in ontogenetic changes and behaviors, we hypothesized that species with immediate habitat transitions and challenges would develop an HPA axis early in development. To test this hypothesis, we studied tadpoles of the dyeing poison frog ( Dendrobates tinctorius ), a species in which tadpoles hatch terrestrially and are transported to pools of water by their parent. We measured the excretion rate and whole-body concentration of corticosterone and the corticosterone response to adrenocorticotropic hormone (ACTH). We found no significant differences in excretion rates and whole-body concentration of corticosterone, nor physiological response to ACTH injection across tadpole development. These findings indicate that the glucocorticoid response is developed early in ontogeny. These findings generally differ from those found in other species of tadpoles, which may suggest the unique ecological pressures of D. tinctorius has shaped the development of its HPA axis. More broadly, this study illustrates how life history strategies and tradeoffs of glucocorticoids impact the timing of the development of the HPA axis. Highlights: The timing of HPA axis development differs across species. We studied the HPA axis across tadpole development in Dendrobates tinctorius . No difference in corticosterone concentration across development.No difference in corticosterone response to ACTH across development.Results suggest an early developed HPA axis is essential for their life history.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Macrophage Depletion Protects Against Cisplatin-Induced Ototoxicity and Nephrotoxicity.

Cisplatin is a widely used and highly effective anti-cancer drug with significant side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced ototoxicity and nephrotoxicity, we used PLX3397, an FDA-approved inhibitor of the colony-stimulating factor 1 receptor (CSF1R), to eliminate tissue-resident macrophages during the course of cisplatin administration. Mice treated with cisplatin alone (cisplatin/vehicle) had significant hearing loss (ototoxicity) as well as kidney injury (nephrotoxicity). Macrophage ablation using PLX3397 resulted in significantly reduced hearing loss measured by auditory brainstem responses (ABR) and distortion-product otoacoustic emissions (DPOAE). Sensory hair cells in the cochlea were protected against cisplatin-induced death in mice treated with PLX3397. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis as well as reduced plasma blood urea nitrogen (BUN) and neutrophil gelatinase-associated lipocalin (NGAL) levels. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together our data indicate that ablation of tissue-resident macrophages represents a novel strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Semi-automated quantification of hair cells in the mature mouse utricle.

The mouse utricle model system is the best-characterized ex vivo preparation for studies of mature mammalian hair cells (HCs). Despite the many advantages of this model system, efficient and reliable quantification of HCs from cultured utricles has been a persistent challenge with this model system. Utricular HCs are commonly quantified by counting immunolabeled HCs in regions of interest (ROIs) placed over an image of the utricle. Our data indicate that the accuracy of HC counts obtained using this method can be impacted by variability in HC density across different regions of the utricle. In addition, the commonly used HC marker myosin 7a results in a diffuse cytoplasmic stain that is not conducive to automated quantification and must be quantified manually, a labor-intensive task. Furthermore, myosin 7a immunoreactivity is retained in dead HCs, resulting in inaccurate quantification of live HCs using this marker. Here we have developed a method for semi-automated quantification of surviving HCs that combines immunoreactivity for the HC-specific transcription factor Pou4f3 with labeling of activated caspase 3/7 (AC3/7) to detect apoptotic HCs. The discrete nuclear Pou4f3 signal allowed us to utilize the binary or threshold function within ImageJ to automate HC quantification. To further streamline this process, we created an ImageJ macro that automates the process from raw image loading to a final quantified image that can be immediately evaluated for accuracy. Within this quantified image, the user can manually correct the quantification via an image overlay indicating the counted HC nuclei. Pou4f3-positive HCs that also express AC3/7 are subtracted to yield accurate counts of surviving HCs. Overall, we present a semi-automated method that is faster than manual HC quantification and identifies surviving HCs with high accuracy.

Single-Cell RNA-Seq of Cisplatin-Treated Adult Stria Vascularis Identifies Cell Type-Specific Regulatory Networks and Novel Therapeutic Gene Targets.

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

Atorvastatin is associated with reduced cisplatin-induced hearing loss.

BACKGROUNDCisplatin is widely used to treat adult and pediatric cancers. It is the most ototoxic drug in clinical use, resulting in permanent hearing loss in approximately 50% of treated patients. There is a major need for therapies that prevent cisplatin-induced hearing loss. Studies in mice suggest that concurrent use of statins reduces cisplatin-induced hearing loss.METHODSWe examined hearing thresholds from 277 adults treated with cisplatin for head and neck cancer. Pretreatment and posttreatment audiograms were collected within 90 days of initiation and completion of cisplatin therapy. The primary outcome measure was a change in hearing as defined by the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE).RESULTSAmong patients on concurrent atorvastatin, 9.7% experienced a CTCAE grade 2 or higher cisplatin-induced hearing loss compared with 29.4% in nonstatin users (P < 0.0001). A mixed-effect model analysis showed that atorvastatin use was significantly associated with reduced cisplatin-induced hearing loss (P ≤ 0.01). An adjusted odds ratio (OR) analysis indicated that an atorvastatin user is 53% less likely to acquire a cisplatin-induced hearing loss than a nonstatin user (OR = 0.47; 95% CI, 0.30-0.78). Three-year survival rates were not different between atorvastatin users and nonstatin users (P > 0.05).CONCLUSIONSOur data indicate that atorvastatin use is associated with reduced incidence and severity of cisplatin-induced hearing loss in adults being treated for head and neck cancer.TRIAL REGISTRATIONClinicalTrials.gov identifier NCT03225157.FUNDINGFunding was provided by the Division of Intramural Research at the National Institute on Deafness and Other Communication Disorders (1 ZIA DC000079, ZIA DC000090).

Ototoxicity and Platinum Uptake Following Cyclic Administration of Platinum-Based Chemotherapeutic Agents.

Cisplatin is a widely used anti-cancer drug used to treat a variety of cancer types. One of the side effects of this life-saving drug is irreversible ototoxicity, resulting in permanent hearing loss in many patients. In order to understand why cisplatin is particularly toxic to the inner ear, we compared the hearing loss and cochlear uptake of cisplatin to that of two related drugs, carboplatin and oxaliplatin. These three drugs are similar in that each contains a core platinum atom; however, carboplatin and oxaliplatin are considered less ototoxic than cisplatin. We delivered these three drugs to mice using a 6-week cyclic drug administration protocol. We performed the experiment twice, once using equimolar concentrations of the drugs and once using concentrations of the drugs more proportional to those used in the clinic. For both concentrations, we detected a significant hearing loss caused by cisplatin and no hearing loss caused by carboplatin or oxaliplatin. Cochlear uptake of each drug was measured using inductively coupled plasma mass spectrometry (ICP-MS) to detect platinum. Cochlear platinum levels were highest in mice treated with cisplatin followed by oxaliplatin, while carboplatin was largely excluded from the cochlea. Even when the drug doses were increased, cochlear platinum remained low in mice treated with oxaliplatin or carboplatin. We also examined drug clearance from the inner ear by measuring platinum levels at 1 h and 24 h after drug administration. Our findings suggest that the reduced cochlear platinum we observed with oxaliplatin and carboplatin were not due to increased clearance of these drugs relative to cisplatin. Taken together, our data indicate that the differential ototoxicity among cisplatin, carboplatin, and oxaliplatin is attributable to differences in cochlear uptake of these three drugs.

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. These auditory brainstem caspase-3 substrates were enriched for proteins previously shown to be cleaved by caspase-3, especially in non-apoptotic contexts. Functional annotation analysis revealed that our caspase-3 substrates were also enriched for proteins associated with several protein categories, including proteins found in extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem was highly enriched for our caspase-3 substrates. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of extracellular vesicle (EV) proteins.

Lovastatin protects against cisplatin-induced hearing loss in mice.

Cisplatin is used to treat a variety of solid tumors in both children and adults. However, cisplatin has serious side-effects, some of which may permanently affect patients' quality of life following treatment, such as ototoxicity. There is currently no FDA-approved therapy for the prevention or treatment of cisplatin-induced hearing loss. Herein we examine the potential for statins to prevent cisplatin-induced ototoxicity. Statins, a class of drugs commonly used to prevent or manage hypercholesterolemia, have been of clinical utility for decades with dependable outcomes and reliable safety profiles in humans. Statins are known to be protective in animal models of noise-induced and age-related hearing loss. Moreover, studies have demonstrated an additive benefit of statins in cancer treatment. In the current study, lovastatin reduces cisplatin-induced hearing loss in adult mice. Lovastatin-mediated protection was significantly greater among female than male mice, and the dose of lovastatin required for protection was different between the sexes. Taken together our data indicate that lovastatin reduces cisplatin-induced hearing loss in mice and suggest that concurrent statin and cisplatin therapy may represent a feasible clinical strategy for reducing cisplatin-induced ototoxicity that should be explored for future clinical use.

Cell-Specific Transcriptional Responses to Heat Shock in the Mouse Utricle Epithelium.

Sensory epithelia of the inner ear contain mechanosensory hair cells (HCs) and glia-like supporting cells (SCs), both of which are required for hearing and balance functions. Each of these cell types has unique responses to ototoxic and cytoprotective stimuli. Non-lethal heat stress in the mammalian utricle induces heat shock proteins (HSPs) and protects against ototoxic drug-induced hair cell death. Induction of HSPs in the utricle demonstrates cell-type specificity at the protein level, with HSP70 induction occurring primarily in SCs, while HSP32 (also known as heme oxygenase 1, HMOX1) is induced primarily in resident macrophages. Neither of these HSPs are robustly induced in HCs, suggesting that HCs may have little capacity for induction of stress-induced protective responses. To determine the transcriptional responses to heat shock of these different cell types, we performed cell-type-specific transcriptional profiling using the RiboTag method, which allows for immunoprecipitation (IP) of actively translating mRNAs from specific cell types. RNA-Seq differential gene expression analyses demonstrated that the RiboTag method identified known cell type-specific markers as well as new markers for HCs and SCs. Gene expression differences suggest that HCs and SCs exhibit differential transcriptional heat shock responses. The chaperonin family member Cct8 was significantly enriched only in heat-shocked HCs, while Hspa1l (HSP70 family), and Hspb1 and Cryab (HSP27 and HSP20 families, respectively) were enriched only in SCs. Together our data indicate that HCs exhibit a limited but unique heat shock response, and SCs exhibit a broader and more robust transcriptional response to protective heat stress.

Exosomes mediate sensory hair cell protection in the inner ear.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.