Global Analysis of Protein Expression of Inner Ear Hair Cells.

The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT: Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that hundreds of proteins are uniquely identified or enriched in HCs, extending previous gene expression analyses to reveal novel HC proteins and isoforms. Importantly, deafness-linked proteins were significantly enriched in HCs, suggesting that this in-depth proteomic analysis of IE sensory cells may hold potential for deafness gene discovery.

Homozygosity for frameshift mutations in XYLT2 result in a spondylo-ocular syndrome with bone fragility, cataracts, and hearing defects.

Heparan and chondroitin/dermatan sulfated proteoglycans have a wide range of roles in cellular and tissue homeostasis including growth factor function, morphogen gradient formation, and co-receptor activity. Proteoglycan assembly initiates with a xylose monosaccharide covalently attached by either xylosyltransferase I or II. Three individuals from two families were found that exhibited similar phenotypes. The index case subjects were two brothers, individuals 1 and 2, who presented with osteoporosis, cataracts, sensorineural hearing loss, and mild learning defects. Whole exome sequence analyses showed that both individuals had a homozygous c.692dup mutation (GenBank: NM_022167.3) in the xylosyltransferase II locus (XYLT2) (MIM: 608125), causing reduced XYLT2 mRNA and low circulating xylosyltransferase (XylT) activity. In an unrelated boy (individual 3) from the second family, we noted low serum XylT activity. Sanger sequencing of XYLT2 in this individual revealed a c.520del mutation in exon 2 that resulted in a frameshift and premature stop codon (p.Ala174Profs(∗)35). Fibroblasts from individuals 1 and 2 showed a range of defects including reduced XylT activity, GAG incorporation of (35)SO4, and heparan sulfate proteoglycan assembly. These studies demonstrate that human XylT2 deficiency results in vertebral compression fractures, sensorineural hearing loss, eye defects, and heart defects, a phenotype that is similar to the autosomal-recessive disorder spondylo-ocular syndrome of unknown cause. This phenotype is different from what has been reported in individuals with other linker enzyme deficiencies. These studies illustrate that the cells of the lens, retina, heart muscle, inner ear, and bone are dependent on XylT2 for proteoglycan assembly in humans.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

The relationship between jugular bulb position and jugular bulb related inner ear dehiscence: a retrospective analysis.

OBJECTIVE: High jugular bulb (HJB) can erode inner ear structures creating a jugular bulb related inner ear dehiscence (JBID). The aim of this study was to analyze the relationship between the position of jugular bulb (JB) and JBID using high-resolution computed tomography (HRCT). MATERIAL AND METHODS: In this retrospective study HRCT images of 552 ears of 276 patients with hearing loss, otogenic vertigo, tinnitus or idiopathic peripheral facial nerve paralysis were analyzed. HJB type-1 was defined when JB dome reached above the inferior part of the round window, but was below the inferior edge of the internal acoustic meatus (IAM). HJB type-2 was defined when the dome of JB was higher than the inferior edge of IAM. The frequencies and types of HJB were evaluated. JBID for each HJB type was determined. Frequencies of JBID eroding the vestibular aqueduct (VA), the cochlear aqueduct and the posterior semicircular canal were examined. RESULTS: HJB type-1 and HJB type-2 were found in 19% (105/552) and in 15.8% (87/552) of studied ears. JBID showed to be in 3.8% (21/552) of all ears. 90.5% (19/21) of JBID revealed eroding of VA. Jugular bulb related cochlear aqueduct dehiscence and jugular bulb related posterior semicircular canal dehiscence were found in one ear each. The frequency of jugular bulb related vestibular aqueduct dehiscence (JBVAD) in patients with HJB reaching above IAM was higher than in patients with HJB lower than IAM. CONCLUSIONS: HJB is common, but JBID is rare. JBID prevalently erodes VA. HJB rising above IAM is most at risk to show JBVAD.

Molecular profile of cochlear immunity in the resident cells of the organ of Corti.

BACKGROUND: The cochlea is the sensory organ of hearing. In the cochlea, the organ of Corti houses sensory cells that are susceptible to pathological insults. While the organ of Corti lacks immune cells, it does have the capacity for immune activity. We hypothesized that resident cells in the organ of Corti were responsible for the stress-induced immune response of the organ of Corti. This study profiled the molecular composition of the immune system in the organ of Corti and examined the immune response of non-immune epithelial cells to acoustic overstimulation. METHODS: Using high-throughput RNA-sequencing and qRT-PCR arrays, we identified immune- and inflammation-related genes in both the cochlear sensory epithelium and the organ of Corti. Using bioinformatics analyses, we cataloged the immune genes expressed. We then examined the response of these genes to acoustic overstimulation and determined how changes in immune gene expression were related to sensory cell damage. RESULTS: The RNA-sequencing analysis reveals robust expression of immune-related genes in the cochlear sensory epithelium. The qRT-PCR array analysis confirms that many of these genes are constitutively expressed in the resident cells of the organ of Corti. Bioinformatics analyses reveal that the genes expressed are linked to the Toll-like receptor signaling pathway. We demonstrate that expression of Toll-like receptor signaling genes is predominantly from the supporting cells in the organ of Corti cells. Importantly, our data demonstrate that these Toll-like receptor pathway genes are able to respond to acoustic trauma and that their expression changes are associated with sensory cell damage. CONCLUSION: The cochlear resident cells in the organ of Corti have immune capacity and participate in the cochlear immune response to acoustic overstimulation.

Autologous meatal skin graft implantation and intratympanic injection of Pseudomonas aeruginosa: a new experimental mouse model of acquired middle ear cholesteatoma.

AIM: To create an experimental model for the biomedical research of middle ear cholesteatoma. METHODS: Cholesteatoma was induced in the right ears of mice. An autologous meatal skin graft was implanted into the middle ear via the tympanic membrane followed by an intratympanic injection of Pseudomonas aeruginosa. Six weeks after surgery, the formation of acquired cholesteatoma was evaluated by macroscopic examination, CT scan, and histological analysis. The expressions of TNF-α, IL-1ß, and IL-6 were measured with real-time PCR. Auditory-evoked brain stem response was used for assessing the changes in hearing levels. RESULTS: None of the mice died during the modeling time. By the sixth postoperative week, cholesteatoma had successfully formed in 23 out of 25 mice, which equals a success rate of 92%. A CT scan revealed that the cholesteatoma was eroding the cochlea. Furthermore, histological analysis demonstrated a cystic structure covered by stratified squamous epithelium and keratin desquamation in the lamellae inside the cystic cavity in the bullae. All mice with experimentally induced cholesteatoma showed hearing impairment and an upregulated expression of TNF-α, IL-1ß, and IL-6. CONCLUSION: The present study successfully developed a mouse model of acquired middle ear cholesteatoma, which provides an extremely valuable tool for auditory and biomedical research. The modeling approach is technically easy and has a high success rate.

Retinal and cochlear toxicity of drugs: new insights into mechanisms and detection.

PURPOSE OF REVIEW: Various medications can modify the physiology of retinal and cochlear neurons and lead to major, sometime permanent, sensory loss. A better knowledge of pathogenic mechanisms and the establishment of relevant monitoring protocols are necessary to prevent permanent sensory impairment. In this article, we review main systemic medications associated with direct neuronal toxicity on the retina and cochlea, their putative pathogenic mechanisms, when identified, as well as current recommendations, when available, for monitoring protocols. RECENT FINDINGS: Pathogenic mechanisms and cellular target of retinotoxic drugs are often not well characterized but a better knowledge of the course of visual defect has recently helped in defining more relevant monitoring protocols especially for antimalarials and vigabatrin. Mechanisms of ototoxicity have recently been better defined, from inner ear entry with the use of fluorescent tracers to evidence for the role of oxidative stress and program cell death pathways. SUMMARY: Experimental and clinical studies have elucidated some of the pathogenic mechanisms, courses and risk factors of retinal toxicity and ototoxicity, which have led to establishment of relevant monitoring protocols. Further studies are, however, warranted to better understand cellular pathways leading to degeneration. These would help to build more efficient preventive intervention and may also contribute to understanding of other degenerative processes such as genetic disorders.

Histopathology of the temporal bone in a case of superior canal dehiscence syndrome.

OBJECTIVES: We describe the histopathologic findings in the temporal bones of a patient who had, during life, received a diagnosis of superior canal dehiscence (SCD) syndrome. METHODS: The patient was found to have SCD syndrome at 59 years of age. She became a temporal bone donor, and died of unrelated causes at 62 years of age. Both temporal bones were prepared in celloidin and examined by light microscopy. RESULTS: The patient developed bilateral aural fullness, pulsatile tinnitus, and difficulty tolerating loud noises after minor head trauma at 53 years of age. The symptoms were worse on the right. She also had Valsalva-induced dizziness and eye movements, as well as sound-induced dizziness (more prominent on the right). Audiometry showed a small air-bone gap of 10 dB in the right ear. Vestibular evoked myogenic potential testing showed an abnormally low threshold of 66 dB on the right, and a computed tomography scan showed dehiscence of the superior canal on the right. Histopathologic examination of the right ear showed a 1.4 x 0.6-mm dehiscence of bone covering the superior canal. Dura was in direct contact with the endosteum and the membranous duct at the level of the dehiscence. No osteoclastic process was evident within the otic capsule bone surrounding the dehiscence. The left ear showed thin but intact bone over the superior canal. Both ears showed focal microdehiscences of the tegmen tympani and tegmen mastoideum. The auditory and vestibular sense organs on both sides appeared normal. No endolymphatic hydrops was observed. CONCLUSIONS: The findings were consistent with the hypothesis put forth by Carey and colleagues that SCD may arise from a failure of postnatal bone development, and that minor trauma may disrupt thin bone or stable dura over the superior canal.

Early hearing-impairment results in crossmodal reorganization of ferret core auditory cortex.

Numerous investigations of cortical crossmodal plasticity, most often in congenital or early-deaf subjects, have indicated that secondary auditory cortical areas reorganize to exhibit visual responsiveness while the core auditory regions are largely spared. However, a recent study of adult-deafened ferrets demonstrated that core auditory cortex was reorganized by the somatosensory modality. Because adult animals have matured beyond their critical period of sensory development and plasticity, it was not known if adult-deafening and early-deafening would generate the same crossmodal results. The present study used young, ototoxically-lesioned ferrets (n = 3) that, after maturation (avg. = 173 days old), showed significant hearing deficits (avg. threshold = 72 dB SPL). Recordings from single-units (n = 132) in core auditory cortex showed that 72% were activated by somatosensory stimulation (compared to 1% in hearing controls). In addition, tracer injection into early hearing-impaired core auditory cortex labeled essentially the same auditory cortical and thalamic projection sources as seen for injections in the hearing controls, indicating that the functional reorganization was not the result of new or latent projections to the cortex. These data, along with similar observations from adult-deafened and adult hearing-impaired animals, support the recently proposed brainstem theory for crossmodal plasticity induced by hearing loss.

Evaluation of ear function after cardiopulmonary bypass with otoacoustic emissions: a pilot study.

OBJECTIVES: During extracorporeal circulation (ECC) there is a great hemodynamic stress with possible impact on the microcirculation, including cochlear one. Previous studies have evaluated the effect of ECC on inner ear with contrasting results. The aim of this study is to evaluate possible modifications of the outer hair cells (OHC) function after open heart surgery (OHS) under ECC with transient evoked (TEOAEs) and distortion product otoacoustic emissions (DPOAEs). METHODS: Ten patients (5 F and 5 M), undergoing OHS with ECC, were subjected to an audiological assessment pre- and postoperatively. We compared the pre-operative and post-operative mean auditory thresholds, mean TEOAEs reproducibility and amplitude, and mean DPOAEs amplitude. Student's t-test was used to compare different values. RESULTS: No significant differences were found between pre- and post-operative audiological assessment both in hearing level and in otoacoustic emissions. CONCLUSION: OHC function seems to be not affected by hemodynamic stress induced by ECC. Further studies on a larger scale will be necessary to confirm our preliminary data.

Quantitative definition of neurobehavior, vision, hearing and brain volumes in macaques congenitally exposed to Zika virus.

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design.

Gender differences in audio-vestibular disorders.

In the last years, the attention to the role of gender in physiopathology and pharmacology of diseases in several medical disciplines is rising; however, the data on the relationship between gender and audio-vestibular disorders are still inconclusive and sometimes confusing. With this letter to the editor, we would like to review the role of gender in audio-vestibular disorders. Literature data show that anatomic variances of the inner ear do exist in men and women and that the different physiology and/or hormonal influence between genders could produce different clinical outcome of routine audiological and vestibular tests. Beyond the epidemiological gender-related differences, the clinical data suggest that the gender has a potential role as an etiopathogenetic factor in audio-vestibular disorders and it is probably responsible for the different clinical features observed between male and female subjects.

GJB2 mutation spectrum in 2,063 Chinese patients with nonsyndromic hearing impairment.

BACKGROUND: Mutations in GJB2 are the most common molecular defects responsible for autosomal recessive nonsyndromic hearing impairment (NSHI). The mutation spectra of this gene vary among different ethnic groups. METHODS: In order to understand the spectrum and frequency of GJB2 mutations in the Chinese population, the coding region of the GJB2 gene from 2063 unrelated patients with NSHI was PCR amplified and sequenced. RESULTS: A total of 23 pathogenic mutations were identified. Among them, five (p.W3X, c.99delT, c.155_c.158delTCTG, c.512_c.513insAACG, and p.Y152X) are novel. Three hundred and seven patients carry two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carry only one mutant allele. Thus, GJB2 mutations account for 17.9% of the mutant alleles in 2063 NSHI patients. Overall, 92.6% (684/739) of the pathogenic mutations are frame-shift truncation or nonsense mutations. The four prevalent mutations; c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG, account for 88.0% of all mutantalleles identified. The frequency of GJB2 mutations (alleles) varies from 4% to 30.4% among different regions of China. It also varies among different sub-ethnic groups. CONCLUSION: In some regions of China, testing of the three most common mutations can identify at least one GJB2 mutant allele in all patients. In other regions such as Tibet, the three most common mutations account for only 16% the GJB2 mutant alleles. Thus, in this region, sequencing of GJB2 would be recommended. In addition, the etiology of more than 80% of the mutant alleles for NSHI in China remains to be identified. Analysis of other NSHI related genes will be necessary.

Bcl-2 gene therapy prevents aminoglycoside-induced degeneration of auditory and vestibular hair cells.

To evaluate the protective effects of bcl-2, we have developed an in vivo model of gentamicin ototoxicity in C57BL/6 mice using intratympanic delivery of gentamicin. Hair cell survival was evaluated using myosin VIIa immunohistochemistry, cytocochleogram and auditory brainstem response (ABR) testing. At 10 days after gentamicin application, a consistent loss of outer hair cells was seen. Mice were pretreated with an adenovector expressing human bcl-2 (Ad.11D.bcl-2) or a control vector (Ad.11D). Seventy-two hours after vector delivery mice were treated with intratympanic gentamicin and evaluated at 10 days after ototoxin delivery. Pretreatment with Ad.11D.bcl-2 resulted in morphologic protection of hair cells and preservation of hearing thresholds measured by ABR.

The Influence of Occupational Noise Exposure on Cardiovascular and Hearing Conditions among Industrial Workers.

This study was conducted to estimate the current prevalence of hypertension, cardiovascular condition and hearing difficulty of workers exposure to occupational noise, and to analyze any associations between these abnormal signs and occupational noise exposure. The subjects included 5205 noise-exposed workers. Workers with high noise exposure were more likely to have a higher threshold value than low exposure ones (P < 0.05). Subjects in the high exposure group had a significantly higher risk of hypertension and hearing loss than the ones in low exposure group. Between the ages of 30 and 45, high-level occupational noise exposure led to a significantly raising risk of both hypertension (Adjusted OR = 1.59, 95% CI, 1.19-2.11) and hearing loss (Adjusted OR = 1.28, 95% CI, 1.03-1.60) when comparing to low-level noise exposure. In male workers, the prevalence of hearing difficulty in high exposure group was approximately 1.2 times worse than in low group (P = 0.006). In addition, exposure to high noise level demonstrated a significant association with hypertension and hearing loss when the duration time to occupational noise was longer than 10 years. Hypertension and hearing difficulty is more prevalent in the noise-exposed group (higher than 85 dB[A]). Steps to reduce workplace noise levels and to improve workplace-based health are thus urgently needed.

Prevalence of permanent childhood hearing loss detected at the universal newborn hearing screen: Systematic review and meta-analysis.

CONTEXT: Permanent childhood hearing loss (PCHL) can affect speech, language, and wider outcomes. Adverse effects are mitigated through universal newborn hearing screening (UNHS) and early intervention. OBJECTIVE: We undertook a systematic review and meta-analysis to estimate prevalence of UNHS-detected PCHL (bilateral loss ≥26 dB HL) and its variation by admission to neonatal intensive care unit (NICU). A secondary objective was to report UNHS programme performance (PROSPERO: CRD42016051267). DATA SOURCES: Multiple electronic databases were interrogated in January 2017, with further reports identified from article citations and unpublished literature (November 2017). STUDY SELECTION: UNHS reports from very highly-developed (VHD) countries with relevant prevalence and performance data; no language or date restrictions. DATA EXTRACTION: Three reviewers independently extracted data and assessed quality. RESULTS: We identified 41 eligible reports from 32 study populations (1799863 screened infants) in 6195 non-duplicate references. Pooled UNHS-detected PCHL prevalence was 1.1 per 1000 screened children (95% confidence interval [CI]: 0.9, 1.3; I2 = 89.2%). This was 6.9 times (95% CI: 3.8, 12.5) higher among those admitted to NICU. Smaller studies were significantly associated with higher prevalences (Egger's test: p = 0.02). Sensitivity and specificity ranged from 89-100% and 92-100% respectively, positive predictive values from 2-84%, with all negative predictive values 100%. LIMITATIONS: Results are generalisable to VHD countries only. Estimates and inferences were limited by available data. CONCLUSIONS: In VHD countries, 1 per 1000 screened newborns require referral to clinical services for PCHL. Prevalence is higher in those admitted to NICU. Improved reporting would support further examination of screen performance and child demographics.

Epidermoid Cyst of the Cerebellopontine Angle Presenting with Contralateral Trigeminal Neuralgia: Extremely Rare Case and Review of Literature.

BACKGROUND: An epidermoid cyst is a rare tumor of the cerebellopontine angle region. It usually presents with ipsilateral compressive symptoms. The contralateral trigeminal neuralgia is an unusual presentation in such cases. We did not find such case reports in the literature. CASE DESCRIPTION: Here, we report a case of a 62-year-old female with a right cerebellopontine angle epidermoid cyst presenting with right hearing impairment and the contralateral trigeminal neuralgia. CONCLUSION: The possible mechanism leading to the contralateral trigeminal neuralgia is discussed here along with the diagnosis and management of the case.

The influence of thyroid hormone deficiency on the development of cochlear nonlinearities.

It is well known that failure to treat severe congenital hypothyroidism leads to profound auditory disability, and it has been suggested that an intracochlear defect, or defects, associated with the condition diminishes the efficacy of an active, physiologically vulnerable nonlinear transduction process commonly referred to as cochlear amplification. We address this question directly by tracking the development of threshold-frequency (tuning) curves and two-tone suppression in hypothyroid, Tshr mutant mice born to hypothyroid dams and comparing those findings with findings observed in euthyroid mice. Like sharp tuning, two-tone suppression is a product of transduction nonlinearity and is a useful indicator of the functional status of cochlear amplification. In contrast to euthyroid mice that acquire sharp tuning, normal two-tone suppression, and adultlike sensitivity by the end of the third postnatal week, as shown in earlier studies, hypothyroid mice remained grossly insensitive to sound throughout life. In addition, tuning was generally broad in hypothyroid mice, tuning curve "tips" were frequently missing, and two-tone suppression was rarely observed. However, unlike tip thresholds, tuning curve "tail" thresholds, a feature that reflects the functional status of passive elements of transduction, acquired normal values over a roughly 2-month postnatal time period. These observations collectively suggest that active transduction micromechanics, at least in the frequency region studied here, are profoundly affected by thyroid hormone and support speculation that abnormal outer hair cell function may be the cause of the primary, enduring peripheral auditory defect associated with profound, congenital hypothyroidism in the Tshr mutant mouse.

[Stem-cell-based approaches for treating inner ear diseases]. / Stammzellbasierte Ansätze zur Therapie von Innenohrerkrankungen.

The capacity of stem cells to regenerate lost tissue cells has gained recognition among physicians. Despite the successful use of blood stem cells for treating blood cancers, other stem cell types have not yet been widely introduced into clinical practice. Therapy options involving stem cells for inner ear diseases consequently have not been implemented. Nonetheless, several reports have recently been published describing the generation of morphologically and immunologically distinctive inner ear cell types-such as hair cells, supporting cells, and spiral ganglion neurons-from stem cells. Although promising, all of these studies still lack functional results regarding hearing restoration or vestibular function.

Transcriptomic analysis of chicken cochleae after gentamicin damage and the involvement of four signaling pathways (Notch, FGF, Wnt and BMP) in hair cell regeneration.

Unlike mammalian hair cells, which are essentially unable to regenerate after damage, avian hair cells have a robust capacity for regeneration. The prerequisite for understanding the above difference is knowing the genetic programming of avian hair cell regeneration. Although the major processes have been known, the precise molecular signaling that induces regeneration remains unclear. To address this issue, we performed a high-throughput transcriptomic analysis of gene expression during hair cell regeneration in the chick cochlea after antibiotic injury in vivo. A total of 16,588 genes were found to be expressed in the cochlea, of which about 1000 genes were differentially expressed among the four groups studied, i.e., 2 days (d) or 3 d post-treatment with gentamicin or physiological saline. The differentially expressed genes were distributed across approximately one hundred signaling pathways, including the Notch, MAPK (FGF), Wnt and TGF-ß (BMP) pathways that have been shown to play important roles in embryonic development. Some differentially expressed genes (2-3 in each pathway) were further verified by qRT-PCR. After blocking Notch, FGF or BMP signaling, the number of regenerating hair cells and mitotic supporting cells increased. However, the opposite effect was observed after suppressing the Wnt pathway or enhancing BMP signaling. To our knowledge, the present study provided a relatively complete dataset of candidate genes and signaling pathways most likely involved in hair cell regeneration and should be a useful start in deciphering the genetic circuitry for inducing hair cell regeneration in the chick cochlea.