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Age-related hearing loss (ARHL) is associated with hair cell apoptosis, but the underlying mechanism of hair cell apoptosis remains unclear. Here, we investigated the expression profiles of long noncoding RNAs (lncRNAs) and mRNAs in an ARHL model created with C57BL/6 J mice using RNA sequencing and found that the expression of several lncRNAs was significantly correlated with apoptosis-associated mRNAs in the cochlear tissues of old mice compared to young mice. We found that lncRNA Mirg was upregulated in the cochlear tissues of old mice compared to young mice and its overexpression promoted apoptosis in House Ear Institute-Organ of Corti 1 (HEI-OC1). H 2 O 2 -induced oxidative stress increased HEI-OC1 cell apoptosis by upregulating lncRNA Mirg. Furthermore, the expression of lncRNA Mirg and Foxp1 showed the highest correlation coefficient in the cochlear tissues of old mice, and lncRNA Mirg promoted HEI-OC1 cell apoptosis by increasing Foxp1 expression. In conclusion, our findings suggest that lncRNA Mirg expression correlates with cell apoptosis-associated mRNAs in the ARHL model created using C57BL/6 J mice and that oxidative stress-induced lncRNA Mirg promotes HEI-OC1 cell apoptosis by increasing Foxp1 expression. These data suggest the potential therapeutic significance of targeting lncRNA Mirg/Foxp1 signaling in ARHL., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ca 2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca 2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca 2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca 2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca 2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca 2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca 2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca 2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca 2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca 2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca 2+ homeostasis in these cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Advances in microelectromechanical systems (MEMS) technologies are enhancing the development of intracochlear delivery devices for the treatment of hearing loss with emerging pharmacological therapies. Direct intracochlear delivery addresses the limitations of systemic and intratympanic delivery. However, optimization of delivery parameters for these devices requires pharmacokinetic assessment of the spatiotemporal drug distribution inside the cochlea. Robust methods of measuring drug concentration in the perilymph have been developed, but lack spatial resolution along the tonotopic axis or require complex physiological measurements. Here we describe an approach for quantifying distribution of fluorescent drug-surrogate probe along the cochlea's sensory epithelium with high spatial resolution enabled by confocal fluorescence imaging. Fluorescence from FM 1-43 FX, a fixable endocytosis marker, was quantified using confocal fluorescence imaging of whole mount sections of the organ of Corti from cochleae resected and fixed at several time points after intracochlear delivery. Intracochlear delivery of FM 1-43 FX near the base of the cochlea produces a base-apex gradient of fluorescence in the row of inner hair cells after 1 h post-delivery that is consistent with diffusion-limited transport along the scala tympani. By 3 h post-delivery there is approximately an order of magnitude decrease in peak average fluorescence intensity, suggesting FM 1-43 FX clearance from both the perilymph and inner hair cells. The increase in fluorescence intensity at 72 h post-delivery compared to 3 h post-delivery may implicate a potential radial transport pathway into the scala media., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Autophagy is a highly evolutionary conserved quality control defense mechanism within cells, which has also been implicated in cell death processes. In the mammalian inner ear, autophagy has been shown to play a role during early morphogenesis as well as in adult cochlear hair cells exposed to ototoxic insults. Mitophagy, a selective autophagic cell process targeting mitochondria, hasn't been studied in the inner ear so far. On this work, we searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC). We first tested for the expression of Pink1/Park2 mRNA in 5-day-old C57BL/6 mice's cochleae using RT-PCR. We focused on the induction of mitophagy in HEI-OC1 cells as well as in the OC and investigated a possible mitophagic potential of the aminoglycoside agent gentamicin. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after a 6-h incubation with a well-described mitochondrial uncoupler and mitophagy-inducing agent: carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Incubation with gentamicin generated no mitochondrial translocation of LC3. Protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after a 24-h CCCP treatment as well as gentamicin. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV. A downregulation of COXIV could also be visualized in the OC after CCCP. A significant oxygen consumption rate (OCR) changed in cells treated with CCCP as well as significant morphological changes of mitochondria by electron microscopy (EM) strengthen this assumption. Gentamicin exposure generated no impact on OCR or mitochondrial morphological changes by EM. Finally, we demonstrated changes in the expression of Atg12 and LC3 proteins in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data indicate that gentamicin had no impact in the activation of mitophagy-neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sound waves are converted into electrical signals by a process of mechano-electrical transduction (MET), which takes place in the hair bundle of cochlear hair cells. In response to the mechanical stimulus of the hair bundle, the tip-links, key components of the MET machinery, are tensioned and the MET channels open, which results in the generation of the cell receptor potential. Tip-links are composed of cadherin-23 (Cdh23) and protocadherin-15 (Pcdh15), both non-conventional cadherins, that form the upper and the lower part of these links, respectively. Here, we review the various Pcdh15 isoforms present in the organ of Corti, their localization in the auditory hair bundles, their involvement in the molecular complex forming the tip-link, and their interactions with transmembrane molecules that are components of the lower MET machinery., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Individual variation in the susceptibility of the auditory system to acoustic overstimulation has been well-documented at both the functional and structural levels. However, the molecular mechanism responsible for this variation is unclear. The current investigation was designed to examine the variation patterns of cochlear gene expression using RNA-seq data and to identify the genes with expression variation that increased following acoustic trauma. This study revealed that the constitutive expressions of cochlear genes displayed diverse levels of gene-specific variation. These variation patterns were altered by acoustic trauma; approximately one-third of the examined genes displayed marked increases in their expression variation. Bioinformatics analyses revealed that the genes that exhibited increased variation were functionally related to cell death, biomolecule metabolism, and membrane function. In contrast, the stable genes were primarily related to basic cellular processes, including protein and macromolecular syntheses and transport. There was no functional overlap between the stable and variable genes. Importantly, we demonstrated that glutamate metabolism is related to the variation in the functional response of the cochlea to acoustic overstimulation. Taken together, the results indicate that our analyses of the individual variations in transcriptome changes of cochlear genes provide important information for the identification of genes that potentially contribute to the generation of individual variation in cochlear responses to acoustic overstimulation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study was to examine the activities of hASCs (Human Adipose tissue Derived Stem Cells) on experimental autoimmune hearing loss (EAHL) and how human stem cells regenerated mouse cochlea cells. We have restored hearing in 19 years old white female with autoimmune hearing loss with autologous adipose tissue derived stem cells and we wish to understand the mechanism of restoration of hearing in animal model. BALB/c mice underwent to develop EAHL; mice with EAHL were given hASCs intraperitoneally once a week for 6 consecutive weeks. ABR were examined over time. The helper type 1 autoreactive responses and T-reg cells were examined. H&E staining or immunostaining with APC conjugated anti-HLA-ABC antibody were conducted. The organ of Corti, stria vascularis, spira ligament and spiral ganglion in stem cell group are normal. In control group, without receiving stem cells, the organ of Corti is replaced by a single layer of cells, atrophy of stria vascularis. Systemic infusion of hASCs significantly improved hearing function and protected hair cells in established EAHL. The hASCs decreased the proliferation of antigen specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine interleukin10 in splenocytes. They also induced the generation of antigen specific CD4(+)CD25(+)Foxp3(+)T-reg cells. The experiment showed the restoration is due to the paracrine activities of human stem cells, since there are newly regenerated mice spiral ganglion cells, not human mesenchymal stem cells derived tissue given by intraperitoneally., (Copyright © 2015. Published by Elsevier B.V.)

Mutations in the connexin 26 gene (GJB2) are the most common genetic cause of deafness, leading to congenital bilateral non-syndromic sensorineural hearing loss. Here we report the generation of a mouse model for a connexin 26 (Cx26) mutation, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the auditory epithelium. We determined that these conditional knockout mice, designated Gjb2-CKO, have a severe hearing loss. Immunocytochemistry of the auditory epithelium confirmed absence of Cx26 in the non-sensory cells. Histology of the organ of Corti and the spiral ganglion neurons (SGNs) performed at ages 1, 3, or 6 months revealed that in Gjb2-CKO mice, the organ of Corti began to degenerate in the basal cochlear turn at an early stage, and the degeneration rapidly spread to the apex. In addition, the density of SGNs in Rosenthal's canal decreased rapidly along a gradient from the base of the cochlea to the apex, where some SGNs survived until at least 6 months of age. Surviving neurons often clustered together and formed clumps of cells in the canal. We then assessed the influence of brain derived neurotrophic factor (BDNF) gene therapy on the SGNs of Gjb2-CKO mice by inoculating Adenovirus with the BDNF gene insert (Ad.BDNF) into the base of the cochlea via the scala tympani or scala media. We determined that over-expression of BDNF beginning around 1 month of age resulted in a significant rescue of neurons in Rosenthal's canal of the cochlear basal turn but not in the middle or apical portions. This data may be used to design therapies for enhancing the SGN physiological status in all GJB2 patients and especially in a sub-group of GJB2 patients where the hearing loss progresses due to ongoing degeneration of the auditory nerve, thereby improving the outcome of cochlear implant therapy in these ears., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Mammalian cochlear supporting cells remain quiescent at postnatal ages and age-dependent changes in supporting cell proliferative capacity are evident. Ectopic Atoh1 expression in neonatal supporting cells converts only a small percentage of these cells into hair cell-like cells. Despite tremendous potential for therapeutics, cellular reprogramming in the mammalian inner ear remains a slow inefficient process that requires weeks, with most cells failing to reprogram. Cellular reprogramming studies in other tissues have shown that epigenetic inhibitors can significantly improve reprogramming efficiency. Very little is known about epigenetic regulation in the mammalian inner ear, and almost nothing is known about the histone modifications. Histone modifications are vital for proper transcriptional regulation, and aberrant histone modifications can cause defects in the regulation of genes required for normal tissue development and maintenance. Our data indicate that cofactors of repressive complexes such as NuRD and PRC2 are present in the neonatal organ of Corti. These NuRD cofactors are present throughout most of the organ of Corti from E18.5 until P4. By P6, these NuRD cofactors are mostly undetectable by immunofluorescence and completely lost by P7, but are detectable again at P8 and continue to be present through P21. The PRC2 enzymatic subunit, EZH2 is also highly present from E18.5 to P0 in the organ of Corti, but lost between P2 and P4. However, EZH2 staining is evident again throughout the organ of Corti by P6 and persists through P21. Our data provide evidence that HDACs, DNA methyltransferases, histone methyltransferases, and histone demethylases are expressed postnatally within the organ of Corti, and may be targets for drug inhibition to increase the capacity, speed, and efficiency of reprogramming a supporting cell into a hair cell., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study tests the hypothesis that pegylated nanoparticles (NPs) could be taken up by the cochlear cells [House Ear Institute-organ of Corti 1 (HEI-OC1) and Stria vascularis K-1 (SVK-1)], through endocytic pathways. Furthermore, the in vitro drug release and the cytotoxicity of Furosemide (FUR)-loaded NPs on these two cochlear cells are investigated. FUR-loaded pegylated NPs are prepared by the emulsion-solvent diffusion method without surfactant. The NPs are characterized for particle mean diameter, polydispersity index (PDI), morphology, percent drug encapsulation efficiency (EE%), and FUR release kinetics. The methyl tetrazolium salt (MTS) and lactate dehydrogenase (LDH) bioassays are used to evaluate in vitro, the cytotoxicity of FUR-loaded NPs and native FUR. The NPs uptake is investigated using confocal microscopy, microplate reader/fluorimetry, and flow cytometry. Spherical NPs with a mean diameter range of 133-210 nm and PDI values varying from 0.037 to 0.41 are produced. The FUR EE% is 86% and the drug is released from the NPs according to the zero-order and Higuchi models. After treatment with blank NPs, the percentage of cell viability and cell death are 95.96% and 8.95%, in HEI-OC1 cells, respectively. The NPs are internalized by HEI-OC1 cells through a clathrin-dependent pathway. In addition, results show that NPs can be taken up via clathrin and cytoskeleton mediated pathways in SVK-1 cells. The internalization of the pegylated NPs can enhance the drug toxicity by necrosis in a dose-dependent and sustained release manner. The formulated NPs provide a promising template for a targeted drug delivery system to the inner ear., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial for inner ear neurosensory development. The proneural TF Atoh1 regulates the differentiation of hair cells (HCs) whereas Neurog1 and Neurod1 regulate specification and differentiation of neurons, respectively, but also affect HC development. Expression of Delta and Jagged ligands in nascent HCs and Notch receptors in supporting cells induce supporting cell differentiation through the regulation of neurogenic bHLH TFs (such as Hes1, Hes5) and suppression of limited Atoh1 expression. In sensorineural hearing loss, HCs are lost followed by supporting cells and progressive degeneration of neurons, at least in rodents. Regaining complete hearing may require reconstituting the organ of Corti from scratch, including the two types of HCs, inner and outer hair cells with the precise sorting of two types of afferent (type I and II) and efferent (lateral and medial olivo-cochlear) innervation. We review effects of bHLH TF dosage and their cross-regulation to differentiate HC types in the organ of Corti. We categorize findings of specific gene expressions in HCs: 1. as markers without meaning for the regeneration task, 2. as stabilizers who are needed to maintain or complete differentiation, and 3. as decision-making genes, expressed and acting early enough to be useful in this process. Only one TF has been characterized that fits the last aspect: Atoh1. We propose that temporal and intensity variations of Atoh1 are naturally modulated to differentiate specific types of HCs. Importantly, the molecular means to modify the Atoh1 expression are at least partially understood and can be readily implemented in the attempts to regenerate specific types of HCs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The avian basilar papilla (BP) is a likely homolog of the auditory sensory epithelium of the mammalian cochlea, the organ of Corti. During mammalian development Fibroblast growth factor receptor-3 (Fgfr3) is known to regulate the differentiation of auditory mechanosensory hair cells (HCs) and supporting cells (SCs), both of which are required for sound detection. Fgfr3 is expressed in developing progenitor cells (PCs) and SCs of both the BP and the organ of Corti; however its role in BP development is unknown. Here we utilized an in vitro whole organ embryonic culture system to examine the role of Fgf signaling in the developing avian cochlea. SU5402 (an antagonist of Fgf signaling) was applied to developing BP cultures at different stages to assay the role of Fgf signaling during HC formation. Similar to the observed effects of inhibition of Fgfr3 in the mammalian cochlea, Fgfr inhibition in the developing BP increased the number of HCs that formed. This increase was not associated with increased proliferation, suggesting that inhibition of the Fgf pathway leads to the direct conversion of PCs or supporting cells into HCs, a process known as transdifferentiation. This also implies that Fgf signaling is required to prevent the conversion of PCs and SCs into HCs. The ability of Fgf signaling to inhibit transdifferentiation suggests that its down-regulation may be essential for the initial steps of HC formation, as well as for the maintenance of SC phenotypes., (Copyright © 2012. Published by Elsevier B.V.)

Besides their prominent function in cellular energy metabolism, the central role of mitochondria has been focused on control of cellular death in last decades. The mitochondrial permeability transition pore (PTP) is involved in the intrinsic pathway of apoptosis via the release of cytochrome c into cytosol. Metformin, a drug widely used in the treatment of type II diabetes, has recently received attention owing to new findings regarding its effect on apoptosis through mitochondrial permeability transition and cytochrome c release. The modulation of PTP is still unknown, but calcium is certainly the most important known inducer. In the present study, the preventive effects of metformin on gentamicin ototoxicity were investigated through the changes of intracellular calcium concentrations using calcium imaging in HEI-OC1 cells. Calcium imaging traced the changes of intracellular calcium concentration after the application of 50 mM of gentamicin in both 100 uM of metformin pretreated group and non-pretreated group. These calcium reactions were compared and analyzed with the results of cell viability test, Hoechst staining, intracellular reactive oxygen species level and expression of caspase-3, and poly-ADP-ribose polymerase (PARP). Continuous increase of intracellular calcium concentration (increase of 380/340 ratio) occurred after application of 50 mM of gentamicin. However, there was no change of intracellular calcium concentration in 100 uM metformin pretreated group. Cell viability was significantly higher in 100 uM metformin pretreated group and also, metformin pretreated HEI-OC1 cells produced less ROS that gentamicin alone treated group. Gentamicin increased cleaved PARP and caspase-3, but metformin inhibited the expression of caspase-3 and cleavage of PARP. This study demonstrated that metformin prevented gentamicin induced apoptosis through the calcium modulating and ROS reducing anti-apoptotic effects., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This review summarizes recent progress in our understanding of the molecular basis of cochlear duct growth, specification of the organ of Corti, and differentiation of the different types of hair cells. Studies of multiple mutations suggest that developing hair cells are involved in stretching the organ of Corti through convergent extension movements. However, Atoh1 null mutants have only undifferentiated and dying organ of Corti precursors but show a near normal extension of the cochlear duct, implying that organ of Corti precursor cells can equally drive this process. Some factors influence cochlear duct growth by regulating the cell cycle and proliferation. Shortened cell cycle and premature cell cycle exit can lead to a shorter organ of Corti with multiple rows of hair cells (e.g., Foxg1 null mice). Other genes affect the initial formation of a cochlear duct with or without affecting the organ of Corti. Such observations are consistent with evolutionary data that suggest some developmental uncoupling of cochlear duct from organ of Corti formation. Positioning the organ of Corti requires multiple genes expressed in the organ of Corti and the flanking region. Several candidate factors have emerged but how they cooperate to specify the organ of Corti and the topology of hair cells remains unclear. Atoh1 is required for differentiation of all hair cells, but regulation of inner versus outer hair cell differentiation is still unidentified. In summary, the emerging molecular complexity of organ of Corti development demands further study before a rational approach towards regeneration of unique types of hair cells in specific positions is possible., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Based on observations that mutations of GATA-3 are responsible for the HDR-syndrome (hypoparathyroidism, deafness, renal defects) and that GATA-transcription factors have an important role to play in inner ear development, we hypothesized that these transcription factors may be involved in regulatory changes of prestin transcription. To prove this, we examined in parallel the expression of mRNA of prestin and Gata-3,-2 and Gata-1 in the organ of Corti during early postnatal development of rats and in organotypic cultures. Remarkable relations are observed between prestin and Gata-3,-2 expression in organ of Corti preparations in vivo and in vitro: (i) Gata-3,-2 expression display similar apical-basal gradients as prestin mRNA levels. (ii) The prestin expression increases between postnatal day two and postnatal day eight by a factor of about four in the apical and middle segments and by a factor of two in the basal part. Highly significant Pearson correlation coefficients were observed between Gata-3,-2 mRNA and prestin levels when the data were evaluated by regression analyses. (iii) Parallel changes of prestin mRNA and Gata-3,-2 mRNA levels were observed in response to thyroid hormone and to gemfibrozil application. These observations suggest a regulatory role played by the Gata-3,-2 transcription factors in prestin expression., (2009 Elsevier B.V. All rights reserved.)

Sex differences in song behavior and in the neural system controlling song in songbirds are well documented but relatively little is known about sex differences in hearing. We recently demonstrated the existence of sex differences in auditory brainstem responses in a songbird species, the zebra finch (Taeniopygia guttata). Many sex differences are regulated by sex steroid hormone action either during ontogeny or in adulthood. As a first step to test the possible implication of sex steroids in the control of sex differences in the zebra finch auditory system, we evaluated via immunocytochemistry whether estrogens are produced and act in the zebra finch inner ear. Specifically we examined the distribution of aromatase, the enzyme converting testosterone into an estrogen, and of estrogen receptors of the alpha subtype (ERalpha) in adult zebra finch inner ears. The anatomy of the basilar papillae was visualized by fluorescein-phalloidin, which delineated the actin structure of hair cells and supporting cells at their apical surface. Whole mount preparations of basilar papillae stained by immunocytochemistry revealed in both males and females an abundant aromatase distribution in the cytoplasm of hair cells, while ERalpha was identified in the nuclei of hair cells and of underlying supporting cells. Double-labeled preparations confirmed the extensive co-localization of aromatase and ERalpha in the vast majority of the hair cells. These results are consistent with studies on non-avian species, suggesting a role for estrogens in auditory function. These findings are also consistent with the notion that estrogens may contribute to a sex difference in hearing. To our knowledge, this is the first demonstration of the presence of aromatase and of the co-localization of aromatase and ERalpha in the sensory epithelium of the inner ear in any animal model.

Ebselen, an organoselenium compound that acts as a glutathione peroxidase mimetic, has been demonstrated to possess antioxidant and anti-inflammatory activities. However, the molecular mechanism underlying this effect is not fully understood in auditory cells. The purpose of the present study is to investigate the protective effect of ebselen against cisplatin-induced toxicity in HEI-OC1 auditory cells, organotypic cultures of cochlear explants from two-day postnatal rats (P(2)) and adult Balb/C mice. Pretreatment with ebselen ameliorated apoptotic death induced by cisplatin in HEI-OC1 cells and organotypic cultures of Corti's organ. Ebselen pretreatment also significantly suppressed cisplatin-induced increases in intracellular reactive oxygen species (ROS), intracellular reactive nitrogen species (RNS) and lipid peroxidation levels. Ebselen dose-dependently increased the expression level of an antioxidant response element (ARE)-luciferase reporter in HEI-OC1 cells through the translocation of Nrf2 into the nucleus. Furthermore, we found that pretreatment with ebselen significantly restored Nrf2 function, whereas it ameliorated the cytotoxicity of cisplatin in cells transfectants with either a pcDNA3.1 (control) or a DN-Nrf2 (dominant-negative) plasmid. We also observed that Nrf2 activation by ebselen increased the expression of phase II antioxidant genes, including heme oxygenase (HO-1), NAD(P)H:quinine oxidoreductase, and gamma-glutamylcysteine synthetase (gamma-GCS). Treatment with ebselen resulted in an increased expression of HO-1 and intranuclear Nrf2 in hair cells of organotypic cultured cochlea. After intraperitoneal injection with cisplatin, auditory brainstem responses (ABRs) threshold was measured on 8th day in Balb/C mice. ABR threshold shift was marked occurred in mice injected with cisplatin (16 mg/kg, n=5; Click and 8-kHz stimuli, p<0.05; 4, 16 and 32 kHz, p<0.01), whereas that of animal group which was treated with cisplatin and ebselen was not significantly changed. These results suggest that ebselen activates the Nrf2-ARE signaling pathway, which ultimately prevents free radical stresses from cisplatin and further contributes to protect auditory sensory hair cells from free radicals produced by cisplatin.

During the development of the inner ear, the Notch cell signaling pathway is responsible for the specification of the pro-sensory domain and influences cell fate decisions. It is assumed that Notch signaling ends during maturity and cannot be reinitiated to alter the fate of new or existing cells in the organ of Corti. This is in contrast to non-mammalian species which reinitiate Delta 1-Notch1 signaling in response to trauma in the auditory epithelium, resulting in hair cell regeneration through transdifferentiation and/or mitosis. We report immunohistochemical data and Western protein analysis showing that in the aminoglycoside-damaged guinea pig organ of Corti, there is an increase in proteins involved in Notch activation occurring within 24h of a chemical hair cell lesion. The signaling response is characterized by the increased presence of Jagged1 ligand in pillar and Deiters cells, Notch1 signal in surviving supporting cell nuclei, and the absence of Jagged2 and Delta-like1. The pro-sensory bHLH protein Atoh1 was absent at all time points following an ototoxic lesion, while the repressor bHLH transcription factors Hes1 and Hes5 were detected in surviving supporting cell nuclei in the former inner and outer hair cell areas, respectively. Notch pathway proteins peaked at 2 weeks, decreased at 1 month, and nearly disappeared by 2 months. These results indicate that the mammalian auditory epithelium retains the ability to regulate Notch signaling and Notch-dependent Hes activity in response to cellular trauma and that the signaling is transient. Additionally, since Hes activity antagonizes the transcription of pro-sensory Atoh1, the presence of Hes after a lesion may prohibit the occurrence of transdifferentiation in the surviving supporting cells.

Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca2+-free solution. L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca2+ concentrations ([Ca2+]i) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca2+ response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca2+]i increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca2+ response via the NO-cGMP-PKG pathway by a feedback mechanism.

Noise-induced hearing loss has been associated with alterations in cochlear blood flow. Our study analyzed the expression of Vascular Endothelial Growth Factor (VEGF) and its functional receptors, Flt-1 and Flk-1, in the cochlear structures of noise-exposed and unexposed guinea pigs. VEGF is a prototypical angiogenic agent, with multiple functions on vascular biology, ranging from vascular permeability to endothelial cell migration, proliferation, differentiation, and survival. Acoustic trauma was induced by a continuous pure tone of 6 kHz, at 120 dB SPL for 30 min. Auditory function was evaluated by electrocochleographic recordings at 2-20 kHz for 7 days. Noise-induced cochlear morphological changes were studied by immunohistochemistry and scanning electron microscopy. The expression of VEGF and its receptors was examined by immunohistochemistry and western blotting analysis. The hearing threshold shift reached a level of 60 dB SPL on day 1 after trauma and underwent a partial recovery over time, reaching a value of about 20 dB SPL on day 7. Outer hair cell loss was more prominent in the area located 14-16 mm from the apex. Increased cochlear VEGF expression was observed in noise-exposed animals, in particular at the level of stria vascularis, spiral ligament, and spiral ganglion cells. No changes were observed in the expression of VEGF-receptors. Our data suggest a role for VEGF in the regulation of the vascular network in the inner ear after acoustic trauma and during auditory recovery, with potentially important clinical and therapeutic implications.

Current models of the lateral K+ recycling pathway in the mammalian cochlea include two multicellular transport networks separated from one another by three interstitial gaps. The first gap is between outer hair cells and Deiters cells, the second is between outer sulcus cells and type II spiral ligament fibrocytes and the third is between intermediate and marginal cells in the stria vascularis. K+ taken up by cells bordering these interstitial spaces is accompanied by Cl-. Maintaining appropriate electrolyte balance and membrane potentials in these cells requires a mechanism for exit of the resorbed Cl-. One possible candidate for regulating this Cl- efflux is ClC-K, a chloride channel previously thought to be kidney specific. Here, we demonstrate the expression of both known isoforms of ClC-K in the organ of Corti, spiral ligament and stria vascularis of the rat cochlea by immunohistochemical, Western blot and RT-PCR analysis. These results indicate a role for ClC-K in mediating Cl- recycling in the cochlea. The widespread expression of both ClC-K isoforms in the cochlea may help to explain the symptoms of Bartter's syndrome Type III, a mutation in the hClC-Kb gene (human homologue of ClC-K2), which results in renal salt wasting without deafness. These data support the hypothesis that both isoforms of ClC-K are co-expressed in some cell membranes and account for the preservation of hearing in the presence of a mutation in only one channel isoform.

Growth factors are known to activate signaling cascades for DNA replication; they participate in the regulation of cell differentiation and are required as positive signals for cell survival. Thus, many of them may be regarded as potential candidates stimulating regeneration processes in the inner ear. We analyzed the expression of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) and their receptor (bFGFR and NGFR)-like immunoreactivity in chick basilar papillae, along with bFGF and NGF mRNA expression. The evaluation was made 1 and 5 days after exposure to wide-band noise with two increasing levels of acoustic energy. For both factors, the immunoreactivity was shown predominantly in the middle part of basilar papilla, in noise-exposed, but not control birds. It was localized in the cytoplasm of hair cells, nuclei of supporting cells and cytoplasm of ganglion cells. Strong immunoreactivity of bFGFR and NGFR was found both in control and noise-exposed animals, with the cell localization similar to that of growth factors. The increase in mRNA expression for bFGF and NGF was found in noise-exposed animals only after lower exposure to noise, on day 5 after exposure (p<0.01). A lack of increased expression after higher exposure could be excused by larger damage of hair cells followed by the increase of mRNA for beta-actin to which the results were referred. The results suggest bFGF and NGF involvement in postinjury regeneration of the basilar papilla.

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

Mice, in which the genetics can be manipulated and the life span is relatively short, enable evaluation of the effects of specific gene expression on cochlear degeneration over time. Antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1) protect cells from toxic, reactive oxygen species and may be involved in age-related degeneration. The effects of SOD1 deletion and over-expression on the cochlea were examined in Sod1-null mice, Sod1 transgenic mice and in age- and genetics-matched controls. Auditory brainstem responses (ABR) were measured and cochleae were histologically examined. The absence of SOD1 resulted in hearing loss at an earlier age than in wildtype or heterozygous mice. The cochleae of the null mice had severe spiral ganglion cell degeneration at 7-9 months of age. The stria vascularis in the aged, null mice was thinner than in the heterozygous or wildtype mice. Over-expression of SOD1 did not protect against hearing loss except at 24 months of age. In conclusion, SOD1 seems important for survival of cochlear neurons and the stria vascularis, however even half the amount is sufficient and an over abundance does not provide much protection from age-related hearing loss.

To quantitate in absolute terms the prestin mRNA levels in the explant culture of rat cochlea, we used competitive RT-PCR with a synthetic internal cRNA standard. Prestin gene expression was found at levels of 100 fg specific mRNA/microg total RNA on postnatal day 3, which corresponds to about 300 copies per outer hair cell (OHC) and is indicative of an intermediate level of expression. Two days of culturing resulted in an increase of prestin mRNA levels and in the formation of an apical-basal gradient (p<0.001). To elucidate the variations the prestin mRNA levels undergo as a result of damage to the organ of Corti, we exposed the explant cultures to ischemia and hypoxia. While total RNA was observed to remain unchanged, the numbers of OHCs and the prestin mRNA levels were found to decrease by about 20% and 35%, respectively, compared to normoxia. In conclusion, we showed that the prestin mRNA levels during in vitro development increase and form an apical-basal gradient within 2 days in culture, similar to the postnatal in vivo development. Hypoxia and ischemia result in a decrease of the prestin mRNA level in parallel with OHC loss. The prestin mRNA level can therefore be used as marker of damage to or loss of OHCs.

Cisplatin-induced ototoxicity is correlated with functional and morphological changes in the organ of Corti, the stria vascularis and the spiral ganglion. However, the cochlear sites of cisplatin uptake and accumulation have not been properly identified. Therefore, we have developed an immunohistochemical method to, indirectly, detect cisplatin in semithin cryosections of the guinea pig cochlea (basal turn) using an antiserum containing antibodies against cisplatin-DNA adducts. Platinated DNA was present in the nuclei of most cells in the organ of Corti and the lateral wall after cisplatin administration. Nuclear immunostaining was most pronounced in the outer hair cells, the marginal cells and the spiral ligament fibrocytes. This study is the first to demonstrate the presence of cisplatin in histological sections of the cochlea.

To investigate the effect of proinflammatory cytokines on spiral ligament (SL) fibrocytes and regulation of cytokines by dexamethasone (Dex), in vitro studies were performed in murine secondary cell cultures. Cultured SL fibrocytes were stimulated with tumor necrosis factor-alpha (TNF-alpha), and the secretion of various mediators was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcribed-polymerase chain reaction (RT-PCR). After stimulation with TNF-alpha, levels of keratinocyte-derived cytokine (KC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) were elevated in the culture supernatant, and their corresponding messenger RNAs were detected in the cultured fibrocytes. When the cultures were incubated with both TNF-alpha and Dex, the levels of KC, MCP-1, MIP-2 and IL-6 were significantly lower than those in cultures treated with TNF-alpha alone. The data suggest that Dex suppresses the inflammatory response in SL fibrocytes. Given that SL fibrocytes play a role in cochlear fluid and ion homeostasis, glucocorticoids may suppress the cochlear malfunction caused by SL inflammation.

The cochlear portion of the inner ear converts movements produced by sound waves into electrical impulses. Transcripts enriched in the cochlea are likely to have an important role in hearing. In this paper, we report that microarray analyses of the Soares NMIE inner ear library revealed cochlear enriched expression of apolipoprotein D (apoD), a glycoprotein and member of the lipocalin family that transport small hydrophobic ligands. The cochlear enriched expression of Apod was validated by quantitative real time PCR analysis. To investigate the function of apoD in the inner ear the transcript and protein were localised in the cochlea. Apod messenger RNA (mRNA) expression was localised to the spiral ligament and spiral limbus, particularly in the suprastrial and supralimbral regions. The apoD protein was detected in the spiral ligament, spiral limbus and also in the outer hair cells of the organ of Corti. Investigation of cell lines exhibiting characteristics of hair and supporting cells revealed no Apod mRNA expression in these cells. This suggests transport of the protein within the cochlea, followed by internalisation into outer hair cells. The spiral limbus and ligament contain subpopulations of fibrocytes that are intimately involved in regulation of ion balance in the cochlear fluids and type I, II and III fibrocytes of the spiral ligament were all shown to be positive for apoD protein. On the basis of these results it was hypothesised that apoD could be involved in maintaining cochlear fluid homeostasis. To determine whether the apoD gene product was important for normal auditory function the hearing ability of an apoD knockout mouse was tested. The mouse was found to have a hearing threshold that was not significantly different to the control strain.

Endogenous production of prostaglandins has been demonstrated in the cochlea, but no information is available on the distribution of the cyclo-oxygenase (COX) enzymes, or prostanoid receptors in the cochlea. The purpose of the present study was to investigate the localization of the FP, EP(1) and EP(3) prostanoid receptors as well as the COX-1 and COX-2 enzymes in the cochlea of guinea pig and man. Cochleas were processed for immunohistochemistry using routine techniques. Appropriate controls comprised incubation with specific blocking peptides, or incubation without primary antibodies. Both in guinea pig and man the FP prostanoid receptor was abundantly distributed in the cochlea, e.g., in stria vascularis, the spiral ligament, spiral ganglion, and organ of Corti. The immunohistochemical staining of the EP(1) and EP(3) receptors in the same structures was significantly weaker and sometimes lacking altogether (e.g., EP(3) receptor in human cochlea). Weak, but mostly consistent immunostaining of the COX-1 enzyme was found in the cochlear structures. The COX-2 enzyme appeared to be lacking. The abundant distribution of the FP receptor in several important cochlear structures both in guinea pig and man suggests a physiological function for PGF(2alpha) in the cochlea. The COX-1 enzyme seems to be constitutively expressed in the cochlea in contrast to COX-2.

Cisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti. To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase. As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels. These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.

OCP1 and OCP2 are the most abundant proteins in the organ of Corti. Their distributions map identically to the epithelial gap-junction system, which unites the supporting cell population. Sequence data imply that OCP1 and OCP2 are subunits of an SCF E3 ubiquitin ligase. Consistent with that hypothesis, electrophoretic mobility-shift assays and pull-down assays with immobilized OCP1 demonstrate the formation of an OCP1-OCP2 complex. Sedimentation equilibrium data indicate that the complex is heterodimeric. The coincidence of the OCP1-OCP2 distribution and the epithelial gap-junction system suggests that one or more connexin isoforms may be targets of an SCF(OCP1) complex. Significantly, immobilized OCP1 binds (35)S-labeled connexin 26 (Cx26) produced by in vitro transcription-translation. Moreover, Cx26 can be co-immunoprecipitated from extracts of the organ of Corti by immobilized anti-OCP1, implying that OCP1 and Cx26 may associate in vivo. Given that lesions in the Cx26 gene (GJB2) are the most common cause of hereditary deafness, the OCP1-Cx26 interaction has substantial biomedical relevance.

The genes for heat shock proteins (Hsps) can be upregulated in response to cellular trauma, resulting in enhanced cell survival and protection. Hsp32, also known as heme oxygenase 1, catalyzes the degradation of heme to produce carbon monoxide and bilirubin, which play a variety of cytoprotective functions at physiological concentrations, and iron, which is rapidly sequestered by the iron-binding protein ferritin. In the present study we examined the expression and localization of Hsp32 in the rat cochlea after heat shock using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. Low levels of constitutive Hsp32 expression were observed in the normal rat cochlea by RT-PCR and Western blot. Hsp32 mRNA (messenger RNA) was present at higher levels in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus. Western blot revealed that Hsp32 protein levels increase in the rat cochlea following heat shock. Immunocytochemistry showed scattered staining of outer hair cells in the organ of Corti of normal untreated rats. Following heat shock Hsp32 is upregulated in outer hair cells and the cells of the stria vascularis. These results suggest a potential role for Hsp32 as a component of the oxidative stress response pathway in the rat cochlea.

Hair cells are specialized mechanoreceptors common to auditory and vestibular sensory organs of mammalian and non-mammalian species. Different hair cells are believed to share common features related to their mechanosensory function. It has been shown that hair cells possess various forms of motile properties that enhance their receptor function. Membrane-based electromotility is a form of hair cell motility observed in isolated outer hair cells (OHCs) of the cochlea. A novel membrane protein, prestin, recently cloned from gerbil and rat tissues, is presumably responsible for electromotility. We cloned prestin from mouse organ of Corti and confirmed strong homology of this protein among different rodent species. We explored whether or not prestin is present in hair cells of the vestibular system. Using reverse transcription-polymerase chain reaction, we demonstrated that prestin is expressed in mouse and rat auditory and vestibular organs, but not in chicken auditory periphery. In situ hybridization and immunolocalization studies confirmed the presence of prestin in OHCs as well as in vestibular hair cells (VHCs) of rodent saccule, utricle and crista ampullaris. However, in the VHCs, staining of varying intensity with anti-prestin antibodies was observed in the cytoplasm, but not in the lateral plasma membrane or in the stereociliary membrane. Whole-cell patch-clamp recordings showed that VHCs do not possess the voltage-dependent capacitance associated with membrane-based electromotility. We conclude that although prestin is expressed in VHCs, it is unlikely that it supports the form of somatic motility observed in OHCs.

An in vivo tracer was used to determine if the reticular lamina and/or the cell membranes abutting the endolymphatic space are temporarily disrupted after intense noise exposure (4-kHz OBN, 108-dB SPL, 1.75 h). Using a double-barreled micropipette, the endolymphatic potential (EP) was recorded and artificial endolymph containing 10% carbon particles was injected into the endolymphatic space either 0 days or 28 days post-exposure. The cochleae were fixed 30-45 min post-injection, then dehydrated, embedded in plastic and dissected as flat preparations. Damage in the organ of Corti (OC) was quantified, the location of carbon was determined, and some OC segments were then sectioned radially. EP averaged 72+/-5 mV in five controls. These cochleae had carbon tracer in the endolymphatic space only. Four of five noise-exposed chinchillas examined 3-4 h post-exposure had a low EP (30+/-6 mV). The cochleae from these 0-day animals had several focal lesions in which nearly all outer hair cells had just degenerated. At these lesions, carbon was attached to cell membranes and debris between the reticular lamina and basilar membrane. By transmission electron microscopy, discontinuities were found in the apical membranes of sensory and supporting cells. Carbon particles were found in the cytoplasm of these cells. Four of five animals examined at 28 days had an average EP of 70+/-11 mV. The cochleae from these animals had multiple lesions in the basal turn, all of which were healed by phalangeal scars or squamous epithelial cells. In these cochleae, no carbon was found within the OC. Acute disruption of the reticular lamina and the apical membranes of sensory and supporting cells from noise appears to be a major mechanism to account for degeneration in the cochlea that spreads or continues for days to weeks post-exposure., (Copyright 2002 Elsevier Science B.V.)

The sensory epithelium of the mammalian cochlea (the organ of Corti) represents an excellent developmental system. The organ of Corti contains two main cell types: the sensory hair cells and the supporting cells which are organized in a defined mosaic pattern. Previous results have demonstrated the participation of Notch signaling in the regulation of the pattern of hair cell differentiation within this sensory mosaic. It has also been shown that the basic helix-loop-helix (bHLH) transcription factor Math1 is a positive regulator of hair cell differentiation. We demonstrated that Hes1 and Hes5, two members of the inhibitory bHLH transcription factors, act as negative regulators of hair cell differentiation. Loss-of-function studies implicating the neurogenic genes Notch1, Jag2, Hes1 and Hes5 generated a significant increase in the number of hair cells. However, their functional interplay within the organ of Corti has not been determined. To clarify the mechanisms that regulate hair cell differentiation, we examined the expression of Notch/Notch ligand system and Math1 in the developing organ of Corti of Hes1- and Hes5-deficient mice. Our study suggests complex specific relationships between Notch signaling, Math1 and Hes1/Hes5 in the control of hair cell differentiation in the developing organ of Corti.

The two most abundant proteins of the organ of Corti, OCP1 and OCP2, are acidic, cytosolic, low molecular weight proteins diffusely distributed within the cytoplasm of supporting cells. A recent study by Henzl et al. (2001) found first, that these two proteins co-localize with connexin 26 along the epithelial gap junction system and second, that OCP2 could participate with OCP1 in an organ of Corti-specific SCF complex (Skp1, cul1in, and Fbp), a ubiquitin ligase complex. Previous study has also implicated OCP2 in the recycling and regulation of intracellular K(+) efflux as well as pH homeostatic mechanisms. In the present study, we document the emergence and distribution features of OCP2 through various stages (weeks 11-28) of gestation in human fetal cochleae. Four fetal cochleae, the cochleae of a normal hearing human adult and a mature rat for positive control were fixed in 4% formalin within 2 h post mortem. Immunohistochemical studies were performed using a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 3-16. Specimens were mounted in paraffin sections. Results show that OCP2 immunoreactivity is evident at a prenatal age of 11 weeks, peaks in expression at the onset of cochlear function at 20 weeks and achieves adult-like patterns of distribution just prior to histological maturation at 28 weeks. Though this protein could be associated with the development, maturation, and electrochemical maintenance of the cochlear gap junction system, the nature of this protein's function in the developing and mature human cochlea remains unclear.

Estrogen receptors have earlier been shown in the normal mouse, rat and human inner ear. If estrogens are important in normal hearing and development of presbyacusis in the normal population is not known. However it is known that patients with Turner syndrome, where a lack of estrogens is one of the main characteristics, commonly develop an early presbyacusis. A 'Turner mouse' has been developed, as a model for the ear problems in Turner syndrome, and it shows otitis media and a premature aging of the hearing. Estrogen receptors exist in an alpha and a beta form. In this study inner ear tissue, from the Turner mouse and an estrogen receptor beta knockout mouse (betaERKO), was investigated regarding estrogen receptor alpha and beta using immunohistochemistry. Results show that the Turner mouse has the same pattern of inner ear labeling, both concerning the estrogen receptor alpha and beta, as that of a normal CBA/Ca mouse, with positive staining in the organ of Corti and spiral ganglion. The betaERKO mice show close to normal inner ear morphology and positive estrogen receptor alpha immunostaining at the same locations as the CBA/Ca mouse.

A nitric oxide (NO)-selective sensor (tip diameter 30 microm) was inserted into the perilymph of the basal turn of the guinea pig cochlea. The basal level and stimulation-induced changes of NO were measured. The mean (+/-S.E.M.) basal level of NO was 273+/-42.9 nM. Following perilymphatic perfusion of the artificial perilymph containing NO synthase (NOS) substrate L-arginine (100 microM) combined with cofactor (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride (100 microM), a rapid and significant increase of NO to a mean concentration of 392+/-32.3 nM (P < 0.01, n = 10) was recorded. In contrast, a significant decrease of mean NO concentration to 180+/-32.7 nM (P < 0.01, n = 10) was observed following the perfusion of the NOS-inhibiting agent N(G)-nitro-L-arginine methyl ester (100 microM). No change in the NO concentration was found following the perfusion of either artificial perilymph or N(G)-monomethyl-D-arginine (100 microM) solution employed as controls. Broadband noise exposure (3 h/day at 120 dBA SPL) for three consecutive days produced an increase in NO concentration to 618+/-60.7 nM (P < 0.05, n = 10) in the perilymph. In addition, by using specific dyes for NO, 4,5-diaminofluoresceine diacetate and for the reactive oxygen species (ROS), dihydrorhodamine 1,2,3, the distribution of NO in the whole mounts of the organ of Corti and the production of ROS in vivo in the organ of Corti were investigated in both control (n = 5) and noise-exposed (n = 5) animals. The more intense NO and ROS fluorescence was observed in both the inner and outer hair cells in the noise-exposed groups. It is proposed that both the basal level and the increase in NO concentration following the addition of substrate (L-arginine) are produced by the constitutive NOS while the elevated NO and ROS following noise exposure indicate that NO may be involved in noise-induced hearing loss.

Midkine (MK) is one of a new family of heparin-binding growth factors involved in the regulation of growth and differentiation. We have analyzed expression of MK in the cochlea using ICR mice within 1 day from birth. The expression of MK in the cochlea was confirmed by Western blotting and immunohistochemistry. Anti-MK immunoreactivity was observed in the stria vascularis, spiral prominence, spiral ganglion, and ganglion nerve fibers. These findings suggest that MK plays a role in the development of the cochlea.

Immunohistochemical data indicate that OCP1 co-localizes exactly with OCP2 in the epithelial gap junction region of the guinea pig organ of Corti (OC). Despite the abundance of OCP1 in the OC, gaining access to its coding sequence -- and, in particular, the 5' end of the coding sequence -- proved unexpectedly challenging. The putative full-length OCP1 cDNA -- 1180 nucleotides in length -- includes a 67 nucleotide 5' leader sequence, 300 codons (including initiation and termination signals), and a 216 nucleotide 3' untranslated region. The cDNA encodes a protein having a predicted molecular weight of 33,700. The inferred amino acid sequence harbors an F-box motif spanning residues 52--91, consistent with a role for OCP1 and OCP2 in the proteasome-mediated degradation of select OC proteins. Although OCP1 displays extensive homology to an F-box protein recently cloned from rat brain (NFB42), clustered sequence non-identities indicate that the two proteins are transcribed from distinct genes. The presumptive human OCP1 gene was identified in the human genome databank. Located on chromosome 1p35, the inferred translation product exhibits 94% identity with the guinea pig OCP1 coding sequence.

Vascular endothelial growth factor (VEGF) is known as an endothelial cell-specific mitogen. There are no reports concerning the presence of VEGF in the inner ear. To gain information, immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF receptors Flt-1 and KDR/Flk-1 was performed on paraffin-sectioned temporal bones from five guinea pigs. Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in spiral ganglion cells. VEGF could also be found in the endothelium of blood vessels, in the spiral ligament and in the organ of Corti. Flt-1 was found in the limbus epithelium, in all supporting cells of the organ of Corti, in Claudius cells, cells of the sulcus and in the spiral ligament. Flk-1 could be detected in some supporting cells of the organ of Corti (inner pillar cells and Deiters' cells). Immunoreactivity to Flk-1 was also found in endothelium of blood vessels and in the spiral ligament. Hair cells showed VEGF immunostaining, but did not contain staining to Flt-1 nor Flk-1. In the stria vascularis any immunoreactivity to all used VEGF and VEGF receptor antibodies could not be detected. The findings were supported by Western blot analysis on inner ear tissues and ovaries from guinea pigs. We may conclude that the growth factor VEGF and both receptors participate in cochlear physiology.

SPARC is a multifunctional extracellular matrix (ECM) glycoprotein that shares partial sequence homology with SC1/hevin. These ECM molecules exhibit calcium-binding properties and modulate cellular interactions. This study examines the expression of SC1 and SPARC mRNA in the developing cochlea of the rat inner ear prior to and after the onset of hearing. At all ages examined, SC1 mRNA is highly expressed in neurons of the spiral ganglion. In contrast, SPARC transcripts are not detected in the spiral ganglion but are enriched in the temporal bone and cartilaginous otic capsule surrounding the cochlea. Both SC1 and SPARC mRNA are expressed in connective tissue elements involved in maintaining ionic homeostasis of cochlear fluids. SC1 mRNA is localized to type III fibrocytes of the spiral ligament (slg) and marginal cells of the stria vascularis, while SPARC mRNA is apparent in the spiral limbus and type I fibrocytes of the slg. At postnatal day 10, SPARC mRNA shows a dramatic change in expression. High levels of SPARC transcripts are induced in Deiters cells (dc) of the organ of Corti. Interestingly, this induction of SPARC mRNA correlates with the onset of hearing. This suggests that SPARC may play a role in calcium regulation in dc when functional maturation of the cochlea is attained and rapid changes in calcium levels are required.

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.

Tubulin, the principal component of microtubules, exists as two polypeptides, termed alpha and beta. Seven isotypes of beta tubulin are known to exist in mammals. The distributions of four beta tubulin isotypes, beta(I), beta(II), beta(III), and beta(IV), have been examined in the adult cochlea by indirect immunofluorescence using isotype-specific antibodies. In the organ of Corti, outer hair cells contained only beta(I) and beta(IV), while inner hair cells contained only beta(I) and beta(II). Inner and outer pillar cells contained beta(II) and beta(IV), but Deiters cells contained those isotypes plus beta(I). Fine fibers in the inner spiral bundle, tunnel crossing fibers, and outer spiral fibers, probably efferent in character, contained beta(I), beta(II), and beta(III), but not beta(IV). In the spiral ganglion, the somas and axons of neurons contained all four isotypes, and the myelination of ganglion cells also contained beta(I). Fibers of the intraganglionic spiral bundle contained beta(I), beta(II), and beta(III). No antibody labeled the dendritic processes of spiral ganglion neurons. The differences in isotype distribution in organ of Corti and neurons described here are consistent with and support the multi-tubulin hypothesis, which states that tubulin isotypes are expressed specifically in different cell types and may therefore have different functions.

It has been suggested that spiral prominence is associated with ion transport, but the characterization of ion channels has not been explored so far. We studied the electrical properties and ion conductances of the spiral prominence epithelial cells (SPECs), which are epithelial cells covering the luminal side of spiral prominence, in the upper turn of neonatal rat cochlea using a whole-cell variant patch clamp technique. The cell capacitance was 16.3+/-2.1 pF (n=33) and the resting membrane potential was -68. 9+/-2.5 mV (n=14) in perilymph-like bath solution. It was found that those SPECs possess a large voltage-activated, outwardly rectifying K(+) current and a small inwardly rectifying K(+) current. The outward K(+) current was activated by depolarizing pulses more positive than -30 mV, and sensitive to tetraethylammonium chloride (20 mM), 4-aminopyridine (10 mM), but not to Ba(2+) (0.5 mM). Tail current analysis revealed that it was primarily K(+)-selective. The time course of activation was well fitted by an exponential function raised to second power. The small inwardly rectifying K(+) current was sensitive to Ba(2+) (0.5 mM), and the Ba(2+)-sensitive current was K(+)-selective. In cell-attached or inside-out patch recordings, no discernible K(+) channel currents were found in the apical membrane of SPECs. Based on these results, we conclude that SPECs have two types of voltage-dependent K(+) currents, which are most likely located in the basolateral membrane.

Recent in vitro studies demonstrated that members of the epidermal growth factor (EGF) family are involved in hair cell replacement in the postnatal mammalian organ of Corti (OC) after ototoxic damage. This suggests a role for the EGF receptor (EGFR) in this process. We examined the expression of EGFR mRNA within the normal postnatal day 3 (P3) and adult rat cochlear epithelium by RT-PCR and examined its cellular localization with non-radioactive in situ hybridization in P3 and adult cochleae. RT-PCR demonstrated that EGFR mRNA is expressed in P3 and adult cochlear epithelium. In situ hybridization localized high levels of EGFR transcripts in the OC, spiral ganglion, Kölliker's organ and detectable levels in the supporting cells and the stria vascularis of P3 cochlea. In the adult cochlea, EGFR transcripts were detected only in the spiral ganglion. Our results support that the EGFR is implicated in the differentiation of several cochlear cell types and in the response of OC to ototoxic damage of the P3 rat. In the adult, it may participate in the maintenance of the mature neurons and its absence in the OC may contribute to the lack of regenerative responses in the adult cochlea.

In the late stages of inner ear development, the relatively undifferentiated cells of Kollicker's organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to beta-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.

Identification of the K+ transporters located in the lateral wall of the cochlea is essential for a better understanding of the mechanisms by which a positive endocochlear potential and a high K+ concentration are achieved in endolymph. In this study, we have determined the distribution of the K+ channel rat ether à go-go (eag) mRNA in the cochlea. After reverse transcription of adult rat cochlear tissues, cDNA was amplified with primers specific to eag channel. The eag mRNA was localized in cochlear tissues by in situ hybridization using specific oligonucleotide probes tailed with digoxigenin conjugated UTP. Eag mRNA was detected in the organ of Corti but mainly in the fibrocytes of the spiral ligament but not in spiral prominence or in stria vascularis. The expression pattern of rat eag transcript in spiral ligament is complementary to the Na+,K+-ATPase distribution in the cochlear lateral wall. The localization of eag mRNA suggests that eag potassium channel may be produced in the corresponding cells. Considering the importance of the K+ gradient in the cochlea, the result reported here suggests that eag channel may play a role in the control of K+ fluxes in the spiral ligament.