Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

Transcriptomic analysis and ednrb expression in cochlear intermediate cells reveal developmental differences between inner ear and skin melanocytes.

In the inner ear, the neural crest gives rise to the glia of the VIII ganglion and two types of melanocytic cells: The pigmented cells of the vestibular system and intermediate cells of the stria vascularis. We analyzed the transcriptome of neonatal intermediate cells in an effort to better understand the development of the stria vascularis. We found that the expression of endothelin receptor B, which is essential for melanocyte development, persists in intermediate cells long after birth. In contrast, skin melanocytes rapidly downregulate the expression of EdnrB. Our findings suggest that endothelins might have co-opted new functions in the inner ear during evolution of the auditory organ.

Cochlear supporting cells function as macrophage-like cells and protect audiosensory receptor hair cells from pathogens.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

The Effects of Mobile Phone Radiofrequency Radiation on Cochlear Stria Marginal Cells in Sprague-Dawley Rats.

To investigate the possible mechanisms for biological effects of 1,800 MHz mobile radiofrequency radiation (RFR), the radiation-specific absorption rate was applied at 2 and 4 W/kg, and the exposure mode was 5 min on and 10 min off (conversation mode). Exposure time was 24 h short-term exposure. Following exposure, to detect cell DNA damage, cell apoptosis, and reactive oxygen species (ROS) generation, the Comet assay test, flow cytometry, DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining, and a fluorescent probe were used, respectively. Our experiments revealed that mobile phone RFR did not cause DNA damage in marginal cells, and the rate of cell apoptosis did not increase (P > 0.05). However, the production of ROS in the 4 W/kg exposure group was greater than that in the control group (P < 0.05). In conclusion, these results suggest that mobile phone energy was insufficient to cause cell DNA damage and cell apoptosis following short-term exposure, but the cumulative effect of mobile phone radiation still requires further confirmation. Activation of the ROS system plays a significant role in the biological effects of RFR. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

TBX1 is required for normal stria vascularis and semicircular canal development.

Little is known about the role of TBX1 in post-otocyst stages of inner ear development. Here, we report on mice with a missense mutation of Tbx1 that are viable with fully developed but abnormally formed inner ears. Mutant mice are deaf due to an undeveloped stria vascularis and show vestibular dysfunction associated with abnormal semicircular canal formation. We show that TBX1 is expressed in endolymph-producing strial marginal cells and vestibular dark cells of the inner ear and is an upstream regulator of Esrrb, which previously was shown to control the developmental fate of these cells. We also show that TBX1 is expressed in sensory cells of the crista ampullaris, which may relate to the semicircular canal abnormalities observed in mutant mice. Inner ears of mutant embryos have a non-resorbed fusion plate in the posterior semicircular canal and a single ampulla connecting anterior and lateral canals. We hypothesize that the TBX1 missense mutation prevents binding with specific co-regulatory proteins. These findings reveal previously unknown functions of TBX1 during later stages of inner ear development.

Cochlear Capillary Pericytes.

Capillary pericytes in the cochlea of mammals are-compared to pericytes in other tissues, like the CNS-relatively poorly researched. To begin with, there is still a considerable debate as to whether the very last precapillary arterioles should-due to their contractile properties-may be considered to be pericytes.However, cochlear capillary pericytes have shifted into the center of attention in the past decade. Most mammals show a considerable number of pericytes in the stria vascularis of the cochlea-up to 1300 in a mouse alone. This high number may be explained by the observation that cochlear capillary pericytes may be differentiated into different subgroups, depending on the immune markers that are expressed by them. Corresponding with these subpopulations, cochlear pericytes fulfill three core functions in the physiology of the cochlea: Formation of the intrastrial blood-fluid barrier-Pericytes monitor the ion, fluid, and nutrient household and aid in the homeostasis thereof. Regulation of cochlear blood flow-By contraction on relaxation, pericytes contribute to the regulation of cochlear blood flow, a paramount function parameter of the cochlea. Immune response-Pericytes actually contribute to the immune response in inflammation of the cochlea. Due to these central roles in the physiology of the cochlea, pericytes actually play a major role in numerous cochlear pathologies, including, but not limited to, sudden sensorineural hearing loss, acoustic trauma, and inflammation of the cochlea.

Isolation, cultivation, and characterization of primary bovine cochlear pericytes: A new in vitro model of stria vascularis.

The study of strial pericytes has gained great interest as they are pivotal for the physiology of stria vascularis. To provide an easily accessible in vitro model, here we described a growth medium-based approach to obtain and cultivate primary bovine cochlear pericytes (BCP) from the stria vascularis of explanted bovine cochleae. We obtained high-quality pericytes in 8-10 days with a > 90% purity after the second passage. Immunocytochemical analysis showed a homogeneous population of cells expressing typical pericyte markers, such as neural/glial antigen 2 (NG2), platelet-derived growth factor receptorß (PDGFRß), α-smooth muscle actin (α-SMA), and negative for the endothelial marker von Willebrand factor. When challenged with tumor necrosis factor or lipopolysaccharide, BCP changed their shape, similarly to human retinal pericytes (HRPC). The sensitivity of BCP to ototoxic drugs was evaluated by challenging with cisplatin or gentamicin for 48 hr. Compared to human retinal endothelial cells and HRPC, cell viability of BCP was significantly lower ( p < 0.05) after the treatment with gentamicin or cisplatin. These data indicate that our protocol provides a simple and reliable method to obtain highly pure strial BCP. Furthermore, BCP are suitable to assess the safety profile of molecules which supposedly exert ototoxic activity, and may represent a valid alternative to in vivo tests.

Differential localizations of the myo-inositol transporters HMIT and SMIT1 in the cochlear stria vascularis.

The cochlear stria vascularis produces endolymph and thereby plays an active role in inner ear homeostasis. We recently reported that the H+/myo-inositol cotransporter (HMIT) gene is expressed in the stria vascularis. Here, we examined the protein localization of HMIT and Na+/myo-inositol cotransporter 1 (SMIT1) in the stria vascularis by immunohistochemistry. HMIT and SMIT1 were detected in the lateral wall of the cochlear duct. HMIT was widely detected throughout the stria vascularis, while SMIT1 was enriched in the strial basal cells. To examine the localization of HMIT in the stria vascularis in more detail, dissociated strial cells were immunostained, which resulted in the detection of HMIT immunoreactivity in marginal cells. These results indicate that HMIT is expressed in marginal cells and basal cells of the stria vascularis, while SMIT1 expression is enriched in basal cells. We speculate that HMIT and SMIT1 may play important roles in the homeostasis of cochlear fluids, for example by participating in pH regulation and osmoregulation.

Electrophysiological properties of strial pericytes and the effect of aspirin on pericyte K+ channels.

The present study was designed to investigate the electrophysiological properties of strial pericytes and the effect of aspirin on pericyte K+ channels. Pericytes were identified by determining their morphological characteristics and using pericyte­associated immunofluorescence techniques. The electrophysiological properties of strial pericytes were observed with a whole­cell patch­clamp technique. Alterations in the outward current of cochlear pericytes in the stria vascularis of guinea pigs were examined following the application of K+ channel retardants. The effects of aspirin on pericyte K+ channels were also evaluated with the whole­cell patch­clamp technique. The results demonstrated that pericytes were desmin positive, and their nuclei were large and surrounded by a small proportion of the cytoplasm. Cytoplasmic processes gradually declined in size as branches grew parallel to the capillary axis. Thus, capillaries were surrounded by tips. The electrophysiological properties of the cochlear pericytes in the stria vascularis of guinea pigs were also determined. The membrane capacitance of the pericytes was 5.9±0.3 pF, while the membrane resistance and resting potential were 2.2±0.3 GΩ and ­30.9±1.2 mV, respectively. The current densities of the pericytes (pA/pF) were 3.2±0.7, 10.6±1.0, 15.7±0.9 and 21.3±1.2 at command voltages of 0, +20, +40, and +60 mV, respectively. The K+ channels were activated when the pericytes were within the range of ­20 mV to +20 mV, particularly at 0 mV. The inhibition rates of the outward current of cochlear pericytes in the stria vascularis of the guinea pigs were determined by administering iberiotoxin (IBTX) and IBTX + 4­aminopyridine. Once the background leakage current was removed, the following inhibition rates were obtained with 3, 10, 30, 300 and 1,000 µmol/l aspirin: 20.8±4.8, 34.1±6.9, 48.2±6.7, 63.6±7.1 and 65.7±8.1%, respectively. The outward current of the cochlear pericytes in the stria vascularis was inhibited by aspirin with a half maximal inhibitory concentration of 24.5±4.5 µmol/l. The membranes of the pericytes in the stria vascularis are characterized by high­conductance calcium­activated K+ (BKCa) and voltage­dependent K+ (KV) channels. The outward current of the cochlear pericytes in the stria vascularis of guinea pigs was inhibited by aspirin in a concentration­dependent manner. In addition, BKCa and KV channels were inhibited by aspirin.

The dual role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy under oxidative stress in rat cochlear marginal cells of the stria vascularis.

Oxidative stress is reported to regulate several apoptotic and necrotic cell death pathways in auditory tissues. Poly(ADP-ribose) polymerase-1 (PARP-1) can be activated under oxidative stress, which is the hallmark of parthanatos. Autophagy, which serves either a pro-survival or pro-death function, can also be stimulated by oxidative stress, but the role of autophagy and its relationship with parthanatos underlying this activation in the inner ear remains unknown. In this study, we established an oxidative stress model in vitro by glucose oxidase/glucose (GO/G), which could continuously generate low concentrations of H2O2 to mimic continuous exposure to H2O2 in physiological conditions, for investigation of oxidative stress-induced cell death mechanisms and the regulatory role of PARP-1 in this process. We observed that GO/G induced stria marginal cells (MCs) death via upregulation of PARP-1 expression, accumulation of polyADP-ribose (PAR) polymers, decline of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF), which all are biochemical features of parthanatos. PARP-1 knockdown rescued GO/G-induced MCs death, as well as abrogated downstream molecular events of PARP-1 activation. In addition, we demonstrated that GO/G stimulated autophagy and PARP-1 knockdown suppressed GO/G-induced autophagy in MCs. Interestingly, autophagy suppression by 3-Methyladenine (3-MA) accelerated GO/G-induced parthanatos, indicating a pro-survival function of autophagy in GO/G-induced MCs death. Taken together, these data suggested that PARP-1 played dual roles by modulating parthanatos and autophagy in oxidative stress-induced MCs death, which may be considered as a promising therapeutic target for ameliorating oxidative stress-related hearing disorders.

The morphological and functional development of the stria vascularis in miniature pigs.

The purpose of this study was to examine the morphological and functional development of the lateral wall of the scala media of the cochlea in miniature pigs; light and transmission electron microscopy and electrophysiology were used for this purpose. We showed that the lateral wall of the scala media of the cochlea appears at embryonic Day 21 (E21) when the cochlear duct begins to form. From E28 to E49, the lateral wall can be distinguished according to its position along the cochlea. At E56, cells in the lateral wall begin to differentiate into three different types. At E70, three cell types, marginal, intermediate and basal, can be clearly distinguished. At E91, the stria vascularis is adult-like and the organ of Corti is also morphologically mature. The average endocochlear potential measured from the second turn of the cochlea (at E98, postnatal Day 1 (P1), P13 and P30) was 71.4±2.5 (n=7), 78.8±1.5 (n=10), 77.3±2.3 (n=10) and 78.0±2.1 mV (n=10), respectively. Our results suggest that in miniature pigs the stria vascularis develops during the embryonic period, concurrent with maturation of the organ of Corti. The magnitude of the endocochlear potential reached its mature level when the stria vascularis was morphologically adult-like at E98. These findings provide a morphological and functional basis for future animal studies using the miniature pig model concerning the pathogenesis of various inner-ear diseases.

Organ of Corti and Stria Vascularis: Is there an Interdependence for Survival?

Cochlear hair cells and the stria vascularis are critical for normal hearing. Hair cells transduce mechanical stimuli into electrical signals, whereas the stria is responsible for generating the endocochlear potential (EP), which is the driving force for hair cell mechanotransduction. We questioned whether hair cells and the stria interdepend for survival by using two mouse models. Atoh1 conditional knockout mice, which lose all hair cells within four weeks after birth, were used to determine whether the absence of hair cells would affect function and survival of stria. We showed that stria morphology and EP remained normal for long time despite a complete loss of all hair cells. We then used a mouse model that has an abnormal stria morphology and function due to mutation of the Mitf gene to determine whether hair cells are able to survive and transduce sound signals without a normal electrochemical environment in the endolymph. A strial defect, reflected by missing intermediate cells in the stria and by reduction of EP, led to systematic outer hair cell death from the base to the apex after postnatal day 18. However, an 18-mV EP was sufficient for outer hair cell survival. Surprisingly, inner hair cell survival was less vulnerable to reduction of the EP. Our studies show that normal function of the stria is essential for adult outer hair cell survival, while the survival and normal function of the stria vascularis do not depend on functional hair cells.

[Study on the electrophsiological properties in the stria vascularis pericytes in cochlear of guinea pig].

OBJECTIVE: The purpose of this paper was to study the electrophysiological properties and the type of potassium channels on cell membrane in the stria vascularis pericytes in cochlear of guinea pig. METHODS: Firstly examined the expression of the stria vascularis pericytes by desmin, a marker of pericytes, in cochlear of guinea pig with immunofluorescent method. Using whole-cell patch clamp recording techniques to observe electrophysiological properties in the cochlear pericytes in stria vascularis of guinea pig. RESULTS: Pericytes were predominately distributed in the capillaries of cochlea.The average membrane capacitance, resistance, and potential of a single pericyte in stria vascularis were(5.9±0.3)pF, (2.2±0.3)GΩ and (-30.9±1.2)mV, respectively by using patch clamp technique. In addition, the average current density of cochlear pericyte was voltage-sensitive (Vh from 0 to + 60 mV, in 20 mV steps). The pericytes exhibited outward current and this property could be blocked by TEA (tetraethylammonium) 1 mmol/L, a large-conductance calcium-activated potassium channel(BKCa)inhibitor and 4-AP (4-aminopyridine) 1 mmol/L, a voltage-dependent K(+) channels(KV) channel blocker. TEA blocked the outward current from (296.2±35.9)pA to (163.7±16.8)pA and 4-AP blocked the outward current from (248.7±39.8)pA to (158.0±38.0)pA. CONCLUSION: These results suggest that pericytes in stria vascularis have BKCa and KV channels.

Endothelin-1 mediated induction of extracellular matrix genes in strial marginal cells underlies strial pathology in Alport mice.

Alport syndrome, a type IV collagen disorder, manifests as glomerular disease associated with hearing loss with thickening of the glomerular and strial capillary basement membranes (SCBMs). We have identified a role for endothelin-1 (ET-1) activation of endothelin A receptors (ETARs) in glomerular pathogenesis. Here we explore whether ET-1 plays a role in strial pathology. Wild type (WT) and Alport mice were treated with the ETAR antagonist, sitaxentan. The stria vascularis was analyzed for SCBM thickness and for extracellular matrix (ECM) proteins. Additional WT and Alport mice were exposed to noise or hypoxia and the stria analyzed for hypoxia-related and ECM genes. A strial marginal cell line cultured under hypoxic conditions, or stimulated with ET-1 was analyzed for expression of hypoxia-related and ECM transcripts. Noise exposure resulted in significantly elevated ABR thresholds in Alport mice relative to wild type littermates. Alport stria showed elevated expression of collagen α1(IV), laminin α2, and laminin α5 proteins relative to WT. SCBM thickening and elevated ECM protein expression was ameliorated by ETAR blockade. Stria from normoxic Alport mice and hypoxic WT mice showed upregulation of hypoxia-related, ECM, and ET-1 transcripts. Both ET-1 stimulation and hypoxia up-regulated ECM transcripts in cultured marginal cells. We conclude that ET-1 mediated activation of ETARs on strial marginal cells results in elevated expression of ECM genes and thickening of the SCBMs in Alport mice. SCBM thickening results in hypoxic stress further elevating ECM and ET-1 gene expression, exacerbating strial pathology.

ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 µM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss.

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9-W18) and show the cochlear expression dynamics of key potassium-regulating proteins. At W12, MITF+/SOX10+/KIT+ neural-crest-derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+-ATPase-positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy.

[Age-related expression of plasma membrane Ca(2+) -ATPase isoform 2 in the cochleas of C57BL/6J mice].

OBJECTIVE: To investigate the location and distribution of plasma membrane Ca²âº -ATPase isoform 2(PMCA2) in the cochleas of C57BL/6J mice at various ages (4w, 14w, 22w, 45w), and to reveal the relationship of PMCA2 and age-related hearing loss (AHL). METHODS: The distribution of PMCA2 in the cochleas of C57BL/6J mice was detected by immunohistochemistry at various ages (4w, 14w, 22w, 45w). Real-time polymerase chain reaction (Rt-PCR) was used to detect the level of PMCA2 mRNA in the cochleas of C57BL/6J mice at the ages of 4, 14, 22 and 45 weeks old respectively. Using SPSS17.0 software for statistical analysis. RESULTS: PMCA2 was mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 was also observed in spiral ligament. Hair cells missed and the number of spiral ganglion cells reduced with age. Expression of PMCA2 in the cochleas of C57BL/6J mice also showed age-related decreasing. The results of Rt-PCR demonstrated the expression of mRNA of gene (Atp2b2) at 14 weeks age was significantly less than 4 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 22 weeks age was significantly less than 14 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 45 weeks age was significantly less than 14 week-old mice cochlears (P<0.01). CONCLUSIONS: PMCA2 is mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 is also observed in spiral ligament. The expression of PMCA2 demonstrates an age-related decrease with age. The mRNA expression level of PMCA2 gene(Atp2b2) in the cochleas of C57BL/6J mice displayed an age-related decrease. PMCA2 transporters may play a critical role in maintaining the normal morphology of the inner ear and it may be related to AHL.

Cellular localization and developmental changes of Zip8, Zip14 and transferrin receptor 1 in the inner ear of rats.

Prior studies have demonstrated that the inner ear can accumulate a variety of essential and potentially toxic heavy metals including manganese, lead, cobalt and cadmium. Metal accumulation is regulated in part by the functionality and affinity of these metals for the different transport systems responsible for uptake across the blood-cochlea barrier and their subsequent uptake into the different cells within the inner ear. Transport of these metals across cell membranes occurs by many of the same transport systems which include DMT1, Zip8 and Zip14. All three metal transporters have been identified in the cochlea based on quantitative PCR analysis. Prior studies in our laboratory examined the localization and developmental changes of DMT1 in rat cochlea and since the two Zip proteins are also likely to contribute to the transport of essential and non-essential divalent cations, we performed immunolabeling experiments in postnatal day three rat pups and adult rats. For comparison, we also immunolabeled the specimens with antibody against transferrin receptor 1 (TfR1) which is important in DMT1-mediated transport of Fe and Mn. Results presented in this paper demonstrate that the cellular and subcellular distribution of both Zip8 and Zip14 within the different components of the inner ear are distinct from that of DMT1. Nuclear localization for both Zip transporters as well as TfR1 was observed. The findings also reveal that the selective distribution of the three proteins was altered during development presumably to meet the changing needs of the cells to maintain normal and functional levels of iron and other essential metals.

Conditioning the cochlea to facilitate survival and integration of exogenous cells into the auditory epithelium.

The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media.

Precise toxigenic ablation of intermediate cells abolishes the "battery" of the cochlear duct.

The extracellular potential of excitable and nonexcitable cells with respect to ground is ∼0 mV. One of the known exceptions in mammals is the cochlear duct, where the potential is ∼80-100 mV, called the endocochlear potential (EP). The EP serves as the "battery" for transduction of sound, contributing toward the sensitivity of the auditory system. The stria vascularis (StV) of the cochlear duct is the station where the EP is generated, but the cell-specific roles in the StV are ill defined. Using the intermediate cell (IC)-specific tyrosinase promoter, under the control of diphtheria toxin (DT), we eliminated and/or halted differentiation of neural crest melanocytes after migration to the StV. The ensuing adult transgenic mice are profoundly deaf. Additionally, the EP was abolished. Expression of melanocyte early marker and Kir4.1 in ICs precedes the onset of pigment synthesis. Activation of DT leads to loss of ICs. Finally, in accord with the distinct embryology of retinal pigmented cells, transgenic mice with toxigenic ablation of neural crest-derived melanocytes have intact visual responses. We assert that the tyrosinase promoter is the distinct target for genetic manipulation of IC-specific genes.