Spiral Ligament Fibrocytes Research Articles

Heptanol Application to the Mouse Round Window: A Model for Studying Cochlear Lateral Wall Regeneration

Identify cells supporting cochlear lateral wall regeneration. Prospective controlled trial. Laboratory. Human presbyacusis occurs, in part, secondary to age-related degeneration of cochlear lateral wall structures such as the stria vascularis and spiral ligament fibrocytes. This degeneration is likely linked to the diminished regenerative capacity of lateral wall cells with age. While lateral wall regeneration is known to occur after an acute insult, this process remains poorly understood and the cells capable of self-replication unidentified. We hypothesized that spiral ligament fibrocytes constitute these proliferative cells. To test the hypothesis, an acute ototoxic insult was created in 65 normal-hearing, young adult mice via cochlear exposure to heptanol. Sacrifice occurred at 1 to 60 days posttreatment. Auditory brainstem responses, 5-ethynyl-2'-deoxyuridine assay, and immunostaining were used to assess regeneration. Posttreatment hearing thresholds were elevated in nearly all treated mice. Selective fibrocyte apoptosis and strial injury were observed at the time of peak hearing loss around 1 to 7 days posttreatment. Cellular proliferation was detected in the region of type II fibrocytes during this time. Hearing thresholds plateaued at 7 days posttreatment followed by a significant recovery of both hearing and morphologic appearance. Permanent outer hair cell degeneration was observed. Heptanol application to the round window of young adult mice is a rapid, selective, and reliable technique for investigating proliferation in the cochlear lateral wall. The data indirectly showed that spiral ligament fibrocytes may be the proliferative cells of the cochlear lateral wall. Further studies of this process are needed.

The expression of PTEN in the development of mouse cochlear lateral wall

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that regulates various cell processes including proliferation, growth, synaptogenesis, neural and glioma stem/progenitor cell renewal. In addition, PTEN can regulate sensory cell proliferation and differentiation of hair bundles in the mammalian cochlea. In this study we use immunofluorescence, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) to reveal the expression of PTEN in the developing cochlear lateral wall, which is crucial for regulating K+ homeostasis. Relatively high levels of PTEN are initially expressed in the marginal cells (MCs) of the lateral wall at embryonic day (E) 17.5 when they start to differentiate. Similarly high levels are subsequently expressed in differentiating root cells (RCs) at postnatal day (P) 3 and then in spiral ligament fibrocytes (SLFs) at P 10. In the mature cochlea, PTEN expression is low or undetectable in MCs and SLFs but it remains high in RCs and their processes. The expression pattern for PTEN in the developing lateral wall suggests that it plays a critical role in the differentiation of the cellular pathways that regulate K+ homeostasis in the cochlea.

Heptanol Application to the Mouse Round Window: A Novel Model for Studying Cochlear Regenerative Potential

Objectives: 1) Introduce a novel model for studying cochlear lateral wall regeneration. 2) Describe implications of this study on research and treatment of human presbycusis. Background: Human presbycusis likely occurs secondary to senile degeneration of the stria vascularis (SV) and spiral ligament fibrocytes (SLFs). SLFs have a limited regenerative capacity. Augmenting this process may play a role in hearing preservation, recovery, or both. Few models currently exist, however, that can selectively and temporarily damage the cochlear lateral wall to study SLF regeneration. Methods: Heptanol, a gap junction uncoupler, was surgically applied to the round window of 25 normal hearing mice, with sacrifice at points 12 hours - 14 days post-treatment. Auditory brainstem response testing, electron microscopy, and immunostaining techniques were used to assess treatment response. Results: Click tone thresholds were significantly elevated (mean +40.5 dB sound pressure level) in all treated mice. Hearing loss plateaued on days 1-7, followed by near complete recovery thereafter. Ultrastructural analysis revealed selective SV injury and SLF apoptosis at the apical, middle, and basal turns. A marked reduction in ion channel immunostaining intensity occurred throughout the lateral wall but resolved by 2 weeks. 3-4 days, SLFs stained positive for EdU, a labeled thymidine analog and marker of cellular proliferation. Conclusions: This is one of the first models described to reliably and selectively induce lateral wall toxicity followed by full hearing recovery and SLF regeneration. Future study of the molecular and genetic expression profiles governing this process will be greatly facilitated by this technique.

The expression of glutamate aspartate transporter (GLAST) within the human cochlea and its distribution in various patient populations

Glutamate plays an important role in the central nervous system as an excitatory neurotransmitter. However, its abundance can lead to excitotoxicity which necessitates the proper function of active glutamate transporters. The glutamate-aspartate transporter (GLAST) has been shown to exist and function within non-human cochlear specimens regulating the inner ear glutamate concentration. In this study, we examined human cochleas from formalin-fixed celloidin-embedded temporal bone specimens of three different types of patients (Meniere's disease, normal controls, and other otopathologic conditions) and examined the differential expression of GLAST in the spiral ligament of the basal, middle, and apical turns of the cochlea. Immunohistochemical staining was performed with polyclonal antibodies against GLAST and image analysis was carried out with the Image J analysis software. In contrast to other studies with non-human specimens, GLAST was expressed in the spiral ligament fibrocytes but was not detected in the satellite cells of the spiral ganglia or supporting cells of the Organ of Corti in the human cochlea. Our data also showed that GLAST expression significantly differs in the basal and apical turns of the cochlea. Lastly, post-hoc analysis showed a difference in the GLAST immunoreactive area of patients with Meniere's disease when compared to that of patients with other otopathologic conditions—such as presbycusis or ototoxicity. These results may potentially lead to further understanding of different disease states that affect hearing.

IL-10-mediated modulation of cochlear inflammation (P6228)

Abstract Inflammatory response is commonly involved in the pathogenesis of various cochlear diseases such as acoustic trauma and cisplatin ototoxicity. Previously, we have demonstrated that the spiral ligament fibrocytes play a pivotal role in cochlear inflammation through secretion of chemokines such as CCL2 and CXCL2 in response to pro-inflammatory signals. In this study, we aim to determine molecular mechanism involved in negative modulation of cochlear inflammation. We found that IL-10 inhibits SLF’s CCL2 up-regulation and migration of THP-1 cells in response to SLF-derived molecules. The SLFs appeared to up-regulate HMOX1 expression via NRF-2 activation. The inhibitory effect of IL-10 was mimicked by CoPP and CORM-3 but was blocked by silencing of IL-10RA and HMOX1. IL-10 was found to inhibit binding of p65 NFκB to the enhancer region of the CCL2 gene. IL-10 deficiency appeared to enhance cochlear inflammation secondary to nontypeable H. influenzae-induced otitis media. Furthermore, we demonstrated that the cochlear macrophages serve as a local source of IL-10, which appeared to be primarily distributed in the stria vascularis. Our results suggest that cochlear macrophage-derived IL-10 modulates cochlear inflammation by inhibition of SLF’s CCL2 up-regulation through HMOX1-mediated CO production. This study is anticipated providing us with a novel strategy for the management of inflammation-associated cochlear diseases.

Mitochondria toxin-induced acute cochlear cell death indicates cellular activity-correlated energy consumption

The different cell types within the cochlea may have a specific contribution to the pathological changes during metabolism failure, which may provide clues for developing novel strategies for inner ear therapy. In order to evaluate activity-correlated cell death during metabolism failure in the cochlea, 3-nitropropionic acid was used to irreversibly inhibit the respiratory chain. Dose-response of the cochlear cells to 3-nitropropionic acid was analyzed in vitro. 3-Nitropropionic acid was administered onto the round window of guinea pigs. Cell death was identified by terminal transferase labeling the free 3'OH breaks in the DNA strands in vivo and propidium iodide nuclear permeation in vitro. As a result, 23.6 and 96.3 % cell death were induced by 10 and 100 mM 3-nitropropionic acid, respectively, in vitro. In the guinea pigs, 500 mM 3-nitropropionic acid induced vestibular dysfunction and severe to profound hearing losses. The cells that are the most sensitive to 3-nitropropionic acid treatment include the stria marginal and intermediate cells, epithelial cells of the Reissner's membrane, and spiral ligament fibrocytes (types II and V). Moderate sensitive cells were satellite fibrocytes of the spiral limbic central zone, osteocytes of the cochlear shell, hair cells, and spiral ganglion cells. Reduction of neurofilament in the soma and periphery processes of spiral ganglion cells occurred after the exposure. These results may be relevant to the mechanisms of injury in sudden onset sensorineural hearing loss and hazardous substance exposure-induced hearing loss.

Erratum: Functional interaction between mesenchymal stem cells and spiral ligament fibrocytes

Erratum: Functional interaction between mesenchymal stem cells and spiral ligament fibrocytes

Functional interaction between mesenchymal stem cells and spiral ligament fibrocytes

Spiral ligament fibrocytes (SLFs) play an important role in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Our previous study showed that transplantation of mesenchymal stem cells (MSCs) into the inner ear of rats with damaged SLFs significantly accelerates hearing recovery compared with rats without MSC transplantation. To elucidate this mechanism of SLF repair and to determine the contribution of transplanted MSCs in this model, we investigated the mutual effects on differentiation and proliferation between MSCs and SLFs in a coculture system. Factors secreted by SLFs had the ability to promote the transdifferentiation of MSCs into SLF-like cells, and the factors secreted by MSCs had a stimulatory effect on the proliferation of SLFs. Cytokine antibody array analysis revealed the involvement of transforming growth factor-β (TGF-β) in SLF proliferation induced by MSCs. In addition, a TGF-β inhibitor reduced SLF proliferation induced by MSC stimulation. Our results suggest that there are two mechanisms of hearing recovery following transplantation of MSCs into the inner ear: 1) MSCs transdifferentiate into SLF-like cells that compensate for lost SLFs, and 2) transplanted MSCs stimulate the regeneration of host SLFs. Both mechanisms contribute to the functional recovery of the damaged SLF network.

Contractility in Type III Cochlear Fibrocytes Is Dependent on Non-muscle Myosin II and Intercellular Gap Junctional Coupling

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

ERK2‐dependent activation of c‐Jun is required for nontypeable H. influenzae‐induced Cxcl2 up‐regulation in the inner ear fibrocytes

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune‐privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of pro‐inflammatory molecules derived from middle ear infection. Since middle ear infection is highly prevalent in children, middle ear infection‐induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable H. influenzae (NTHi), a major otitis media pathogen, and up‐regulate a monocyte‐attracting chemokine through TLR2‐dependent NF‐κB activation. Here, we aimed to determine molecular mechanism involved in NTHi‐induced cochlear infiltration of polymorphonuclear cells (PMNs). The spiral ligament fibrocytes (SLFs) were found to release Cxcl2 in response to NTHi via activation of c‐Jun, not NF‐κB, leading to PMN recruitment to the cochlea. We also demonstrated that MEK1/ERK2 signaling pathway is required for NTHi‐induced Cxcl2 up‐regulation in the SLFs. Two AP‐1 motifs in the 5′‐flanking region of Cxcl2 appeared to function as a NTHi‐responsive element, and the proximal AP‐1 motif was found to have a higher binding affinity to NTHi‐activated c‐Jun than the distal one. Our results will enable us to better understand the molecular pathogenesis of middle ear infection‐induced inner ear inflammation. [Supported in part by NIH grants DC8696, DC5025, and DC 6276]

ERK2-Dependent Activation of c-Jun Is Required for Nontypeable Haemophilus influenzae-Induced CXCL2 Upregulation in Inner Ear Fibrocytes

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.

Expression and distribution of creatine transporter and creatine kinase (brain isoform) in developing and mature rat cochlear tissues

Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0-P14) and adult (P22-P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K(+) cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine-phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.

Subcellular Distribution and Relative Expression of Fibrocyte Markers in the CD/1 Mouse Cochlea Assessed by Semiquantitative Immunogold Electron Microscopy

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Developmental and Cell-Specific Expression of Thyroid Hormone Transporters in the Mouse Cochlea

Thyroid hormone is essential for the development of the cochlea and auditory function. Cochlear response tissues, which express thyroid hormone receptor β (encoded by Thrb), include the greater epithelial ridge and sensory epithelium residing inside the bony labyrinth. However, these response tissues lack direct blood flow, implying that mechanisms exist to shuttle hormone from the circulation to target tissues. Therefore, we investigated expression of candidate thyroid hormone transporters L-type amino acid transporter 1 (Lat1), monocarboxylate transporter (Mct)8, Mct10, and organic anion transporting polypeptide 1c1 (Oatp1c1) in mouse cochlear development by in situ hybridization and immunofluorescence analysis. L-type amino acid transporter 1 localized to cochlear blood vessels and transiently to sensory hair cells. Mct8 localized to the greater epithelial ridge, tympanic border cells underlying the sensory epithelium, spiral ligament fibrocytes, and spiral ganglion neurons, partly overlapping with the Thrb expression pattern. Mct10 was detected in a highly restricted pattern in the outer sulcus epithelium and weakly in tympanic border cells and hair cells. Organic anion transporting polypeptide 1c1 localized primarily to fibrocytes in vascularized tissues of the spiral limbus and spiral ligament and to tympanic border cells. Investigation of hypothyroid Tshr(-/-) mice showed that transporter expression was delayed consistent with retardation of cochlear tissue maturation but not with compensatory responses to hypothyroidism. The results demonstrate specific expression of thyroid hormone transporters in the cochlea and suggest that a network of thyroid hormone transport underlies cochlear development.

Relative Time Course of Degeneration of Different Cochlear Structures in the CD/1 Mouse Model of Accelerated Aging

Presbycusis (age-related hearing loss) can result from various cochlear pathologies. We have studied the time course of degeneration in a mouse that shows accelerated presbycusis, the CD/1 mouse, as a possible model to investigate stem-cell strategies to prevent or ameliorate presbycusic changes. CD/1 mice from 0 to 72 weeks old were examined by light and electron microscopy. Early pathological changes were detected in basal turn spiral ligament fibrocytes and spiral ganglion, but the latter was variable as both satellite cells and neurons were normal in some cochleae. Light microscopic counts in the spiral ligament of 20-week-old mice revealed that of the five main types (types I-V), only type V fibrocytes showed no reduction in numbers compared with 3-week-old animals, and type IV showed the greatest losses. However, all types of fibrocyte showed subtle damage when examined using electron microscopy, in the form of swollen mitochondria, as early as 2 weeks. The extent of mitochondrial damage showed a degree of correspondence with the light microscopic pattern of fibrocyte loss in that types III and IV fibrocytes had the most abnormal mitochondria and type V the least, especially at early stages. By 10-15 weeks, ultrastructural features of fibrocyte damage were similar to longer term changes reported in gerbils. Stria vascularis, spiral ganglion and hair cells showed few consistent early signs of damage but became increasingly affected, lagging behind the fibrocyte damage. Our data suggest that fibrocyte pathology may precede other presbycusic changes; breakdown of homeostatic mechanisms to which they contribute may cause the subsequent degeneration of the hair cells. Overall, there were many similarities to presbycusic changes in other rodents and humans. Therefore, the features of accelerated aging in this mouse make it a suitable model for rapidly assessing possible strategies to prevent or ameliorate presbycusic changes.

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media

BackgroundOtitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.MethodsLive NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.ResultsTranstympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the in vitro data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation in vivo. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.ConclusionsTaken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.

Improving the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO 4 to quench autofluorescence in the rat inner ear

Fluorescent tags and fluorophore-conjugated molecular probes have been extensively employed in histological studies to demonstrate nanoparticle distribution in inner ear cell populations. However, autofluorescence that exists in the rodent cochleae disturbs visualization of the fluorescent tags and fluorophore labeling. In the present work, we aimed to improve the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO 4 to quench autofluorescence in the rat inner ear. The in vivo study was performed on eight- to nine-month-old rats using confocal laser scanning microscopy, and the in vitro study was carried out with DiI-tagged poly(ethylene glycol) and poly(capro-lactone) polymersomes and different fluorescent-labeling agents using a spectrofluorometer. The nanoparticles were intratympanically administered using either an osmotic pump or transtympanic injection. Abundant autofluorescence was detected in spiral ganglion cells (SGCs), stria marginal cells, spiral ligament fibrocytes (SL) and the subcuticular cytoplasm of inner hair cells (IHCs). Sparsely distributed faint autofluorescence was also visualized in outer hair cells (OHCs). The autofluorescence was eliminated by treatment with 1 mM CuSO 4 (in 0.01 M ammonium acetate buffer) for 70–90 min, while the fluorescent tag in the nanoparticle was absolutely preserved and the labeling fluorescence signals of the molecular probes were mostly retained.

The Membrane Properties of Cochlear Root Cells are Consistent with Roles in Potassium Recirculation and Spatial Buffering

Auditory transduction, amplification, and hair cell survival depend on the regulation of extracellular [K(+)] in the cochlea. K(+) is removed from the vicinity of sensory hair cells by epithelial cells, and may be distributed through the epithelial cell syncytium, reminiscent of "spatial buffering" in glia. Hypothetically, K(+) is then transferred from the epithelial syncytium into the connective tissue syncytium within the cochlear lateral wall, enabling recirculation of K(+) back into endolymph. This may involve secretion of K(+) from epithelial root cells, and its re-uptake via transporters into spiral ligament fibrocytes. The molecular basis of this secretion is not known. Using a combination of approaches we demonstrated that the resting conductance in guinea pig root cells was dominated by K(+) channels, most likely composed of the Kir4.1 subunit. Dye injections revealed extensive intercellular gap junctional coupling, and delineated the root cell processes that penetrated the spiral ligament. Following uncoupling using 1-octanol, individual cells had Ba(2+)-sensitive weakly rectifying currents. In the basal (high-frequency encoding) cochlear region K(+) loads are predicted to be the highest, and root cells in this region had the largest surface area and the highest current density, consistent with their role in K(+) secretion. Kir4.1 was localized within root cells by immunofluorescence, and specifically to root cell process membranes by immunogold labeling. These results support a role for root cells in cochlear K(+) regulation, and suggest that channels composed of Kir4.1 subunits may mediate K(+) secretion from the epithelial gap junction network.

Yin and Yang: CCL2 and IL‐10 in Otitis Media‐induced Inner Ear Inflammation

Otitis media (OM) may lead to inner ear inflammation resulting in sensorineural hearing loss. Here, we aim to determine the role of spiral ligament fibrocytes (SLFs)‐derived CCL2 and exogenous IL‐10 in inner ear inflammation, secondary to nontypeable H. influenzae (NTHI)‐induced OM. THP‐1 cells actively migrated in response to the conditioned medium of NTHI‐exposed SLFs. This migratory activity was inhibited by the CC chemokine inhibitor and by the deficiency of CCL2 in SLFs. The CCR2 inhibitor and CCR2 deficiency inhibited migration of monocyte‐like cells of the splenocytes in response to NTHI‐induced SLF‐derived CCL2. qRT‐PCR showed that recombinant IL‐10 inhibits SLF's NTHI‐induced up‐regulation of CCL2 expression. Luciferase assays showed that silencing of IL‐10RA in SLFs suppressed the inhibitory effect of IL‐10 on NTHI‐induced CCL2 up‐regulation. In contrast, IL‐10 deficiency of SLFs hardly affected NTHI‐induced CCL2 expression. Transtympanic inoculation of live NTHI led to inner ear inflammation in 10 out of 14 murine ears (71%). Unexpectedly, CCL2 deficiency equivocally decreased the incidence of OM‐induced inner ear inflammation (60%), indicating a compensatory effect of other chemokines in vivo. Taken together, we suggest that NTHI‐induced SLF‐derived CCL2 contributes to OM‐induced inner ear inflammation, which is negatively regulated by IL‐10. [Supported in part by DC008696 and DC006276]

Intense noise exposure activates JNK signal via generation of nitric oxide in cochlear spiral ligament fibrocytes

Intense noise exposure activates JNK signal via generation of nitric oxide in cochlear spiral ligament fibrocytes