Development of cochlear spiral ligament fibrocytes of the common marmoset, a nonhuman model animal.

Spiral ligament fibrocytes generate potassium gradients, which hair cells require to convert mechanical sound waves into electrical palsy. Together with the stria vascularis, they regulate endolymph electrolyte homeostasis. Developing spiral ligament fibrocytes and generating endocochlear potential with an appropriate endolymph ion composition are essential for hearing. Understanding spiral ligament fibrocyte development is useful for studying age-related and genetic hearing loss, as well as for regenerative therapy and cochlear immunology. Despite interspecies differences, most studies of cochlear development have been conducted in rodent models due to the difficulty of using human fetal samples. This study investigated the cochlear development of spiral ligament fibrocytes in a small New World monkey species, the common marmoset (Callithrix jacchus). We examined the developmental expression of specific genes in spiral ligament fibrocytes, including those essential for the generation of endolymphatic potential. Our results showed that this animal model of spiral ligament fibrocyte development is similar to that of humans and is a suitable alternative for the analysis of human cochlear development. The time course established in this study will be useful for studying the primate-specific developmental biology of the inner ear, which may lead to novel treatment strategies for human hearing loss.

Electrical and Immunohistochemical Properties of Cochlear Fibrocytes in 3D Cell Culture and in the Excised Spiral Ligament of Mice.

Fibrocyte degeneration in the cochlear lateral wall is one possible pathology of age-related metabolic hearing loss (presbycusis). Within the lateral wall fibrocytes play a role in potassium recycling and maintenance of the endocochlear potential. It has been proposed that cell replacement therapy could prevent fibrocyte degeneration in the CD/1 mouse model of hearing loss. For this to work, the replacement fibrocytes would need to take over the structural and physiological role of those lost. We have grown lateral wall fibrocytes from neonatal CD/1 mice in a 3D-collagen gel culture with the aim of assessing their functional similarity to native lateral wall fibrocytes, the latter in a slice preparation and in excised spiral ligament pieces. We have compared cultured and native fibrocytes using both immuno-labelling of characteristic proteins and single cell electrophysiology. Cultured fibrocytes exhibited rounded cell bodies with extending processes. They labelled with marker antibodies targeting aquaporin 1 and calcium-binding protein S-100, precluding an unambiguous identification of fibrocyte type. In whole-cell voltage clamp, both native and cultured fibrocytes exhibited non-specific currents and voltage-dependent K+ currents. The non-specific currents from gel-cultured and excised spiral ligament fibrocytes were partially and reversibly blocked by external TEA (10 mM). The TEA-sensitive current had a mean reversal potential of + 26 mV, suggesting a permeability sequence of Na+ > K+. These findings indicate that 3D-cultured fibrocytes share a number of characteristics with native spiral ligament fibrocytes and thus might represent a suitable population for transplantation therapy aimed at treating age-related hearing loss.

Cdk5 regulatory subunit-associated protein 1 knockout mice show hearing loss phenotypically similar to age-related hearing loss.

Mitochondrial dysfunction is associated with aging and age-related hearing loss (AHL). However, the precise mechanisms underlying the pathophysiology of hearing loss remain unclear. Cdk5 regulatory subunit-associated protein 1 (CDK5RAP1) enables efficient intramitochondrial translation by catalyzing the deposition of 2-methylthio modifications on mitochondrial tRNAs. Here we investigated the effect of defective mitochondrial protein translation on hearing and AHL in a Cdk5rap1 deficiency C57BL/6 mouse model. Compared to control C57BL/6 mice, Cdk5rap1-knockout female mice displayed hearing loss phenotypically similar to AHL from an early age. The premature hearing loss in Cdk5rap1-knockout mice was associated with the degeneration of the spiral ligament and reduction of endocochlear potentials following the loss of auditory sensory cells. Furthermore, cultured primary mouse embryonic fibroblasts displayed early onset of cellular senescence associated with high oxidative stress and cell death. These results indicate that the CDK5RAP1 deficiency-induced defective mitochondrial translation might cause early hearing loss through the induction of cellular senescence and cochlear dysfunction in the inner ear. Our results suggest that the accumulation of dysfunctional mitochondria might promote AHL progression. Furthermore, our findings suggest that mitochondrial dysfunction and dysregulated mitochondrial tRNA modifications mechanistically cause AHL. Understanding the mechanisms underlying AHL will guide future clinical investigations and interventions in the attempt to mitigate the consequences of AHL.

Neutrophils infiltrate into the spiral ligament but not the stria vascularis in the cochlea during lipopolysaccharide-induced inflammation.

It has been challenging to apply intravital imaging for monitoring the inner ear, as the anatomical location and intricate structure hamper the access of imaging instruments to the inner ear of live mice. By employing intravital imaging of the cochlea in live mice with two-photon microscopy, we investigated neutrophil infiltration into the cochlea tissue and its characteristics under a lipopolysaccharide (LPS)-induced inflammatory state. Methods: Cochlea inflammation was induced by LPS injection to the middle ear. Using two-photon intravital microscopy with specifically designed surgical exteriorization of the cochlea in live mice, we investigated the dynamic features of neutrophils in the lateral wall of the cochlea. The molecular expression pattern of the cochlea lateral wall was also investigated during the LPS-induce inflammation. Results: Despite the contention of whether neutrophils are recruited to the spiral ligament (SL) during inflammation, we observed that LPS-induced inflammation of the middle ear, which mimics acute otitis media, triggered neutrophil migration to the SL in the lateral wall. Notably, massive neutrophil infiltration to the SL occurred 2 days after LPS inoculation, but there was no neutrophil infiltration into the stria vascularis (SV) region. At 1 day after LPS-induced cochlear inflammation, increased mRNA expression of interleukin-1ß, interleukin-6 were identified in both the SL and SV, while the ICAM-1 mRNA expression increased only in the SL. The differential reactivity of ICAM-1 is likely responsible for the different neutrophil recruitment pattern in the cochlea. Conclusion: Intravital imaging of the cochlea revealed that neutrophil recruitment and infiltration during inflammation are spatially controlled and exclusively observed in the SL but not in the SV and organ of Corti.

Bone Marrow Stromal Cells Accelerate Hearing Recovery via Regeneration or Maintenance of Cochlear Fibrocytes in Mouse Spiral Ligaments.

Mammalian cochleae have limited capacity for regeneration, which is one of the major difficulties in the treatment of sensorineural hearing loss. In the current study, we examined the potential of bone marrow-derived stromal cells (BMSCs) for functional restoration of mouse cochleae through regeneration or maintenance of cochlear fibrocytes in the spiral ligament (SL). We used a mouse model of degeneration of cochlear fibrocytes in the SL using local application of 3-nitropropionic acid (3-NP), in which disruption of the gap junction network in the SL resulted in the reduction of the endocochlear potential (EP). Mouse BMSCs were infused into the posterior semicircular canal 7 days after 3-NP application. Transplanted BMSCs were frequently observed in the cochlear fluid space 4 weeks after transplantation, although a few transplants had migrated into the cochlear tissues including the SL. BMSC-treated cochleae exhibited higher cell densities in the SL and greater EP levels than the control ones. Immunohistochemistry further demonstrated the restoration of functional proteins in the SL. Significant recovery in thresholds of auditory brainstem responses following BMSC transplantation was found only at 40 kHz in a mild degeneration model. Our cumulative findings indicated that BMSCs accelerated regeneration or maintenance of fibrocytes in damaged SLs, leading to partial functional restoration of the mouse cochleae. Anat Rec, 303:478-486, 2020. © 2019 American Association for Anatomy.

Early Hearing Loss upon Disruption of Slc4a10 in C57BL/6 Mice.

The unique composition of the endolymph with a high extracellular K+ concentration is essential for sensory transduction in the inner ear. It is secreted by a specialized epithelium, the stria vascularis, that is connected to the fibrocyte meshwork of the spiral ligament in the lateral wall of the cochlea via gap junctions. In this study, we show that in mice the expression of the bicarbonate transporter Slc4a10/Ncbe/Nbcn2 in spiral ligament fibrocytes starts shortly before hearing onset. Its disruption in a C57BL/6 background results in early onset progressive hearing loss. This hearing loss is characterized by a reduced endocochlear potential from hearing onset onward and progressive degeneration of outer hair cells. Notably, the expression of a related bicarbonate transporter, i.e., Slc4a7/Nbcn1, is also lost in spiral ligament fibrocytes of Slc4a10 knockout mice. The histological analysis of the spiral ligament of Slc4a10 knockout mice does not reveal overt fibrocyte loss as reported for Slc4a7 knockout mice. The ultrastructural analysis, however, shows mitochondrial alterations in fibrocytes of Slc4a10 knockout mice. Our data suggest that Slc4a10 and Slc4a7 are functionally related and essential for inner ear homeostasis.

Disruption of Gap Junction-Mediated Intercellular Communication in the Spiral Ligament Causes Hearing and Outer Hair Cell Loss in the Cochlea of Mice.

It is well-known that outer hair cell (OHC) loss occurs in the cochlea of animal models of permanent hearing loss induced by intense noise exposure. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear spiral ligament prior to noise-induced sudden hearing loss. The goal of the present study was to evaluate the mechanism underlying cochlear OHC loss after sudden hearing loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after in vitro exposure to 4-hydroxynonenal (a product of lipid peroxidation), H2O2, SIN-1 (peroxynitrite generator), and carbenoxolone (a gap junction inhibitor). Interestingly, in vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament. However, no significant OHC loss was observed in vivo in animals treated with 4-hydroxynonenal and SIN-1. Taken together, our data suggest that disruption of GJIC in the cochlear lateral wall structures is an important cause of cochlear OHC loss in models of hearing loss, including those induced by noise.

Three-dimensional reconstruction of root cells and interdental cells in the rat inner ear by serial section scanning electron microscopy.

In the cochlea, a high K+ environment in the endolymph is essential for the maintenance of normal hearing function, and the transport of K+ ions through gap junctions of the cochlear epithelium is thought to play an important role in endolymphatic homeostasis. The aim of the present study was to demonstrate the three-dimensional (3D) ultrastructure of spiral ligament root cells and interdental cells, which are located at both ends of the gap junction system of the cochlea epithelium. Serial semi-thin sections of plastic-embedded rat cochlea were mounted on glass slides, stained with uranyl acetate and lead citrate, and observed by scanning electron microscopy (SEM) using the backscattered electron (BSE) mode. 3D reconstruction of BSE images of serial sections revealed that the root cells were linked together to form a branched structure like an elaborate "tree root" in the spiral ligament. The interdental cells were also connected to each other, forming a comb-shaped cellular network with a number of cellular strands in the spiral limbus. Furthermore, TEM studies of ultra-thin sections revealed the rich presence of gap junctions in both root cells and interdental cells. These findings suggest the possibility that both root cells and interdental cells contribute to K+ circulation as the end portion of the epithelial cell gap junction system of the cochlea.

Spiral Form of the Human Cochlea Results from Spatial Constraints.

The human inner ear has an intricate spiral shape often compared to shells of mollusks, particularly to the nautilus shell. It has inspired many functional hearing theories. The reasons for this complex geometry remain unresolved. We digitized 138 human cochleae at microscopic resolution and observed an astonishing interindividual variability in the shape. A 3D analytical cochlear model was developed that fits the analyzed data with high precision. The cochlear geometry neither matched a proposed function, namely sound focusing similar to a whispering gallery, nor did it have the form of a nautilus. Instead, the innate cochlear blueprint and its actual ontogenetic variants were determined by spatial constraints and resulted from an efficient packing of the cochlear duct within the petrous bone. The analytical model predicts well the individual 3D cochlear geometry from few clinical measures and represents a clinical tool for an individualized approach to neurosensory restoration with cochlear implants.

Calpain inhibitor alleviates permanent hearing loss induced by intense noise by preventing disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament.

Our previous studies demonstrated that intense noise-induced hearing loss might be at least in part due to an oxidative stress-induced decrease in the level of gap junction-composing protein connexins in the spiral ligament (SL) of the cochlear lateral wall structures in mice. Further, an in vivo exposure of mice to intense noise activates calpain in the cochlear SL. Based on these studies, we sought to determine whether a calpain inhibitor would prevent an intense noise exposure from causing hearing loss, disruption of gap junction-mediated intercellular communication (GJIC) in the SL. An exposure of mice to intense noise (8-Hz octave band noise, 110-dB sound pressure level, 1h) produced permanent hearing loss and cochlear hair cell death. The results of an ex vivo assay using gap-fluorescence recovery after photobleaching of dissected lateral wall structures revealed that the intense noise disrupted GJIC in the cochlear SL at day-7 post exposure. A prior intracochlear injection of the calpain inhibitor PD150606 significantly abolished this noise-induced hearing loss on days 5 and 7 post exposure. Similarly, PD150606 prevented noise-induced hair cell death and the GJIC disruption on day-7 post exposure. The intense noise temporarily enhanced the gene expression of calpain subtypes Capn1 and Capn2 immediately after exposure. Taken together, our data suggest that calpain inhibitor alleviated the noise-induced hearing loss, at least in part, by preventing disruption of GJIC in the cochlear SL. It possible that calpain inhibitors would be useful as a candidate of therapeutic drugs for sudden sensorineural hearing loss.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall.

Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

Age-related morphological changes in the basement membrane in the stria vascularis of C57BL/6 mice.

Basement membrane anionic sites (BMAS) are involved in the selective transport of electrically charged macromolecules in cochlear capillaries. Using cationic polyethyleneimine (PEI), we examined age-related changes in BMAS in the cochleae of C57BL/6 mice. The mice were grouped according to age as follows: 3 days, 4 weeks, 8 weeks, 6 months, and 12 months. In the right bony labyrinths, widths of the stria vascularis were measured in paraffin-embedded sections using light microscopy. The left bony labyrinths were immersed in a 0.5 % cationic PEI solution and embedded in epoxy resin. Ultrathin sections of the left cochlea were examined using transmission electron microscopy. A significant difference in stria vascularis width was observed between the 4-week-old and 12-month-old mice. The PEI distribution in the capillary and epithelial basement membranes (BMs) of the cochlea was observed. In all animals, PEI particles were evenly distributed in the capillary BM of the spiral ligament and in the subepithelial BM of Reissner's membrane. In the stria vascularis, PEI particles were evenly distributed in the capillary BM in 3-day-old mice. In 4- and 8-week-old mice, PEI particle sizes were markedly lower than those observed in 3-day-old mice. In 6- and 12-month-old mice, PEI particles were hardly detected in the strial capillary BM. In the strial capillary BM in these mice, the laminae rarae externa and interna disappeared, but the lamina densa became larger. We speculated that age-related changes of strial capillary BMAS may affect electrically charged macromolecule transport systems in the stria vascularis of C57BL/6 mice.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall / 华中科技大学学报（医学）（英德文版）

Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear spiral ligament.

The endocochlear potential in the inner ear is essential for hearing ability, and maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication (GJ-IC) in the lateral wall structures of the cochlea. Noise-induced hearing loss is known at least in part due to disruption of GJ-IC resulting from an oxidative stress-induced decrease in connexins (Cxs) level in the lateral wall structures. The purpose of this study was to investigate, using primary cultures of fibrocytes from the cochlear spiral ligament of mice, the mechanism underlying GJ-IC disruption induced by 4-hydroxynonenal (4-HNE), which is formed as a mediator of oxidative stress. An exposure to 4-HNE produced the following events: i.e., an increase in 4-HNE-adducted proteins; a decrease in the protein levels of Cx43, ß-catenin, and Cx43/ß-catenin complex along with intracellular translocation of this complex from the cell membrane to the cytoplasm; enhanced calpain-dependent degradation of endogenous α-fodrin; and disruption of GJ-IC. The 4-HNE-induced decrease in these protein levels and disruption of GJ-IC were most completely abolished by the calpain inhibitor PD150606. Taken together, our data suggest that 4-HNE disrupted GJ-IC through calpain-mediated degradation of Cx43 and ß-catenin in primary cultures of fibrocytes derived from the cochlear spiral ligament.

The paracrine effect of mesenchymal human stem cells restored hearing in ß-tubulin induced autoimmune sensorineural hearing loss.

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.

Histopathology of the human inner ear in Alström's syndrome.

Alström's syndrome is an autosomal recessive syndromic genetic disorder caused by mutations in the ALMS1 gene. Sensorineural hearing loss occurs in greater than 85% of patients. Histopathology of the inner ear abnormalities in the human has not previously been fully described. Histopathology of the inner ear in Alström's syndrome is presented in 2 genetically confirmed cases. The predominant histopathologic correlates of the sensorineural loss were degeneration of the organ of Corti, both inner and outer hair cells, degeneration of spiral ganglion cells, and atrophy of the stria vascularis and spiral ligament.

Lysine-specific demethylase 1 inhibitors protect cochlear spiral ganglion neurons against cisplatin-induced damage.

Cisplatin is a widely used chemotherapeutic drug, but one of its side effects is ototoxicity. Epigenetic-related drugs, such as lysine-specific demethylase 1 (LSD1) inhibitors, have been reported to protect against cisplatin-induced hair cell loss by preventing demethylation of histone H3K4 (H3K4me2). However, the protective effect of LSD1 inhibitors in spiral ganglion neurons (SGNs) remains unclear. To investigate whether LSD1 inhibitors exert similar protective effects on SGNs, we treated mouse cochlear explant cultures with LSD1 inhibitors (2PCPA, S2101, or CBB1007) together with cisplatin. Low concentrations of cisplatin damaged SGNs much more than high concentrations, and blocking the demethylation of H3K4me2 with LSD1 inhibitors prevented the SGNs from injury. Reactive oxygen species are also involved in the injury process, and LSD1 inhibitors protected SGNs by increasing the expression level of the antioxidant gene Slc7a11 and decreasing the level of the pro-oxidant gene lactoperoxidase (Lpo). Our findings show that LSD1 inhibitors prevent cisplatin-induced SGN loss by regulating the demethylation of H3K4 and preventing increases of reactive oxygen species levels, which might provide a potential therapeutic strategy for cisplatin-induced hearing loss.

NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K⁺ transport that controls the electrochemical properties in the mammalian cochlea.

Unidirectional K(+) transport across the lateral cochlear wall contributes to the endocochlear potential (EP) of +80 mV in the endolymph, a property essential for hearing. The wall comprises two epithelial layers, the syncytium and the marginal cells. The basolateral surface of the former and the apical membranes of the latter face the perilymph and the endolymph, respectively. Intrastrial space (IS), an extracellular compartment between the two layers, exhibits low [K(+)] and a potential similar to the EP. This IS potential (ISP) dominates the EP and represents a K(+) diffusion potential elicited by a large K(+) gradient across the syncytial apical surface. The K(+) gradient depends on the unidirectional K(+) transport driven by Na(+),K(+)-ATPases on the basolateral surface of each layer and the concomitant Na(+),K(+),2Cl(-)-cotransporters (NKCCs) in the marginal cell layer. The NKCCs coexpressed with the Na(+),K(+)-ATPases in the syncytial layer also seem to participate in the K(+) transport. To test this hypothesis, we examined the electrochemical properties of the lateral wall with electrodes measuring [K(+)] and potential. Blocking NKCCs by perilymphatic perfusion of bumetanide suppressed the ISP. Unexpectedly and unlike the inhibition of the syncytial Na(+),K(+)-ATPases, the perfusion barely altered the electrochemical properties of the syncytium but markedly augmented [K(+)] of the IS. Consequently, the K(+) gradient decreased and the ISP declined. These observations resembled those when the marginal cells' Na(+),K(+)-ATPases or NKCCs were blocked with vascularly applied inhibitors. It is plausible that NKCCs in the marginal cells are affected by the perilymphatically perfused bumetanide, and these transporters, but not those in the syncytium, mediate the unidirectional K(+) transport.

Disruption of ion-trafficking system in the cochlear spiral ligament prior to permanent hearing loss induced by exposure to intense noise: possible involvement of 4-hydroxy-2-nonenal as a mediator of oxidative stress.

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na(+), K(+)-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K(+)-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na(+), K(+)-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, N(ω)-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na(+), K(+)-ATPase α1, as well as Na(+), K(+)-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na(+), K(+)-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na(+), K(+)-ATPase activity in the cochlear lateral wall structures.