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.

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.

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.

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.

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.

Sox10 expressing cells in the lateral wall of the aged mouse and human cochlea.

Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1-3 month-old) and aged (2-2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10(+) cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10(+) marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10(+) cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10(+) cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10(+) cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear.

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.

Quantitative polarized light microscopy of unstained mammalian cochlear sections.

Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo.

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-ß (TGF-ß) in SLF proliferation induced by MSCs. In addition, a TGF-ß 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.

Late-phase recovery in the cochlear lateral wall following severe degeneration by acute energy failure.

We previously reported a model of acute cochlear energy failure using a mitochondrial toxin, 3-nitropropionic acid (3-NP), to study mechanisms of inner ear disorders such as inner ear ischemia. In this model, the main cause of hearing loss is apoptosis of fibrocytes in the cochlear lateral wall. Here, we analyzed the time course of structural and hearing level changes in the cochlea from the acute phase to the chronic phase up to 2 months after surgery. Hearing levels as determined by auditory brainstem response (ABR) thresholds exceeded the maximum acoustic output (>87 dBSPL) of the system at all frequencies 1 day after 3-NP treatment. Histology showed nearly complete loss of fibrocytes 2 weeks after 3-NP treatment. However, after 2 months, ABR showed significant recovery at low frequency (8 kHz) in four of five rats treated with 3-NP. ABR thresholds at 20 kHz occasionally showed some recovery. At 40 kHz, recovery of ABR thresholds was not observed. Histology of 3-NP-treated rats revealed partial recovery of the lateral wall and the regenerated fibrocytes in the spiral ligament expressed Na/K-ATPase in the cochlear basal turn 2 months after 3-NP treatment. These results indicate that ABR recovery is caused by regeneration of the cochlear lateral wall. Our findings demonstrate the recoverable capacity of the cochlear lateral wall that leads to functional recovery after severe damage.

Acoustic overstimulation activates 5'-AMP-activated protein kinase through a temporary decrease in ATP level in the cochlear spiral ligament prior to permanent hearing loss in mice.

Inner ear disorders are known to be elicited by mitochondrial dysfunction, which decreases the ATP level in the inner ear. 5'-AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by metabolic stress and by an increase in the AMP/ATP ratio. To elucidate the involvement of AMPK-derived signals in noise-induced hearing loss, we investigated whether in vivo acoustic overstimulation would activate AMPK in the cochlea of mice. Std-ddY mice were exposed to 8kHz octave band noise at a 90-, 110- or 120-dB sound pressure level (SPL) for 2h. Exposure to the noise at 110 or 120dB SPL produced outer hair cell death in the organ of Corti and permanent hearing loss. Exposure to the noise at 120-dB SPL elevated the level of the phospho-AMPK α-subunit (p-AMPKα), without affecting the protein level of this subunit, immediately and at 12-h post-exposure in the lateral wall structures including the spiral ligament and stria vascularis. In the hair cells and spiral ganglion cells, no marked change in the level of p-AMPKα was observed at any time post-exposure. The level of phospho-c-Jun N-terminal kinase (p-JNK) was increased in the lateral wall structures at 2- to 4-h post-exposure at 120dB SPL. Noise exposure significantly, but temporarily, decreased the ATP level in the spiral ligament, in an SPL-dependent manner at 110dB and above. Likewise, elevation of p-AMPKα and p-JNK levels was also observed in the lateral wall structures post-exposure to noise at an SPL of 110dB and above. Taken together, our data suggest that AMPK and JNK were activated by ATP depletion in the cochlear spiral ligament prior to permanent hearing loss induced by in vivo acoustic overstimulation.

Connexin32 can restore hearing in connexin26 deficient mice.

Functional gap junction channels composed of certain connexin proteins are essential for the function of the cochlea. Homozygous deficiency in the Gjb2 (mice) or GJB2 (human) gene coding for connexin26 (Cx26) in the cochlea leads to hearing impairment in mice and humans, respectively. Here we have studied the functional equivalence of Cx26 and connexin32 (Cx32) isoforms in the cochlea. We analyzed a conditional mouse mutant in which the Gjb2 coding DNA was exchanged by LacZ DNA coding for the reporter protein beta-galactosidase. This allowed us to follow the unrestricted and cell type specific expression of Gjb2 promoter activity. After inner ear specific, Otogelin-Cre recombinase mediated deletion of the loxP-site-flanked LacZ coding DNA, transcription of the Gjb1 gene, coding for Cx32 was activated by the Gjb2 promoter. Interbreeding of these mice with conditional Gjb2 null mice resulted in animals in which Cx32 instead of Cx26 protein is expressed in the non-sensory epithelial network of the cochlea. When we analyzed the auditory function in these mice, we found that the expression of Cx32 protein is sufficient to support hearing in the absence of Cx26. Thus Cx32 can functionally replace Cx26 in the mouse cochlea resulting in almost normal hearing.

Functional expression of P2X4 receptor in capillary endothelial cells of the cochlear spiral ligament and its role in regulating the capillary diameter.

The cochlear lateral wall generates the endocochlear potential (EP), which creates a driving force for the hair cell transduction current and is essential for normal hearing. Blood flow at the cochlear lateral wall is critically important for maintaining the EP. The vulnerability of the EP to hypoxia suggests that the blood flow in the cochlear lateral wall is dynamically and precisely regulated to meet the changing metabolic needs of the cochlear lateral wall. It has been reported that ATP, an important extracellular signaling molecule, plays an essential role in regulating cochlear blood flow. However, the cellular mechanism underlying ATP-induced regional blood flow changes has not been investigated. In the current study, we demonstrate that 1) the P2X4 receptor is expressed in endothelial cells (ECs) of spiral ligament (SL) capillaries. 2) ATP elicits a characteristic current through P2X4 on ECs in a dose-dependent manner (EC(50) = 0.16 mM). The ATP current has a reversal potential at ∼0 mV; is inhibited by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD), LaCl(3), pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), and extracellular acidosis; and is less sensitive to α,ß-methyleneadenosine 5'-triphosphate (α,ß-MeATP) and 2'- and 3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP). 3) ATP elicits a transient increase of intracellular Ca(2+) in ECs. 4) In accordance with the above in vitro findings, perilymphatic ATP (1 mM) caused dilation in SL capillaries in vivo by 11.5%. N(ω)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nonselective inhibitor of nitric oxide synthase, or 5-BDBD, the specific P2X4 inhibitor, significantly blocked the dilation. These findings support our hypothesis that extracellular ATP regulates cochlear lateral blood flow through P2X4 activation in ECs.

Mechanism underlying the protective effect of tempol and Nω-nitro-L-arginine methyl ester on acoustic injury: possible involvement of c-Jun N-terminal kinase pathway and connexin26 in the cochlear spiral ligament.

There is evidence that reactive oxygen species (ROS) are formed in the cochlea during acoustic injury. However, very little is known about the involvement of ROS signals in the spiral ligament (SL) during such injury. The purpose of this study was to determine the effect of the multifunctional antioxidant tempol and the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) on acoustic injury and the c-Jun N-terminal kinase (JNK) pathway in the SL. Exposure of adult mice to noise (8-kHz octave band, 110-dB SPL for 1 h) produced permanent hearing loss. Noise exposure increased not only the formation of a protein modified by 4-hydroxynonenal and formation of nitrotyrosine, but also the level of phospho-JNK in the SL. Pretreatment with tempol or L-NAME was effective in protecting the noise-exposed animals from hearing loss, as well as in abolishing the noise-induced activation of the JNK signaling pathway. Interestingly, noise exposure caused a dramatic decrease in connexin26 level in the SL. This decrease was prevented by tempol or L-NAME. Taken together, our data suggest that noise-induced hearing loss is due at least in part to ROS / nitric oxide-mediated activation of the JNK pathway and down-regulation of connexin26 in the SL of mice.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

CONCLUSIONS: We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. OBJECTIVE: To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. METHODS: The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. RESULTS: The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Cellular characterization of Connexin26 and Connnexin30 expression in the cochlear lateral wall.

Gap junctions in the cochlear lateral wall, which consists of the stria vascularis (SV) and spiral ligament (SPL), are important for generating a positive endocochlear potential and high potassium concentration in the endolymph. In this study, the cellular expression of connexin 26 (Cx26) and Cx30 in the cochlear lateral wall of rats and guinea pigs was examined by immunofluorescent staining and confocal microscopy. Co-labeling for Kir4.1 revealed that the stria intermediate cells had extensive labeling for Cx26 and Cx30 with a leaf-like distribution. Cx26 and Cx30 also co-distributed hexagonally around the basal cells. However, no labeling was observed in the marginal cells. In the SPL, punctate Cx26 and Cx30 labeling was distributed along vertical lines orthogonal to the cochlear longitudinal direction. Intense labeling for Cx26 and Cx30 was found in type II fibrocytes in the spiral prominence and central region, but Cx26 labeling was absent in the middle region just beneath the SV, where only Cx30 labeling was observed. Outer sulcus (OS) cells and their root processes also exhibited intense labeling for Cx26 and Cx30. Neither Cx26 nor Cx30 was immunopositive in the hyaline region beneath the OS, in the subcentral region (type IV fibrocytes), or in the tension (type III) fibrocytes beneath the bone. Cx26 and Cx30 labeling was also absent in the lateral wall blood vessels. Thus, Cx26 and Cx30 have distinct cell-specific distributions in the SV and SPL, suggesting that they can form different pathways for transporting ions/nutrients in the cochlear lateral wall.