Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Techniques of celloidin removal from temporal bone sections.

OBJECTIVES: We sought to determine whether the technique of celloidin removal influences the results of immunostaining in celloidin-embedded cochleae. METHODS: We compared four protocols of celloidin removal, including those using clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide. By optimally fixing our tissue (perfused mice), and keeping constant the fixative type (formalin plus acetic acid), fixation time (25 hours), and decalcification time (ethylenediaminetetraacetic acid for 7 days), we determined whether the technique of celloidin removal influenced the immunostaining results. Six antibodies were used with each removal method: prostaglandin D synthase, sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase), aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament. RESULTS: Clove oil, acetone, and ether-alcohol resulted in incomplete removal of the celloidin, thereby negatively affecting the results of immunostaining. The methanol-sodium hydroxide method was effective in completely removing the celloidin; it produced the cleanest and most reproducible immunostaining for all six antibodies. CONCLUSIONS: Freshly prepared methanol saturated with sodium hydroxide and diluted 1:2 with methanol was the best solvent for removing celloidin from mouse temporal bone sections, resulting in consistent and reproducible immunostaining with the six antibodies tested.

Immunocytochemical traits of type IV fibrocytes and their possible relations to cochlear function and pathology.

One of the more consistent and least understood changes in the aging human cochlea is the progressive loss of fibrocytes within the spiral ligament. This report presents an animal model for type IV fibrocyte loss, along with immunocytochemical evidence that noise-induced loss of these cells may account for previously unexplained hearing losses. The remarkably low threshold for noise-induced loss of type IV fibrocytes, approximately 24 dB less than the threshold for adjacent hair cell destruction, may account for the prevalence of missing fibrocytes in humans. In mice, changes in the spectrum of traumatizing noise had little effect upon the site of loss of the fibrocytes, suggesting that the primary site of damage that induced the loss was the basal-most cochlear turn, a site expected to be damaged by all three noise bands. Type IV fibrocytes were found to immunostain for connective tissue growth factor (CTGF) and for transforming growth factor beta receptor 3, a receptor that is known to activate CTGF expression. Type IV fibrocytes lack immunostaining for adenosine triphosphatase and connexins that are key players in potassium ion uptake and transmission, which suggests that they play little, if any, role in potassium recycling from perilymphatic space to the endolymphatic space. Consequently, their loss probably does not directly reduce this process. Immunostaining for a receptor for CTGF, low-density-lipoprotein-related protein 1, indicated that CTGF acts as an autocrine and a paracrine agent within the cochlea. The lack of CTGF paracrine effects following noise-induced loss of type IV fibrocytes may account for previously unexplained hearing losses.

CARMA3 mediates lysophosphatidic acid-stimulated cytokine secretion by bronchial epithelial cells.

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.

Cooperative function of Tbx1 and Brn4 in the periotic mesenchyme is necessary for cochlea formation.

The T-box transcription factor TBX1 has been identified as the major gene responsible for the etiology of velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS). Conductive hearing loss occurs in a majority of patients with this syndrome, while sensorineural deafness has also been reported in some cases. Mutations in POU3F4/BRN4, a POU domain transcription factor, cause DFN3, an X-linked nonsyndromic form of deafness characterized by mixed conductive and sensorineural hearing loss. Inactivation of the murine orthologues of these genes causes similar defects to those seen in humans and has provided excellent models for the study of inner ear development. Tbx1 and Brn4 are expressed in the mesenchymal cells surrounding the otic vesicle and have been shown to play roles in cochlear outgrowth. Furthermore, expression of Brn4 is reduced in Tbx1 null mutants, suggesting a possible genetic interaction between these genes. To test whether Tbx1 and Brn4 function in a common pathway, mice mutant for both genes were generated and analyzed for inner ear defects. Brn4-;Tbx1+/- mutants displayed a significant reduction in the number of turns of the cochlea compared to Brn4- or Tbx1+/- mice. In addition, Brn4-;Tbx1+/- mice displayed structural defects in the apical cochlea indicative of Mondini dysplasia found in patients with either VCFS/DGS or DFN3. These data establish a genetic interaction between Tbx1 and Brn4 relevant to human disease and indicate a function of these genes in signaling from the periotic mesenchyme to the otic vesicle to direct proper coiling of the cochlear duct.

3p-- syndrome defines a hearing loss locus in 3p25.3.

Deletions affecting the terminal end of chromosome 3p result in a characteristic set of clinical features termed 3p-- syndrome. Bilateral, sensorineural hearing loss (SNHL) has been found in some but not all cases, suggesting the possibility that it is due to loss of a critical gene in band 3p25. To date, no genetic locus in this region has been shown to cause human hearing loss. However, the ATP2B2 gene is located in 3p25.3, and haploinsufficiency of the mouse homolog results in SNHL with similar severity. We compared auditory test results with fine deletion mapping in seven previously unreported 3p-- syndrome patients and identified a 1.38Mb region in 3p25.3 in which deletions were associated with moderate to severe, bilateral SNHL. This novel hearing loss locus contains 18 genes, including ATP2B2. ATP2B2 encodes the plasma membrane calcium pump PMCA2. We used immunohistochemistry in human cochlear sections to show that PMCA2 is located in the stereocilia of hair cells, suggesting its function in the auditory system is conserved between humans and mice. Although other genes in this region remain candidates, we conclude that haploinsufficiency of ATP2B2 is the most likely cause of SNHL in 3p-- syndrome.

Cochleosaccular dysplasia associated with a connexin 26 mutation in keratitis-ichthyosis-deafness syndrome.

OBJECTIVE: The objective of this study was to characterize the temporal bone phenotype associated with a mutation of GJB2 (encoding connexin 26). STUDY DESIGN: The authors conducted correlative clinical, molecular genetic, and postmortem histopathologic analysis. METHODS: The study subject was a male infant with keratitis-ichthyosis-deafness (KID) syndrome. We performed a nucleotide sequence analysis of GJB2 and a histopathologic analysis of the temporal bones. RESULTS: The subject was heterozygous for G45E, a previously reported KID syndrome mutation of GJB2. The primary inner ear abnormality was dysplasia of the cochlear and saccular neuroepithelium. CONCLUSIONS: GJB2 mutations can cause deafness in KID syndrome, and possibly in other GJB2 mutant phenotypes, by disrupting cochlear differentiation.

Osteoprotegrin knockout mice demonstrate abnormal remodeling of the otic capsule and progressive hearing loss.

OBJECTIVES: The otic capsule, when compared with other bones in the body, is unique in that it undergoes no significant remodeling of bone after development. We previously demonstrated that osteoprotegerin (OPG), which inhibits formation and function of osteoclasts, is produced at high levels in the inner ear of normal mice and secreted into the perilymph from where it diffuses into the surrounding otic capsule bone through a lacunocanalicular system. To test our hypothesis that the high level of OPG may be important in the inhibition of otic capsule remodeling, we studied the light microscopic histology of the otic capsule in OPG knockout mice for evidence of abnormal remodeling of bone. We also tested the hearing in OPG knockout mice to determine whether OPG and its influence on surrounding bone is important for auditory function. METHODS: Temporal bone histopathology and pathophysiology were compared in homozygous OPG knockout mice and C57BL/6 (B6) mice, the background strain for the knockouts. Auditory function in age-matched animals from each group was evaluated at approximately 4-week intervals from 8 to 21 weeks using frequency-specific auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE). After each of the last three evaluations, the cochleae from one mouse of each group were harvested, processed, and examined by light microscopy. RESULTS: Osteoprotegerin knockout mice demonstrated abnormal remodeling of bone within the otic capsule with multiple foci showing osteoclastic bone resorption and formation of new bone. Such changes were not seen in the age-matched B6 controls. The active bone remodeling process in the knockout animals showed many similarities to otosclerosis seen in human temporal bones. Over the time period that we monitored, auditory function was significantly and progressively compromised in the knockout animals relative to B6 controls. At the earliest age of test (8 wk), the loss was apparent as a mild, high-frequency reduction in sensitivity by ABR. In contrast, DPOAE losses in the knockouts were substantial even at 8 weeks, and by 21 weeks, these losses exceeded our equipment limits. Results of ABR testing showed hearing sensitivity changes in the animals of the background strain were confined largely to the high frequencies, whereas OPG knockouts demonstrated substantial low-frequency shifts in addition to those at high frequencies. CONCLUSIONS: The histopathological and pathophysiological findings in OPG knockout mice support the hypothesis that OPG is important in the inhibition of bone remodeling within the otic capsule and the maintenance of normal auditory function. This mouse may provide a valuable animal model of human otosclerosis.

Distribution of type IV collagen in the cochlea in Alport syndrome.

OBJECTIVE: To determine the distribution of alpha1, alpha3, and alpha5 chains of type IV collagen in the cochlea in Alport syndrome. DESIGN: Case-control study. PATIENTS: Two patients with sensorineural hearing loss due to Alport syndrome. Both patients had known mutations in the COL4A5 gene. MAIN OUTCOME MEASURES: Immunostaining was used to study the distribution of type IV collagen (alpha1, alpha3, and alpha5 chains) within the cochlea. Immunostaining was also performed in the cochlear tissues of an unaffected individual used as a control. RESULTS: In the control ear, alpha1 staining was observed in the basement membrane overlying the basilar membrane, in the basement membrane of cochlear blood vessels and Schwann cells, and within the spiral limbus. In the control ear, we also observed strong staining for alpha3 and alpha5 chains in the basement membrane overlying the basilar membrane and within the spiral ligament. In both cases with Alport syndrome, no immunostaining was observed for alpha3 or alpha5 chains within the cochlea, whereas alpha1 staining was present in locations similar to that seen in the control ear. CONCLUSIONS: The results indicate that isotype switching does not occur within the cochlea in Alport syndrome. The results are also consistent with the hypothesis that the sensorineural hearing loss in Alport syndrome may be due to alterations in cochlear micromechanics and/or dysfunction of the spiral ligament.

Osteoprotegerin in the inner ear may inhibit bone remodeling in the otic capsule.

OBJECTIVES: To elucidate factors that may be responsible for the inhibition of remodeling of bone within the otic capsule. METHODS: Expression of osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (RANK), and RANK ligand (RANKL) were assayed in samples of bone obtained from the otic capsule, calvarium, and femur, and from the soft tissue within the cochlea using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) in mice. Immunostaining was used for histologic localization of the gene products. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the amount of OPG within perilymph, serum, and cerebrospinal fluid. The micro-anatomy of the interface between the otic capsule and the fluid spaces of the cochlea was investigated by brightfield and phase-contrast microscopy and by three-dimensional reconstruction in the mouse and human. RESULTS: OPG, a powerful inhibitor of bone remodeling, was expressed at extremely high levels within the soft tissue of the cochlea and was present in the perilymph at very high concentrations. The OPG produced within the inner ear may diffuse into the surrounding otic capsule, where it may be responsible for inhibition of bone turnover. Our anatomic studies revealed an extensive system of interconnected canaliculi within the otic capsule that had direct openings into the fluid spaces of the inner ear, thus providing a possible anatomic route for the diffusion of OPG from the inner ear into the surrounding bone. CONCLUSION: OPG, a potent inhibitor of osteoclast formation and function, is expressed at high levels within the inner ear and is secreted into the perilymph and the surrounding bone and may serve to inhibit active bone remodeling within the otic capsule, especially immediately adjacent to the cochlea. By this means, the cochlear soft tissue may control the nature of the surrounding petrous bone.

Survival of adult spiral ganglion neurons requires erbB receptor signaling in the inner ear.

Degeneration of cochlear sensory neurons is an important cause of hearing loss, but the mechanisms that maintain the survival of adult cochlear sensory neurons are not clearly defined. We now provide evidence implicating the neuregulin (NRG)-erbB receptor signaling pathway in this process. We found that NRG1 is expressed by spiral ganglion neurons (SGNs), whereas erbB2 and erbB3 are expressed by supporting cells of the organ of Corti, suggesting that these molecules mediate interactions between these cells. Transgenic mice in which erbB signaling in adult supporting cells is disrupted by expression of a dominant-negative erbB receptor show severe hearing loss and 80% postnatal loss of type-I SGNs without concomitant loss of the sensory cells that they contact. Quantitative RT-PCR analysis of neurotrophic factor expression shows a specific downregulation in expression of neurotrophin-3 (NT3) in the transgenic cochleas before the onset of neuronal death. Because NT3 is critical for survival of type I SGNs during development, these results suggest that it plays similar roles in the adult. Together, the data indicate that adult cochlear supporting cells provide critical trophic support to the neurons, that survival of postnatal cochlear sensory neurons depends on reciprocal interactions between neurons and supporting cells, and that these interactions are mediated by NRG and neurotrophins.

Clinical implications of inflammatory cytokines in the cochlea: a technical note.

HYPOTHESIS: Establishing the presence of critical cellular stress response components in cochlear cells can contribute to a better understanding of cochlear cell biology and pathology. BACKGROUND: Inflammatory cytokines and related proteins play critical roles in a variety of cellular processes, but to date, little is known about the identity and cellular localization of these compounds within the ear. Cytokines are autocrine, which means that cells that produce them have corresponding surface receptors. The presence of these receptors makes the cells vulnerable to disruption by circulating or local sources of cytokines and related ligands. Such disruptions may explain previously poorly understood cochlear pathologies. METHODS: The messenger RNA precursors that encode inflammatory cytokines and related proteins are identified in the inner ear by using reverse transcriptase-polymerase chain reaction. Cochlear cells that contain the corresponding proteins are identified by immunostaining. RESULTS: Messenger RNA for interleukin-1alpha, tumor necrosis factor alpha, NFkappaB P65 and P50, and IkappaBalpha was found in cochlear tissue. Cells that immunostained most conspicuously for cytokine production are Type I fibrocytes and root cells located within the spiral ligament. CONCLUSION: Production of inflammatory cytokines by the above-mentioned cells indicates that they are vulnerable to disruption by extra-cochlear sources of cytokines and associated ligands. These cells play critical roles in cochlear function, and their disruption could induce hearing loss. These findings suggest that systemic or local production of inflammatory ligands may play roles in a number of causes of deafness, including immune mediated hearing loss, sudden hearing loss, and sensorineural hearing loss associated with otosclerosis, otitis media, and bacterial meningitis.