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PubMed2005

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection.

CONCLUSIONS: In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. OBJECTIVE: To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. MATERIAL AND METHODS: This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. RESULTS: mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.

DNA Mutational Analysis↗
PubMed1999

Expression of membrane-bound and cytosolic guanylyl cyclases in the rat inner ear.

Membrane-bound guanylyl cyclases (GCs) are peptide hormone receptors whereas the cytosolic isoforms are receptors for nitric oxide. In the inner ear, the membrane-bound GCs may be involved in the regulation of fluid homeostasis and the cytosolic forms possibly play a role in signal processing and regulation of local blood flow. In this comprehensive study, we examined, qualitatively and quantitatively, the transcription pattern of all known GC isoforms in the inner ear from rat by RT-PCR. The tissues used were endolymphatic sac, stria vascularis, organ of Corti, organ of Corti outer hair cells, cochlear nerve, Reissner's membrane, vestibular dark cells, and vestibular sensory cells. We show that multiple particulate (GC-A, GC-B, GC-D, GC-E, GC-F and GC-G) and several subunits of the heterodimeric cytosolic GCs (alpha1, alpha2, beta1 and beta2) are expressed, albeit at highly different levels. GC-C was not found. GC-A and the soluble subunits alpha1 and beta1 were transcribed ubiquitously. GC-B was present in all tissues except stria vascularis, which contained GC-A and traces of GC-E and GC-G. GC-B was by far the predominant membrane-bound isoform in the organ of Corti (86%), Reissner's membrane (75%) and the vestibulum (80%). Surprisingly, GC-E, a retinal isoform, was detected in significant amounts in the cochlear nerve (8%) and in the organ of Corti (4%). Although the cytosolic GC is a heterodimer composed of an alpha and a beta subunit, the mRNA transcription of these subunits was not stoichiometric. Particularly in the vestibulum, the transcription of the beta1 subunits was at least four-fold higher than of the alpha1 subunit. The data are compatible with earlier suggestions that membrane receptor GCs may be involved in the control of inner ear electrolyte and fluid composition whereas NO-stimulated GC isoforms mainly participate in the regulation of blood flow and supporting cell physiology.

Animals↗
PubMed1976

Experimental viral infections of the inner ear. I. Acute infections of the newborn hamster labyrinth.

Acute viral infections of the inner ear were produced in neonatal hamsters. Viruses were inoculated percutaneously through the temporal cartilage into the endolymphatic and perilymphatic spaces of the labyrinth or were inoculated intracerebrally to reach the perilymphatic spaces via the cochlear aqueduct. Selective vulnerability of inner ear structures was demonstrated using a variety of viruses. Influenza virus infected only the mesenchymal cells of the perilymphatic channels of the cochlea; mumps virus infected principally endolymphatic structures; herpes simplex virus infected primarily the sensory cells of the labyrinth; and rubeola and vaccinia viruses infected both perilymphatic and endolymphatic cells.

Acute Disease↗
PubMed1996

Localization of the CD15-epitope in the inner ear of the developing mouse.

The expression of the 3-fucosyl-N-acetyl-lactosamine (CD15) epitope during the embryonic development of the mouse inner ear has been immunohistochemically studied on paraffin sections from day 8.5 to day 19 of gestation. Consecutive steps of morphological development are accompanied by up- and down-regulation of CD15 expression and changing cell patterns. Expression levels are reduced once structural maturation has been accomplished. Since CD15 has been described as a determinant that is expressed during critical developmental steps, it is speculated that the changes in the epitope expression in the inner ear reflect relevant stages of differentiation. This system may thus serve as a model for understanding inner ear development.

Animals↗
PubMed2004

Partially overlapping expression of Gata2 and Gata3 during inner ear development.

Gata2 and Gata3 belong to the Gata family of transcription factors in vertebrates that bind to a consensus "GATA" DNA sequence. The Gata3 gene is one of the earliest markers for the developing mouse inner ear. Ear morphogenesis is blocked in Gata3-deficient embryos, whereas nothing was known of the role of Gata2 in mouse inner ear. Here, we have compared the expression patterns of Gata2 and Gata3 during normal inner ear development and investigated their relationship in mice where either Gata3 or Gata2 has been inactivated. The expression of the two Gata genes is highly overlapping at embryonic day (E)10.5 but becomes increasingly distinct later. Whereas Gata2 is predominantly expressed in the dorsal vestibular system, Gata3 was detected mainly in the ventral cochlear duct and ganglion. No phenotypic abnormalities were observed in the inner ear of Gata2-/- embryos before lethality at E10.5 and Gata3 expression was unchanged. In contrast, a delay and strong reduction of Gata2 expression was detected in Gata3-/- otic epithelium.

Animals↗
PubMed1998

Identification of mRNA transcripts and immunohistochemical localization of Na/H exchanger isoforms in gerbil inner ear.

Recent physiological and pharmacological studies have implicated involvement of the Na/H exchanger (NHE) in regulating inner ear ion homeostasis, but the cellular distribution of this membrane transporter remains unknown. Here reverse transcription and the polymerase chain reaction (RT-PCR) were employed to screen adult gerbil inner ears for mRNA transcripts encoding the four best characterized isoforms of NHE. PCR products spanning selected segments of NHE mRNAs were cloned and sequenced. The putative housekeeping gene NHE-1 was found to be expressed and the 459 bp product shared 98.7% amino acid homology with rat sequence. NHE-2, NHE-3 and NHE-4 cDNA transcripts likewise were detected and the PCR products shared 100, 99.4 and 88.9% amino acid homology, respectively, with their rat counterparts. In addition, the cellular distribution of NHE isoforms 1 and 3 was mapped in the gerbil inner ear by immunostaining with polyclonal antisera against rat antigens. In the cochlea, the antiserum against NHE-1 reacted strongly at the basolateral membrane of strial marginal cells as well as with inner and outer hair cells and spiral ganglion neurons. Less intense staining for NHE-1 was present in subpopulations of fibrocytes in the spiral limbus and in inferior and superior areas of the spiral ligament. In the vestibular system dark and transitional cells expressed abundant NHE-1 as did hair cells and vestibular ganglia neurons. Immunostaining with the antiserum against NHE-3 was limited to the apical surface of marginal cells in the stria vascularis. Based on these data, NHE-1 likely functions primarily to maintain intracellular pH levels in cells where it is found in high abundance. NHE-3, on the other hand, possibly participates in the vectorial transcellular movement of Na+ by strial marginal cells thus helping to maintain the extremely low Na+ level in cochlear endolymph.

Animals↗
PubMed1999

Growth factor therapy to the damaged inner ear: clinical prospects.

Most hearing loss results from lesions of the sensory cells and/or of the neurons of the auditory part of the inner ear. There is currently no treatment able to stop the progression of a hearing loss or to restore a lost auditory function. In this paper, we review the progress which has been made with respect to the regeneration and the protection of the hair cells and of the auditory neurons in the cochlea. In particular, we emphasize the control by growth factors of the protection/repair mechanisms of the neurosensory structures within the inner ear, in the prospect of the possible clinical use of these molecules. Finally, we discuss the different approaches which can be used to deliver these therapeutic agents to the inner ear.

Animals↗
PubMed1988

Calbindin (CaBP 28 kDa) appearance and distribution during development of the mouse inner ear.

Previous reports of the distribution of calbindin, a 28 kDa vitamin D-induced calcium-binding protein, in the mammalian peripheral vestibular system postulated that this protein was involved in the calcium-dependent mechanisms occurring in the hair cells and ganglion cells. In this study, we examined the possibility of a relationship between the presence of calbindin and neurotransmission by comparing calbindin appearance in the inner ear to the sequence of synaptogenesis. Calbindin distribution was studied by immunocytochemistry, in the developing mouse inner ear from gestational day 12 to postnatal day 40. During the early development, calbindin was localized in non-neuronal structures: Kolliker's organ, spiral limbus and crista supporting cells; and in cochlear and vestibular ganglion neurons and sensory cells. At later stages and in the adult, no reactivity was observed in the non-neuronal cell populations and only certain sensory and nerve cells remained stained: inner hair cells, outer hair cells, vestibular hair cells of the apex of the cristae and of the striola in the maculae, all Corti's ganglion neurons and some vestibular ganglion neurons. The sequence of appearance of calbindin immunoreactivity in the sensory and nerve cells was not completely parallel to the maturation sequence of the inner ear, especially synaptogenesis. The dual distribution of calbindin during development and its expression in specific sensory and nerve cells opens new perspectives on its role in the inner ear.

Aging↗
PubMed1990

Inner ear autoantibodies in patients with rapidly progressive sensorineural hearing loss.

Recognition of immune-mediated sensorineural deafness that responds to immunosuppressive therapy has led to a search for a diagnostic assay to identify inner ear autoantibodies. Without a confirmed diagnosis of autoimmune disease, many patients have undergone inappropriate immunosuppressive treatment or developed irreversible inner ear damage. Serum from patients with progressive sensorineural hearing loss (n = 54), ulcerative colitis (N = 5), normal controls (N = 14), and animals with experimental autoimmune sensorineural hearing loss (EASNHL) were analyzed by Western blot against fresh bovine inner ear antigen preparations. The hearing loss group (19 [35%]) showed a single-or double-band migrating at 68,000 molecular weight (MW), differing from the normal group (1 of 14 [7%]) which showed a similar band (P = .031). Upon analysis by two-dimensional gel electrophoresis both the EASNHL guinea pigs and a patient reacted against identical components of inner ear antigen. These results suggest an autoimmune basis for disease in patients reacting against the 68,000 MW antigen.

Adult↗
PubMed1992

Developmentally-regulated coexpression of vimentin and cytokeratins in the rat inner ear.

In the present study the expression of vimentin-type intermediate filament proteins and cytokeratins was studied immunohistochemically in the rat inner ear from 12 days postconception up to 40 days after birth. With the use of a broad spectrum monoclonal antibody, cytokeratin expression was found to be present in the whole epithelial lining except for the sensory cells, throughout all the developmental stages examined. Vimentin was detected in the mesenchymal cells, the mesenchyme-derived tissues and the intermediate cells of the stria vascularis, confirming their origin from melanocyte precursor cells. In addition, the coexpression of vimentin and cytokeratins in the epithelial lining of the membranous inner ear was found to be developmentally regulated. During the final stages of differentiation, vimentin expression disappeared from the majority of the cell types. In the mature cochlea the coexpression of vimentin and cytokeratins was still found in the supporting cells of the organ of Corti, in the cells of Claudius and in external sulcus cells. As far as we could conclude from this study, the sensory cells showed only vimentin expression but not cytokeratin expression. A possible relationship between vimentin expression in adult epithelial cells of the inner ear and a specialised function of these cells is discussed.

Animals↗
PubMed1989

[The lymphocyte transformation test for detecting immunologic inner ear deafness].

The basis and performance of the lymphocyte transformation test for proving an immunologically induced inner ear hearing loss are described and documented. In 6 out of 14 patients with inner ear hearing loss of unknown origin an immunological etiology could be elaborated by using this test. The relevance of this test as well and its further application by using collagen type II as antigen are discussed.

Adolescent↗
PubMed2003

Gap junctions in the inner ear: comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals.

The distribution and size of gap junctions (GJ) in the sensory epithelia of the inner ear have been examined in a reptile (gecko), birds (chicken and owl), and mammals (mouse, guinea pig, gerbil, and bat), and the connexin composition of GJs in the mammalian inner ear has been assessed. Freeze fracture revealed a common pattern of GJ distribution in auditory and vestibular sensory epithelia in the different vertebrate classes. In all these tissues, GJs are numerous, often occupying more than 25% of the plasma membrane area of supporting cells and sometimes composed of more than 100,000 channels. Screening for 12 members of the connexin family in the mammalian inner ear by RT-PCR, Western blotting, and immunohistochemistry revealed four connexin isotypes, cx26, cx30, cx31, and cx43, in the cochlea and three, cx26, cx30, and cx43, in the vestibular organs. With antibodies characterised for their specificity, cx26 and cx30 colocalised in supporting cells of the organ of Corti, in the basal cell region of the stria vascularis, and in type 1 fibrocytes of the spiral ligament. No other connexin was detected in these regions. Cx31 was localised among type 2 fibrocytes below the spiral prominence, a region where cx30 was not expressed and cx26 expression appeared to be low. Cx43 was detected only in the region of "tension fibrocytes" lining the inner aspect of the otic capsule. This suggests separate functional compartments in the cochlea. In addition to cx26 and cx30, cx43 was detected in supporting cells of the vestibular sensory epithelia. Where cx26 and cx30 were colocalised, double immunogold labelling of thin sections showed both cx26 and cx30 evenly distributed in individual GJ plaques, a pattern consistent with the presence of heteromeric connexons. Coimmunoprecipitation of cochlear membrane proteins solubilised with a procedure that preserves the oligomeric structure of connexons confirmed the presence of heteromeric cx26/cx30 connexons. Heteromeric cx26/cx30 connexons may be unique to the inner ear, which could be one factor underlying the non-syndromic character of the deafness caused by mutations in cx26.

Animals↗
PubMed1997

Cochlear implants in children with congenital inner ear malformations.

OBJECTIVE: To describe clinical experiences with multichannel cochlear implantation in children with inner ear malformations, including surgical indications and techniques, imaging findings, and outcomes. DESIGN: A retrospective review of a series of 10 consecutive cases with a mean follow-up of 29 months, as well as a review of the literature. SETTING: Academic referral center. SUBJECTS: Ten children who underwent multichannel cochlear implantation for inner ear malformations. High-resolution computed tomographic scans demonstrated a common cavity deformity in 3, an incomplete cochlear partition in 4, and an enlarged vestibule in 1. Two had membranous anomalies as indicated by cerebrospinal fluid gushers at surgery, but the results of imaging were normal. INTERVENTION: All subjects received multichannel cochlear implants. Two subjects underwent mastoid obliteration at the time of implantation owing to preoperative recurrent meningitis or chronic otitis media with episodes of clinical mastoiditis. MAIN OUTCOME MEASURES: The 10 subjects were evaluated for electrode insertion and stability and auditory function for up to 7 years. RESULTS: All 22 electrodes are functional in each child with an incomplete partition, an enlarged vestibule, or a membranous anomaly. Of 3 subjects with common cavities, 2 had full insertion of electrodes and 1 had 16 electrodes inserted. All subjects had speech awareness thresholds detected at 25 dB or better. Three (75%) of the 4 subjects with at least 30 months of experience, including 1 subject with a common cavity, have developed open-set word recognition. CONCLUSIONS: Electrode insertion and hearing results in children with an incomplete partition, an enlarged vestibule, or a membranous anomaly are similar to those in children with normal cochleas. Specific surgical techniques are effective for children with a common cavity, and the results are less certain. Cerebrospinal fluid gushers were encountered frequently but were not difficult to control.

Adolescent↗
PubMed2002

Atrial natriuretic peptide receptor upregulation in the rat inner ear.

The purpose of this study was to further examine whether fluid homeostasis in the endolymphatic system could be regulated by a locally effective paracrine system involving atrial natriuretic peptides (ANPs) and their receptors. We assessed the biologic activity of the 3 ANP receptors (ANP-A, ANP-B, ANP-C) in the rat inner ear by measuring receptor upregulation after inner ear administration of ANPs. After appropriate anesthesia, female Lewis rats were injected with ANP via the round window. The animals were sacrificed 24 hours later, and RNA was isolated for reverse transcription-polymerase chain reaction (RT-PCR). Electrophoresis of RT-PCR products showed the presence of all 3 ANP receptor genes in both injected and control animals. Gene expression was significantly higher 24 hours after injection. These findings demonstrate that ANP receptors in the inner ear can be upregulated after injection of ANPs.

Animals↗
PubMed2003

Pluripotent stem cells from the adult mouse inner ear.

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.

Animals↗
PubMedDate not supplied

Electron probe determination of relative ion distribution in the inner ear.

The relative amounts of potassium and chlorine present in various parts of the inner ear were studied by using an energy dispersive X-ray spectrometer in conjunction with a scanning electron microscope. Whereas the amount of chlorine was high in all compartments investigated, potassium was high in endolymphatic spaces and low in perilymphatic spaces. High contents of potassium and chlorine were also found in the tectorial membrane, in the inner sulcus and in the cupula of the semicircular canal. It is concluded that the tectorial membrane and the cupula do not present a barrier to ions and that therefore the sensory hairs in the inner ear are exposed to the ionic environment provided by the endolymph.

Animals↗
PubMed1999

Characterization of G-protein betagamma expression in inner ear.

Heterotrimeric guanine nucleotide binding proteins (G-proteins) are composed of a diverse set of alpha, beta, and gamma subunits, which couple cell surface receptors to intracellular effectors, such as adenylyl cyclase, phospholipase Cbeta, and ion channels. Both the Galpha and the Gbetagamma dimers mediate effector activity and are believed to contribute to the complexity of the signaling pathway. Molecular and immunocytochemical techniques were employed to determine diversity of Gbeta and Ggamma subunit expression in the murine inner ear. PCR-based assessment of lambdaZAP unidirectional cDNA libraries, representing the cochlea and inner ear hair cells, indicated all five known Gbeta subunits were present in the cochlea, while only a subset of Ggamma isoforms were found. New or novel G-protein beta and gamma subunits were not detected. cDNAs representing Gbeta1-4 and Ggamma2, Ggamma3, Ggamma5, Ggamma8olf subunit transcripts were isolated. In addition, cDNAs corresponding to the Gbeta5 and Ggamma11 isoforms exhibited restricted expression to inner and outer hair cells, respectively. Antisera specific for Gbeta3, Gbeta4, Ggamma3, Ggamma5 and Ggamma11 stained spiral ganglion and neurosensory hair cells. A unique finding was the variable topological distribution of Ggamma3 in the spiral ganglion cells along the cochlear axis. Collectively, our results demonstrate a complementary as well as differential distribution pattern for Gbeta and Ggamma isoforms exists in the inner ear. The co-localization of various G-protein isoforms within the same cell type suggests specific combinatorial Gbeta and Ggamma subunit associations may preferentially be formed. Thus, the detection of multiple subunits presumably reflects the extent of the functional diversity of inner ear signaling pathways and should provide specificity of G-protein mediated pathways.

Animals↗
PubMedDate not supplied

Effect of anti-inflammatory drugs on collagen-induced autoimmune inner ear disease.

Animals that had collagen-induced autoimmune inner ear disease were treated with anti-inflammatory drugs: Solu-Medrol (steroid), sulindac (nonsteroid), or a combination of both. Temporal bones from drug-treated animals were examined for histopathologic and immunohistochemical changes, and sera were examined for levels of circulating antibody to type II collagen. Therapy was beneficial to the animals whether the drugs were administered alone or in combination; however, fewer lesions were observed in animals given either drug alone. Further, animals treated with steroid alone showed the least amount of inner ear damage. Immunohistochemical changes and serum levels of antibodies against type II collagen correlated with the pathologic changes. These findings suggest that both steroidal and nonsteroidal anti-inflammatory drugs may have potential therapeutic value in the treatment of autoimmune ear disease.

Animals↗