Delta/Notch-Like EGF-Related Receptor (DNER) is Expressed in Hair Cells and Neurons in the Developing and Adult Mouse Inner Ear

The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER−/− mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.     

Immunolocalization of tenascin in the chinchilla inner ear

Tenascin was immunolocalized in the chinchilla cochlea and vestibular system to better understand the functional morphology of the inner ear. Inner ear tissues were fixed with acetone, decalcified and cryosectioned. Indirect immunofluorescence, using antibodies directed against human tenascin epitopes, were used to detect tenascin. As a positive control, tenascin immunoreactivity was found in kidney, cortical mesangial cells and the extracellular matrix of glomeruli and medullary tubule interstitial spaces, concurring with previously reported results. In the cochlea, tenascin immunoreactivity was present in osteocytes, the mesothelial cells underlying the basilar membrane (BM) and within the fibrous matrix of the BM. Greater reactivity was observed in the mesothelial cells than in the fibrous matrix of the BM. In the vestibular system, tenascin immunoreactivity formed a diffuse band directly beneath the basal lamina of the ampullary and otoconial organs. Tenascin immunoreactivity was also observed in cup-shaped regions between the type I vestibular hair cells and their surrounding VIII nerve calyces in the ampullary and otoconial organs. This is the first report of the anatomical distribution of tenascin in the adult, mammalian inner ear, other than our previously published abstract P.A. Santi and D. Swartz, Soc. Neurosci. Abstr. 23 (1997) 731.     

TAK1 Expression in the Cochlea: A Specific Marker for Adult Supporting Cells

Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β and BMP signaling, TAK1 mediates the activation of the c-Jun N-terminal kinase signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt/Fz signaling in mesenchymal stem cells. Expression of TAK1 in the cochlea has not been defined. Data mining of previously published murine cochlear gene expression databases indicated that TAK1, along with TAK1 interacting proteins 1 (TAB1), and 2 (TAB2), is expressed in the developing and adult cochlea. The expression of TAK1 in the developing cochlea was confirmed using RT-PCR and immunohistochemistry. Immunolabeling of TAK1 in embryonic, neonatal, and mature cochleas via DAB chromogenic and fluorescent immunohistochemistry indicated that TAK1 is broadly expressed in both the developing otocyst and periotic mesenchyme at E12.5 but becomes more restricted to specific types of supporting cells as the organ of Corti matures. By P1, TAK1 immunolabeling is found in cells of the stria vascularis, hair cells, supporting cells, and Kölliker’s organ. By P16, TAK1 labeling is limited to cochlear supporting cells. In the adult cochlea, TAK1 immunostaining is only present in the cytoplasm of Deiters’ cells, pillar cells, inner phalangeal cells, and inner border cells, with no expression in any other cochlear cell types. While the role of TAK1 in the inner ear is unclear, TAK1 expression may be used as a novel marker for specific sub-populations of supporting cells.     

Efficient transfer of embryonic stem cells into the cochlea via a non-invasive vestibular route

CONCLUSION: Cell transplantation into the utriculus provides an efficient and non-invasive route to introduce embryonic stem (ES) cells into the vestibular and cochlear portions of the inner ear. OBJECTIVE: The transfer of stem cells into the inner ear for therapeutic purposes is an important approach to cure damage to the cochlea and vestibulum. A key issue is to provide an entry point for cell transplants into the inner ear that does not affect its physiologic functions. The aim of this study was to examine the feasibility of transferring ES cells into the inner ear via the utriculus. MATERIALS AND METHODS: ES cells were injected via utriculostomy into the mouse inner ear. The distribution of the injected cells was determined using a beta-galactosidase marker gene expressed by the ES cells. RESULTS: Injected ES cells were found within the perilymph of the scala tympani and vestibuli. Moreover, ES cells were detected close to the cochlear sensory epithelium and spiral limbus.     

Distribution of frequenin in the mouse inner ear during development, comparison with other calcium-binding proteins and synaptophysin

Frequenin is a calcium-binding protein previously implicated in the regulation of neurotransmission. We report its immunocytochemical detection in the mouse inner ear, in the adult, and during embryonic (E) and postnatal (P) development. The distribution of frequenin was compared with those of other calcium-binding proteins (calbindin, calretinin, parvalbumin) and synaptophysin. In the adult mouse inner ear, frequenin immunostaining was observed in the afferent neuronal systems (vestibular and cochlear neurons, their processes and endings) and in the vestibular and cochlear efferent nerve terminals. Frequenin colocalized with synaptophysin in well characterized presynaptic compartments, such as the vestibular and cochlear efferent endings, and in putative presynaptic compartments, such as the apical part of the vestibular calyces. Frequenin was not found in vestibular hair cells and in cochlear inner and outer hair cells. During development, frequenin immunoreactivity was first detected on E11 in the neurons of the statoacoustic ganglion. On E14, frequenin was detected in the afferent neurites innervating the vestibular sensory epithelium, along with synaptophysin. On E16, frequenin was detected in the afferent neurites below the inner hair cells in the organ of Corti. The timing of frequenin detection in vestibular and cochlear afferent neurites was consistent with their sequences of maturation, and was earlier than synaptogenesis. Thus in the inner ear, frequenin is a very early marker of differentiated and growing neurons and is present in presynaptic and postsynaptic compartments.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

CONCLUSION: Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. OBJECTIVES: At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. MATERIALS AND METHODS: Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. RESULTS: Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

Conclusion. Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. Objectives. At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. Materials and methods. Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. Results. Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

The potential use of stem cells for cochlear repair

In light of the currently defined characteristics of stem cells, a re-evaluation of hair cell regeneration in birds suggests that there may be a stem cell population located in the inner ear. It is yet to be determined if the mammalian cochlea contains stem cells, but the presence of a mammalian vestibular stem cell population would not appear to be out of the realm of possibility. This paper reviews the latest advances in stem cell biology and suggests that stem cells may be an appropriate biological tool to be used for cochlear repair. The potential use of several types of stem cells, including embryonic, neural and hematopoietic stem cells, as agents for cochlear repair is examined.     

In vivo adenoviral transduction of the neonatal rat cochlea and middle ear

Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a beta-galactosidase (beta-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of beta-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of beta-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of beta-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express beta-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.     

Comparison of the vascular innervation of the rat cochlea and vestibular system

In order to gain a better understanding of the neuronal and local control of inner ear blood flow, the vascular innervation to the rat cochlea and vestibular system was examined. Specimens were removed in toto beginning at the basilar artery extending to the anterior inferior cerebellar artery, labyrinthine artery, common cochlear artery, modiolar artery and anterior vestibular artery. When possible the vessels were dissected in continuity through the cribrose area. The vestibular endorgans were also removed. Specimens were examined using immunohistochemical techniques for the presence of vasoactive intestinal peptide, neuronal nitric oxide synthase, neuropeptide-Y, substance P and calcitonin gene related peptide. Results show that the vasculature to the cochlea and vestibular portion of the inner ear receive similar types of nonadrenergic innervation, that within the vestibular endorgans, only CGRP and SP were found in the neuroepithelium or in association with vessels, and that within the vestibular system, the majority of the vascular innervation appears to stop at or near the cribrose area. In the cochlea however, it extends to include the radiating arterioles. These findings suggest that cochlear blood flow is under finer control and that neuronally induced changes in blood flow may have a more global effect in the vestibular periphery.     

A novel vestibular approach for gene transfer into the inner ear

The aim of gene transfer to the cochlea and vestibular organ is to protect the inner ear from different disorders. Although various vectors for gene delivery have been used with some success, there remains a need for a reliable transfer of genes into the inner ear without damaging cochlear function. Here, we have tested a novel application method for gene transfer into the rat inner ear in vivo using herpes simplex virus type-1(HSV-1)-based amplicon vectors. Our goal was to find an entry route into the inner ear that leaves its function intact. Besides other non-invasive and invasive application techniques, we applied the viral vector via injection through a small opening of the utriculus. Using this method, efficient -galactosidase reporter gene expression was achieved in nearly all neurons in the vestibulum and cochlea, without functional hearing deficits. At the time point of maximal expression 5 days after injection, -galactosidase activity was also observed in axonal fibres and synaptic endings close to inner and outer hair cells. Our results thus describe an efficient and reliable protocol for short-term expression of potential therapeutic genes in the neuronal compartment of the inner ear.     

Migration of R28 Retinal Precursor Cells into Cochlear and Vestibular Organs

Introduction Permanent cochlear and vestibular damage can becaused by a number of factors including: ototoxic drugs,acoustic trauma, genetic disorders, aging, anoxia, viralinfection, bacterial infection and more (Ding and Salvi,2005; Ding et al, 1999, Sal…     

内耳畸形聋儿人工耳蜗植入术常见类型及手术并发症

目的 探讨内耳畸形聋儿实施人工耳蜗植入术时常见的类型及并发症。方法 回顾性分析电子耳蜗植入术病历资料170例,对其中的32例双侧内耳畸形患者加以畸形类型及手术并发症总结。结果 ①人工耳蜗植入患儿内耳畸形所占比例(32/170,18.8%)明显高于其他文献报道;②32例内耳畸形中,大前庭导水管23例(占全部畸形数71.3%),大前庭导水管伴其他类型畸形者5例(并发Mondini畸形4例,并发外半规管未发育1例),Mondini畸形2例,Mondini畸形并发外半规管未发育前庭腔扩大1例,耳蜗CT影像疑似为“三叉”无法分类1例;③术中发生严重井喷3例(耳蜗CT影像疑似为“三叉”畸形、Mondini畸形并发外半规管未发育前庭腔扩大1例,及大前庭导水管并发Mondini畸形1例);④耳蜗影像疑似为“三叉”患者,术中发生严重井喷,电极植入困难,4个电极不能植入,术后听力未改善,半年后行对侧耳植入成功;⑤Mondini畸形并发外半规管未发育前庭腔扩大患儿术后半年并发脑脊液耳鼻漏、反复脑膜炎发作,术后1年行手术探查,后治愈。结论 ①人工耳蜗植入常见的内耳畸形包括,大前庭导水管综合征及其相伴发或单发的各类内耳畸形;②内耳畸形非人工耳蜗植入术的绝对禁忌证,但术中严重井喷多见,电极植入不完全多见,术后脑脊液耳鼻漏并发脑膜炎也多发生于畸形耳蜗,术前详细的影像学检查可以对各类畸形进行详细分类,并在术前对手术难度有充分的准备,可以减少相关并发症的发生。     

Gentamicin tympanoclysis: effects on the labyrinthine sensory cells

OBJECTIVES: The objective of the study was to determine whether a selective vestibular hair cell toxicity with sparing of the cochlear hair cells could be achieved by infusing different concentrations of gentamicin into the middle ears of adult cats. STUDY DESIGN: Prospective experimental animal study treating only the left ear of each cat, the right ear serving as individual control. METHODS: Gentamicin solution at concentrations of either 30 or 3 mg/mL was infused daily into the left middle ear of adult cats until overt ataxia occurred. After 1 month or 6 months, each cat was killed and its temporal bones prepared for optical microscopy. RESULTS: Animals treated with 30 mg/mL gentamicin until ataxic required a median of five daily doses. These animals had clear-cut cochlear basal turn hair cell losses accompanying toxic lesions in the utricle and cristae. In contrast, animals treated with 3 mg/mL gentamicin until ataxic required an average of 19 daily doses. These animals had lesions restricted to the utricle and cristae with sparing of the cochlea hair cells. Animals that failed to develop ataxia manifested neither lesions of the cochlear nor vestibular hair cells. CONCLUSION: Gentamicin tympanoclysis in the cat animal model, using a dilute solution and continued once daily until clinical ataxia occurs, is capable of producing selective vestibular hair cell toxicity while sparing cochlea hair cells.     

Pattern of hair cell loss and delayed peripheral neuron degeneration in inner ear by a high-dose intratympanic gentamicin

To gain insights into the ototoxic effects of aminoglycoside antibiotics (AmAn) and delayed peripheral ganglion neuron death in the inner ear, experimental animal models were widely used with several different approaches including AmAn systemic injections, combination treatment of AmAn and diuretics, or local application of AmAn. In these approaches, systemic AmAn treatment alone usually causes incomplete damage to hair cells in the inner ear. Co-administration of diuretic and AmAn can completely destroy the cochlear hair cells, but it is impossible to damage the vestibular system. Only the approach of AmAn local application can selectively eliminate most sensory hair cells in the inner ear. Therefore, AmAn local application is more suitable for studies for complete hair cell destructions in cochlear and vestibular system and the following delayed peripheral ganglion neuron death. In current studies, guinea pigs were unilaterally treated with a high concentration of gentamicin (GM, 40 mg/ml) through the tympanic membrane into the middle ear cavity. Auditory functions and vestibular functions were measured before and after GM treatment. The loss of hair cells and delayed degeneration of ganglion neurons in both cochlear and vestibular system were quantified 30 days or 60 days after treatment. The results showed that both auditory and vestibular functions were completely abolished after GM treatment. The sensory hair cells were totally missing in the cochlea, and severely destroyed in vestibular end-organs. The delayed spiral ganglion neuron death 60 days after the deafening procedure was over 50%. However, no obvious pathological changes were observed in vestibular ganglion neurons 60 days post-treatment. These results indicated that a high concentration of gentamycin delivered to the middle ear cavity can destroy most sensory hair cells in the inner ear that subsequently causes the delayed spiral ganglion neuron degeneration. This model might be useful for studies of hair cell regenerations, delayed degeneration of peripheral auditory neurons, and/or vestibular compensation. In addition, a potential problem of ABR recording for unilateral deafness and issues about vestibular compensation are also discussed.     

Intermediate filaments in the newborn inner ear of the mouse

The presence of intermediate filaments in the inner ear of the newborn mouse was analyzed with immunofluorescence techniques using antibodies against the five classes of intermediate filaments: cytokeratins, vimentin, desmin, neurofilaments and glial fibrillary acid protein (GFA). Neurofilaments were found in all nerve fibers from the ganglion cell to the hair cell. In the vestibular ganglion two subpopulations of ganglion cells were identified: a minor part staining intensively with neurofilament and the major part of cells lacking this immunofluorescence. Vimentin occurred in a number of supporting structures in the membranous labyrinth, but not in vestibular or cochlear ganglion cells. Cytokeratins, desmin or GFA were not identified in the inner ear.     

Inner ear anomalies in Waardenburg's syndrome associated with Hirschsprung's disease

Congenital inner ear anomalies are reported in temporal bones of a 22-month-old boy with Waardenburg's syndrome and Hirschsprung's disease. Although no changes in the central auditory pathway were identified, peripheral lesions of the cochlear and vestibular membranous labyrinth were observed. Bilateral atrophy of the organ of Corti and stria, and a sparsity of spiral ganglion cells were observed in the cochlea. Degeneration of the vestibular end organs, including a loss of Scarpa's ganglion cells, was also seen. This is the first report of temporal bone histopathology associated with Waardenburg's syndrome and Hirschsprung's disease. The pathoembryology of these inner ear anomalies associated with aganglionosis of the colon supports the hypothesis that Waardenburg's syndrome and Hirschsprung's disease are hereditary defects of neural crest cells.