Expression of epidermal growth factor, epidermal growth factor receptor and transforming growth factor-alpha in the human fetal inner ear

The expression of epidermal growth factor (EGF), EGF receptor and transforming growth factor (TGF)-alpha was analyzed in the human fetal inner ear using immuno-histochemical methods. EGF receptor was observed only in 9.5-week-old fetal vestibular epithelia. In 14- and 16-week-old fetuses, EGF receptor could not be detected. TGF-alpha was observed strongly in the 9- and 11-week-old vestibular epithelia, whereas only trace amounts were detectable in the 14- and 16-week-old vestibular epithelia. These findings suggest that EGF and TGF-alpha probably have a mitogenic effect in the sensory epithelia of the fetal inner ear, especially at early stages of development.     

Inner ear hair cell regeneration in a mammal: Identification of a triggering factor

Recent observations have shown that mammals possess a limited capacity for regeneration of inner ear sensory epithelia. It is clear, however, that a mitogenic growth factor will be necessary to up-regulate this capacity before clinical application becomes feasible. This study used in vitro cultures of adult mouse vestibular organs for assessing the mitogenic effect of transforming growth factor alpha (TGF-α). Sixty-one utricles and cristae were maintained in culture for 7 to 8 days. Neomycin was used to damage the hair cells. Autoradiography permitted identification of any cell which had undergone mitosis during the culture period. The proliferative response was compared in organs exposed to TGF-alpha and those maintained in the basic culture medium only. The results demonstrated that TGF-alpha significantly increased cell proliferation in the sensory epithelia and also in the marginal zones surrounding them. This finding provides a scientific basis for the concept that inner ear hair cell damage in humans may someday be reversible pharmacologically.     

Expression of calretinin by fetal otocyst cells after transplantation into damaged rat utricle explants

Severe damage by acoustic overstimulation or ototoxins induces inner ear hair cell loss, resulting in permanent hearing loss and balance disorders because hair cell regeneration scarcely occurs in the inner ear sensory organs of mammals. In this study, to evaluate the possibilities of cell transplantation therapy for damaged inner ear sensory organs, dissociated cell cultures of fetal otocyst cells (FOCs) were established from embryonic day 12.5 (E12.5) rat inner ears, and transplanted into gentamicin-treated explants of vestibular sensory epithelia. Two weeks after transplantation, immunohistochemical analysis demonstrated that some of the grafted FOCs survived within the vestibular sensory epithelia and expressed epitopes of calretinin. one of the hair cell marker proteins. These findings indicate that FOCs have the potential to migrate into damaged vestibular epithelia and differentiate into hair cell immunophenotypes. Cell transplantation therapy may be available for functional regeneration in inner ear diseases.     

Robust Postmortem Survival of Murine Vestibular and Cochlear Stem Cells

Potential treatment strategies of neurodegenerative and other diseases with stem cells derived from nonembryonic tissues are much less subjected to ethical criticism than embryonic stem cell-based approaches. Here we report the isolation of inner ear stem cells, which may be useful in cell replacement therapies for hearing loss, after protracted postmortem intervals. We found that neonatal murine inner ear tissues, including vestibular and cochlear sensory epithelia, display remarkably robust cellular survival, even 10 days postmortem. Similarly, isolation of sphere-forming stem cells was possible up to 10 days postmortem. We detected no difference in the proliferation and differentiation potential between stem cells isolated directly after death and up to 5 days postmortem. At longer postmortem intervals, we observed that the potency of sphere-derived cells to spontaneously differentiate into mature cell types diminishes prior to the cells losing their potential for self-renewal. Three-week-old mice also displayed sphere-forming stem cells in all inner ear tissues investigated up to 5 days postmortem. In summary, our results demonstrate that postmortem murine inner ear tissue is suited for isolation of stem cells.     

A novel technique for inducing local inner ear damage

With significant development of mouse genomics and the availability of transgenic and knockout mice, the mouse will be the preferred animal model for inner ear research. However, few studies have used mice as experimental animals for examination of hair cell degeneration, because of their relative resistance to ototoxic agents and difficulties in surgical treatment. This study presents a model for induction of apoptotic cell death in sensory epithelia of the mouse inner ear using injection of neomycin into the posterior semicircular canal. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay revealed that local application of neomycin produced sufficient induction of apoptotic cell death in both auditory and vestibular epithelia over a definite time course. Supplementation of the general caspase-inhibitor significantly reduced induction of TUNEL-positive cells, indicating caspase-dependency of apoptotic cell death observed in the present model. In addition, the approach to the posterior semicircular canal was an easy technique, and sham-operation induced no significant induction of TUNEL-positive cells. This model, hence, enables the use of various genetic tools in studies for mechanisms of hair cell apoptosis.     

Induction of cell proliferation and beta-catenin expression in rat utricles in vitro

Limited regenerative response occurs in mammalian vestibular epithelia, although vestibular hair cell regeneration in mammals has been demonstrated under a variety of experimental conditions. Beta-catenin is known to play an important role in both cell-cell adhesion and signal transduction associated with cell proliferation. This study evaluated cell proliferation activity in mammalian vestibular epithelia in organotypic culture and examined the involvement of beta-catenin in cell proliferation in vestibular epithelia. After 72 h of culture, utricles with or without induction of cell proliferation were examined. Cell proliferation was induced by brief exposure to forskolin and supplementation with fetal bovine serum. Cell proliferation activity was assessed by bromodeoxyuridine (BrdU) labeling. Immunohistochemistry was employed for analysis of cellular distribution of beta-catenin. In utricles cultured without induction of cell proliferation. BrdU labeling was not found in vestibular epithelia. Expression of beta-catenin was found in the area corresponding to the distribution of adherens junctions in vestibular epithelia. However, BrdU labeling was identified in sensory epithelia of utricles following induction of cell proliferation, although the number of BrdU-positive cells in sensory epithelia was very limited. Accumulation of beta-catenin was occasionally found in proliferating cells in sensory epithelia; however, it was not always found in BrdU-positive cells. The present findings suggest that beta-catenin may play a role in the induction of cell proliferation in mammal vestibular epithelia.     

Charakterisierung von Stammzellen des neonatalen auditorischen Sinnesepithels

Background

In contrast to regenerating hair cell-bearing organs of nonmammalian vertebrates the adult mammalian organ of Corti appears to have lost its ability to maintain stem cells. The result is a lack of regenerative ability and irreversible hearing loss following auditory hair cell death. Unexpectedly, the neonatal auditory sensory epithelium has recently been shown to harbor cells with stem cell features. The origin of these cells within the cochlea’s sensory epithelium is unknown.

Material And Methods

We applied a modified neurosphere assay to identify stem cells within distinct subregions of the neonatal mouse auditory sensory epithelium. Sphere cells were characterized by multiple markers and morphologic techniques.

Results

Our data reveal that both the greater and the lesser epithelial ridge contribute to the sphere-forming stem cell population derived from the auditory sensory epithelium. These self-renewing sphere cells express a variety of markers for neural and otic progenitor cells and mature inner ear cell types.

Conclusion

Stem cells can be isolated from specific regions of the auditory sensory epithelium. The distinct features of these cells imply a potential application in the development of a cell replacement therapy to regenerate the damaged sensory epithelium.     

豚鼠前庭感觉上皮损伤中细胞凋亡的观察

细胞凋亡对细胞增殖、器官发生和功能维持起着重要作用。一定剂量的庆大霉素连续注射，造成豚鼠前庭器官损伤。采用半薄切片，透射电镜（TEM）和TUNEL（TdT－modidedbiotin-dUTPNick-endlabeling，末端脱氧核苷酸转移酶介导的生物素标记）原位杂交技术，特异标记DNA片段3′－OH末端，原位显示凋亡细胞。在半薄切片和TEM观察中发现两种类型的细胞损伤方式：①毛细胞肿胀，胞浆空泡化，细胞体从顶端表面挤出；②毛细胞在上皮内变性，显示出细胞凋亡的形态特征，包括细胞核凝缩，核膜消失，成碎块状，并由支持细胞吞噬。原位杂交显示：细胞凋亡标记阳性细胞主要分布在上皮表层，较高水平标记主要发生于给药后第3到7天。提示细胞凋亡是内耳前庭感觉细胞损伤的一种重要方式，凋亡的主动发生可能是一种潜在的介入方式来减少氨基甙类抗生素对毛细胞造成的急性损伤，并与感觉上皮损伤后的修复过程有关。     

Spatiotemporal expression of otogelin in the developing and adult mouse inner ear

Using a PCR-based subtractive method on cDNA from 2-day-old mouse cochlea, we identified a gene encoding otogelin, Otog, an inner ear specific glycoprotein expressed in all acellular structures. Here, we provide evidence that otogelin is detected as early as embryonic day 10 in the otic vesicle. At this stage, otogelin is detected in the epithelial cells which do not overlap with the myosin VIIA-expressing cells, namely the precursors of the hair cells, thus arguing for an early commitment of the two cell populations. Analysis of otogelin spatiotemporal cell distribution allows a molecular tracing for the contribution of the cochlear and vestibular inner ear supporting cells to the formation of the acellular structures. Throughout embryonic and adult life, the expression of the otogelin gene as monitored by LacZ inserted into Otog, and the abundance of the protein are greater in the vestibule than in the cochlea. In adult, otogelin is still produced by the vestibular supporting cells, which argues for a continuous process of otogelin renewal in the otoconial membranes and cupulae. In contrast, in the tectorial membrane, otogelin should be a long-lasting protein since both the otogelin gene and protein were almost undetectable in adult cochlear cells. The data are consistent with the requirement for otogelin in the attachment of the otoconial membranes and cupulae to their corresponding sensory epithelia as revealed in Otog -/- mice.     

Immunohistochemical localization of nonerythroid spectrin (fodrin) in the sensory cells of the vestibular end organs of the rat and guinea pig

Indirect immunofluorescence method was used to study presence and localization of nonerythroid spectrin (fodrin) in the vestibular sensory epithelia. Cryosections of the vestibular organs were treated by monoclonal antibody (mAb) reacting with mammalian fodrin. Strong mAb labelling was observed in the cuticular plate of the vestibular hair cells. The stereocilia were nonreactive and supporting cells showed only a weak reaction. Fodrin seems to have similar localization in the inner ear hair cells as actin (except stereocilia) and it appears to be the major component of the membrane skeleton in the inner ear hair cells. Fodrin-associated membrane skeleton may be involved in the hair cell function in several different ways.     

Inner ear effects of topical gentamicin treatment in patients with Menière's disease

Several efforts have been made to treat patients with Menière's disease with ototoxic drugs. Lately the aminoglycoside antibiotic, gentamicin, has been favored. Several reports have proved its efficacy. In a series of 12 patients treated at the Karolinska Hospital in Stockholm, two patients failed to show improvement regarding their vertigo in spite of repeated courses of treatment. These patients both underwent labyrinthectomy where their vestibular sensory epithelia as well as portions of the cochlea were removed. The findings in these two cases were quite unanimous in that the vestibular sensory epithelia were retained showing advanced signs of degeneration. The organ of Corti in the cochlea was totally absent in all specimens examined. These findings indicate that gentamicin seems to affect the cochlea more than the vestibular sensory epithelia that also was clinically proven since both patients totally lost their hearing in the affected ear. Possible routes and actions of the drug in the inner ear are discussed.     

BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of <Emphasis Type="Italic">Opsanus tau</Emphasis>

We report on a new xyloside conjugated to BODIPY, BX and its utility to prime fluorescent glycosaminoglycans (BX-GAGs) within the inner ear in vivo. When BX is administered directly into the endolymphatic space of the oyster toadfish (Opsanus tau) inner ear, fluorescent BX-GAGs are primed and become visible in the sensory epithelia of the semicircular canals, utricle, and saccule. Confocal and 2-photon microscopy of vestibular organs fixed 4 h following BX treatment, reveal BX-GAGs constituting glycocalyces that envelop hair cell kinocilium, nerve fibers, and capillaries. In the presence of GAG-specific enzymes, the BX-GAG signals are diminished, suggesting that chondroitin sulfates are the primary GAGs primed by BX. Results are consistent with similar click-xylosides in CHO cell lines, where the xyloside enters the Golgi and preferentially initiates chondroitin sulfate B production. Introduction of BX produces a temporary block of hair cell mechanoelectrical transduction (MET) currents in the crista, reduction in background discharge rate of afferent neurons, and a reduction in sensitivity to physiological stimulation. A six-degree-of-freedom pharmacokinetic mathematical model has been applied to interpret the time course and spatial distribution of BX and BX-GAGs. Results demonstrate a new optical approach to study GAG biology in the inner ear, for tracking synthesis and localization in real time.     

Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery

Sensorineural losses of hearing and vestibular sensation due to hair cell dysfunction are among the most common disabilities. Recent preclinical research demonstrates that treatment of the inner ear with a variety of compounds, including gene therapy agents, may elicit regeneration and/or repair of hair cells in animals exposed to ototoxic medications or other insults to the inner ear. Delivery of gene therapy may also offer a means for treatment of hereditary hearing loss. However, injection of a fluid volume sufficient to deliver an adequate dose of a pharmacologic agent could, in theory, cause inner ear trauma that compromises functional outcome. The primary goal of the present study was to assess that risk in rhesus monkeys, which closely approximates humans with regard to middle and inner ear anatomy. Secondary goals were to identify the best delivery route into the primate ear from among two common surgical approaches (i.e., via an oval window stapedotomy and via the round window) and to determine the relative volumes of rhesus, rodent, and human labyrinths for extrapolation of results to other species. We measured hearing and vestibular functions before and 2, 4, and 8 weeks after unilateral injection of phosphate-buffered saline vehicle (PBSV) into the perilymphatic space of normal rhesus monkeys at volumes sufficient to deliver an atoh1 gene therapy vector. To isolate effects of injection, PBSV without vector was used. Assays included behavioral observation, auditory brainstem responses, distortion product otoacoustic emissions, and scleral coil measurement of vestibulo-ocular reflexes during whole-body rotation in darkness. Three groups (N = 3 each) were studied. Group A received a 10 μL transmastoid/trans-stapes injection via a laser stapedotomy. Group B received a 10 μL transmastoid/trans-round window injection. Group C received a 30 μL transmastoid/trans-round window injection. We also measured inner ear fluid space volume via 3D reconstruction of computed tomography (CT) images of adult C57BL6 mouse, rat, rhesus macaque, and human temporal bones (N = 3 each). Injection was well tolerated by all animals, with eight of nine exhibiting no signs of disequilibrium and one animal exhibiting transient disequilibrium that resolved spontaneously by 24 h after surgery. Physiologic results at the final, 8-week post-injection measurement showed that injection was well tolerated. Compared to its pretreatment values, no treated ear’s ABR threshold had worsened by more than 5 dB at any stimulus frequency; distortion product otoacoustic emissions remained detectable above the noise floor for every treated ear (mean, SD and maximum deviation from baseline: ?1.3, 9.0, and ?18 dB, respectively); and no animal exhibited a reduction of more than 3 % in vestibulo-ocular reflex gain during high-acceleration, whole-body, passive yaw rotations in darkness toward the treated side. All control ears and all operated ears with definite histologic evidence of injection through the intended site showed similar findings, with intact hair cells in all five inner ear sensory epithelia and intact auditory/vestibular neurons. The relative volumes of mouse, rat, rhesus, and human inner ears as measured by CT were (mean ± SD) 2.5 ± 0.1, 5.5 ± 0.4, 59.4 ± 4.7 and 191.1 ± 4.7 μL. These results indicate that injection of PBSV at volumes sufficient for gene therapy delivery can be accomplished without destruction of inner ear structures required for hearing and vestibular sensation.     

鸡内耳感觉上皮细胞的自然增殖状态

为研究出生后鸟纲动物内耳感觉上皮细胞是否存在持续的细胞增殖，采用８天龄正常鸡１０只（２只做阴性对照），按１００ｍｇ／ｋｇ剂量腹腔注射ＤＮＡ合成的前体物质５－溴脱氧尿核苷（５－ｂｒｏｍｏｄ－ｅｘｙｕｒｉｄｉｎｅ，ＢｒｄＵ），６小时后处死动物，取颞骨制备石蜡切片，结合免疫细胞化学技术，对鸡基底乳头和前庭器官中的细胞增殖活动进行观察。结果发现，鸡基底乳头切片中未见阳性标记细胞，而椭圆囊斑、球囊斑及壶腹嵴感觉上皮中存在着数量较少的标记支持细胞和标记毛细胞。结论：出生后的鸟纲动物前庭器官存在着较低水平的持续增殖。提示：出生后鸟纲动物前庭器官可能保持着潜在的对抗病理损伤的修复能力。     

鸡内耳感觉上皮细胞的自然增殖状态

为研究出生后鸟纲动物内耳感觉上皮细胞是否存在持续的细胞增殖，采用８天龄正常鸡１０只（２只做阴性对照），按１００ｍｇ／ｋｇ剂量腹腔注射ＤＮＡ合成的前体物质５－溴脱氧尿核苷，６小时后处死动物，取颞骨制备石蜡切片，结合免疫细胞化学技术，对鸡基底乳头和前庭器官中的细胞增殖活动进行观察。结果发现，鸡基底乳头切片中未见阳性标记细胞，而椭圆囊斑、球囊斑及壶腹嵴感觉上皮中存在着数量较少的标记支持细胞和标记毛细胞。     

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.     

CD44 is a Marker for the Outer Pillar Cells in the Early Postnatal Mouse Inner Ear

Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters’ cells in the Fgfr3 ^{P244R/ +} mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.     

Expression of transient receptor potential channel melastin (TRPM) 1–8 and TRPA1 (ankyrin) in mouse inner ear

Conclusions: It has been shown that TRPMs may play a functional role in sensory cell physiology, fluid homeostasis, sensory cell death, and thermosensation in the inner ear, while TRPA1 plays an important role in sensory transduction. Objective: To study expression of TRPM1–8 and TRPA1 in the mouse inner ear. Materials and methods: The localization of TRPM1–8 and TRPA1 in the inner ear of normal and gentamicin-treated CBA/J mice was investigated by immunohistochemistry. Results: The stria vascularis displayed a positive immunofluorescent reaction to TRPM1, 2, 3, 6, and 7. In the organ of Corti, outer and inner hair cells (OHCs and IHCs) showed positive immunofluorescence to TRPM1, 2, 3, 6, 7, and 8. Spiral ganglion cells were immunoreactive to TRPM1, 2, 3, 6, 7, and 8. The nerve fibers in the spiral ganglion cells and the nerves innervating the OHCs or IHCs were noticeably immunofluorescent to TRPM8 and TRPA1. In the vestibular end organs, vestibular sensory cells showed immunofluorescence to TRPM1, 2, 3, 6, and 7. The vestibular dark cells showed immunofluorescence to TRPM1, 3, 6, and 7; only the apical portion reacted to TRPM4. The nerve fibers innervating the vestibular sensory cells were distinctly reactive to TRPM8 and TRPA1, while the vestibular ganglion cells reacted to TRPM1, 2, 3, 6, 7, and 8.