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.     

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.     

Mutant mice reveal the molecular and cellular basis for specific sensory connections to inner ear epithelia and primary nuclei of the brain

We review the in vivo evidence for afferent fiber guidance to the inner ear sensory epithelia and the central nuclei of termination. Specifically, we highlight our current molecular understanding for the role of hair cells and sensory epithelia in guiding afferents, how disruption of certain signals can alter fiber pathways, even in the presence of normal hair cells, and what role neurotrophins play in fiber guidance of sensory neurons to hair cells. The data suggest that the neurotrophin BDNF is the most important molecule known for inner ear afferent fiber guidance to hair cells in vivo. This suggestion is based on experiments on Ntf3 transgenic mice expressing BDNF under Ntf3 promoter that show deviations of fiber growth in the ear to areas that express BDNF but have no hair cells. However, fiber growth can occur in the absence of BDNF as demonstrated by double mutants for BDNF and Bax. We directly tested the significance of hair cells or sensory epithelia for fiber guidance in mutants that lose hair cells (Pou4f3) or do not form a posterior crista (Fgf10). While these data emphasize the role played by BDNF, normally released from hair cells, there is some limited capacity for directed growth even in the absence of hair cells, BDNF, or sensory epithelia. This directed growth may rely on semaphorins or other matrix proteins because targeted ablation of the sema3 docking site on the sema receptor Npn1 results in targeting errors of fibers even in the presence of hair cells and BDNF. Overall, our data support the notion that targeting of the afferent processes in the ear is molecularly distinct from targeting processes in the central nuclei. This conclusion is derived from data that show no recognizable central projection deviation, even if fibers are massively rerouted in the periphery, as in Ntf3(tgBDNF) mice in which vestibular fibers project to the cochlea.     

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.     

Supernumerary outer hair cells arise external to the last row of sensory cells in the organ of corti

During the development of the mammalian inner ear, the number of hair cells produced is highly regulated and remains constant throughout life. The mechanism underlying this regulation is beginning to be understood although many aspects still remain obscure. When late embryonic or early postnatal rat organs of Corti were cultured, the production of supernumerary hair cells was observed. This overproduction of sensory cells could be modulated by the addition of several growth factors. In this study, we examined explants of rat organs of Corti that produced supernumerary hair cells. In the supernumerary hair cell region, up to two rows of inner hair cells and five rows of outer hair cells were observed. Morphological evaluation of these specimens revealed that less mature hair cells were located in the most external rows of these sensory cells. When a supernumerary hair cell was produced, a supporting cell (i.e. Deiters' cell) was also produced, strongly suggesting that the conversion of a Deiters' cell into a hair cell was not the mechanism that produced these extra hair cells. Based on these results, we propose that prosensory cells located at the external edge of the organ of Corti retain a capacity to form hair cells and that it is these prosensory cells that differentiate into supernumerary hair cells and Deiters' cells.     

Comparison of the inner ear ultrastructure between teleost fishes using different channels for communication.

The anatomy and ultrastructure of the inner ear of three species of gouramis which differ widely in acoustic behavior were studied using scanning electron microscopy. Of the three species. Trichopsis possess a pectoral sound-producing mechanism while Macropodus and Betta lack sonic organs. The general structure of the inner ear and the shapes of the sensory epithelia are very similar, although they do differ on the posterior part of the saccular macula which is more S-shaped in Trichopsis and Macropodus than in Betta. The maculae on the three species do not differ either in ciliary bundle type (cells with long kinocilia on the periphery of the maculae and cells with short kinocilia in the central region) or in hair cell orientation pattern. Quantitative measurements of hair cell densities and the size of the sensory epithelia of the saccule did not show significant differences between species. Data presented correlate with physiological results from other investigators showing similar auditory sensitivity in Trichopsis and Macropodus. The similarity in structure and function of the inner ears of gouramis on one hand, and the occurrence of sound-generating organs in just one genus, suggests that hearing evolved prior to vocalization and thus acoustic communication in this taxon.     

Blockade of c-Jun N-terminal kinase pathway attenuates gentamicin-induced cochlear and vestibular hair cell death

The ototoxic action of aminoglycoside antibiotics leading to the loss of hair cells of the inner ear is well documented. However, the molecular mechanisms are poorly defined. We have previously shown that in neomycin-exposed organotypic cultures of the cochlea, the c-Jun N-terminal kinase (JNK) pathway--associated with stress, injury and apoptosis--is activated in hair cells and leads to their death. We have also shown that hair cell death can be attenuated by CEP-1347, an inhibitor of JNK signalling [Pirvola et al., J. Neurosci. 20 (2000) 43-50]. In the present study, we demonstrate that gentamicin-induced ototoxicity leads to JNK activation and apoptosis in the inner ear hair cells in vivo. We also show that systemic administration of CEP-1347 attenuates gentamicin-induced decrease of auditory sensitivity and cochlear hair cell damage. In addition, CEP-1347 treatment reduces the extent of hair cell loss in the ampullary cristae after gentamicin intoxication. Particularly, the inner hair cells of the cochlea and type I hair cells of the vestibular organs are protected. We have previously shown that also acoustic overstimulation leads to apoptosis of cochlear hair cells and that CEP-1347 can attenuate noise-induced sensory cell loss. These results suggest that activation of the JNK cascade may be a common molecular outcome of cellular stress in the inner ear sensory epithelia, and that attenuation of the lesion can be provided by inhibiting JNK activation.     

ErbB Expression: The Mouse Inner Ear and Maturation of the Mitogenic Response to Heregulin

In humans, hair cell loss often leads to hearing and balance impairments. Hair cell replacement is vigorous and spontaneous in avians and nonmammalian vertebrates. In mammals, in contrast, it occurs at a very low rate, or not at all, presumably because of a very low level of supporting cell proliferation following injury. Heregulin (HRG), a member of the epidermal growth factor (EGF) family of growth factors, is reported to be a potent mitogen for neonatal rat vestibular sensory epithelium, but its effects in adults are unknown. We report here that HRG- stimulates cell proliferation in organotypic cultures of neonatal, but not adult, mouse utricular sensory epithelia. Our findings support the idea that the proliferative capabilities of the adult mammalian vestibular sensory epithelia differ significantly from that seen in neonatal animals. Immunohistochemistry reveals that HRG-binding receptors (erbBs 2–4) and erbB1 are widely expressed in vestibular and auditory sensory epithelia in neonatal and adult mouse inner ear. The distribution of erbBs in the neonatal and adult mouse ear is consistent with the EGF receptor/ligand family regulating diverse cellular processes in the inner ear, including cell proliferation and differentiation. Present address (Mette Kirkegaard): Department of Zoophysiology, Bld. 131, Universitetsparken, University of Aarhus, Denmark     

Insulin-like growth factor binding protein (IGFBP)-mediated hair cell survival on the mouse utricle exposed to neomycin: the roles of IGFBP-4 and IGFBP-5

CONCLUSION: This study suggests for the first time that 1) IGF-I, IGFBP-4, and -5 alone and IGF-I+IGFBP-5 mixture stimulated hair cell survival and prevented neomycin-induced hair cell loss in the sensory epithelial culture of mouse utricles, 2) When administered together, IGFBP-4 diminished the effect of IGF-I, 3) In P3-5 mice utricle, IGF-I, IGFBP-4, and IGFBP-5 are expressed in the cytoplasm of hair cells. And Insulin/IGF-I Receptor is expressed in the nucleus of hair cells. OBJECTIVES: Several growth factors have been demonstrated to protect auditory sensory cells in vitro and in vivo from aminoglycoside toxicity. IGF-I is one of the most well-known mitogenic and protective substance working in the inner ear. However, there are no reports available regarding the function of IGFBPs in the inner ear. In the present study, the effects of IGFBP-4 and -5 on hair cell survival were investigated in mouse utriclular organ cultures. MATERIALS AND METHODS: The amount of cellular damage and cell viability in vestibular organs were assessed by counting hair cells stained with a rhodamine-phalloidin probe. The expressions of IGFBP-4, IGFBP-5, IGF-IR, and IGF-I were localized by immunohistochemistry. RESULTS: When treated with IGF-I, IGFBP-4, or IGFBP-5 for 24 h, explant culture showed hair cell survival rates of 136+/-18%, 140+/-15%, and 133+/-6%, respectively, compared to controls. Neomycin (1 mM) induced hair cell loss resulted in 45+/-17% of hair cell survival. However, pre-treatment of IGF-I, IGFBP-4, or -5 before neomycin insult showed survival rates of 113+/-14%, 98+/-8%, and 73+/-24%, respectively. Similar to IGF-I, IGFBP-4 and IGFBP-5 were significantly protective. IGFBP-4 and -5 immunoreactivities were observed in the cytoplasm of normal explanted vestibular hair cells as well as in the P3 mouse utricular hair cells in vivo.     

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.     

Structure and function in the saccule of the goldfish (Carassius auratus): a model of diversity in the non-amniote ear

The vertebrate inner ear is comprised of a remarkable diversity of cell types, including several types of sensory hair cells. In amniotes (reptiles, birds, and mammals), the morphological and physiological characteristics that distinguish these cell types have been well documented, while cellular variation in the ears of non-amniotes (all other vertebrate groups) has remained underrecognized. Since non-amniotes have become increasingly popular models for developmental and genetic research, a more comprehensive understanding of structure and function in the inner ears of these species is warranted. This paper first reviews the large body of data describing the morphology and physiology of hair cells and afferent neurons in the inner ear of the goldfish (Carassius auratus). In particular, we examine the structure of the goldfish saccule, an endorgan that has been the subject of numerous investigations on audition. New data on the structural variation of synaptic bodies in saccular hair cells are also presented, and the functional implications of these data are discussed. Finally, we conclude that hair cell structure varies along the length of the goldfish saccule in a manner consistent with known physiological characteristics of the endorgan. The saccule provides an excellent model for investigating structure-function relationships in the vertebrate inner ear, as well as the development of auditory and vestibular sensory epithelia.     

Transplantation of neural stem cells into explants of rat inner ear

Damage and loss of hair cells in the inner ear is the most frequent cause of hearing loss and balance disorders. Mammalian hair cells do not regenerate in the conventional ways. To regenerate the hair cell in the mammalian inner ear we transplanted neural stem cells into explants of rat inner ear. The stem cells integrated successfully into the sensory epithelium of the vestibular organs, but not into the organ of Corti. This method is useful to investigate efficient ways to transplant stem cells into the inner ear.     

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。     

Embryonic Inner Ear Cells Reaggregate into Specific Patterns In Vitro

The sensory epithelia of the mammalian inner ear consist of a highly precise pattern of sensory hair cells and supporting cells. The mechanisms regulating this patterning are only beginning to be determined. The present study describes a method for culturing dissociated embryonic inner ear cells and the resulting patterning that occurs in these cultures. The results indicate that developing inner ear cells aggregate into precise patterns on a two-dimensional substrate, suggesting that intrinsic patterning mechanisms remain active in vitro. Using antibodies and scanning electron microscopy to detect hair cells and nonsensory cells, it was determined that only a subset of aggregates contained sensory hair cells. The hair cells were organized into specific patterns and surrounded by supporting cells, similar to the in vivo pattern. Additionally, hair cells increased their immunoreactivity and number of stereocilia over time, suggesting that hair cells continue to mature in vitro. Thus, the study reveals that the cells of the developing inner ear provide the necessary signals that direct sensory hair cells and supporting cells to reassociate into very precise patterns in vitro and that these patterns are reminiscent of the patterning that occurs in vivo.     

Extracorporeal preservation. Organ culture of the post-natal mammalian inner ear.

Postnatal (newborn and mature) inner ear organs from CBA/CBA mouse and guinea pig were analysed concerning hair cell survival in an in vitro system. After only a few days in the artificial surroundings a transformation of hair cell characteristics occurred, in the form of either loss of sensory hairs (cochlear hair cells) or hair fusion (vestibular hair cells), although the hair cell itselt survived for a considerably longer time. Intracellular myelin figures became evident after 2--3 days in culture. However, a considerable individual variation among hair cells was observed concerning the ultrastructure of the cell at this stag in vitro. Completely normal hair cells could in rare cases be recognised after 1 week in organ culture (cochlear inner hair cells of the CBA/CBA mouse). Loss of the surface structures of the hair cells is likely to constitute an irreversible transformation not in agreement with the true hair cell characteristics.     

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear

CONCLUSION: The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. OBJECTIVE: To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. MATERIAL AND METHODS: Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. RESULTS: Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.     

Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

The target-derived factors necessary for promoting initial outgrowth from the statoacoustic ganglion (SAG) to the inner ear have not been fully characterized. In the present study, conditioned medium from embryonic Immortomouse inner ear cell lines that maintain many characteristics of developing inner ear sensory epithelia were screened for neurite-promoting activity. Conditioned medium found to be positive for promoting SAG neurite outgrowth and neuronal survival was then tested for the presence of chemokines, molecules that have not previously been investigated for promoting SAG outgrowth. One candidate molecule, monocyte chemotactic protein 1 (MCP-1), was detected in the conditioned medium and subsequently localized to mouse hair cells by immunocytochemistry. In vitro studies demonstrated that function-blocking MCP-1 antibodies decreased the amount of SAG neurite outgrowth induced by the conditioned medium and that subsequent addition of MCP-1 protein was able to promote outgrowth when added to the antibody-treated conditioned medium. The use of the Immortomouse cell lines proved valuable in identifying this candidate cofactor that promotes outgrowth of early-stage SAG nerve fibers and is expressed in embryonic hair cells.