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.     

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.     

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.     

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.     

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.     

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.     

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.     

豚鼠内耳椭圆囊斑体外原代培养

目的探讨豚鼠内耳椭圆囊斑体外正常培养状态及其在体外用氨基糖苷类抗生素庆大霉素后的毒性损伤特征,以期建立前庭外周器官体外实验模型。方法豚鼠被随机分成三组:①正常未培养对照组;②正常原代培养组,利用体外组织培养方法对豚鼠内耳椭圆囊斑进行正常原代培养;③庆大霉素损伤原代培养组。正常原代培养组和庆大霉素损伤原代培养组在培养期间每天用相差显微镜观察生长晕,正常培养组在培养的第5天和第10天固定用石蜡包埋切片光镜观察。庆大霉素组在48小时用树脂包埋半薄切片观察。结果正常原代培养组在培养10天过程中,用相差显微镜观察椭圆囊斑在培养第1～4天无明显生长晕,在培养第5天明显从椭圆囊边缘长出。随培养时间延长,生长晕不断扩大,以成纤维细胞为主。庆大霉素损伤原代培养组在培养2天内每天用相差显微镜观察椭圆囊斑未见明显生长晕。正常原代培养至第5和第10天,石蜡包埋切片显示支持细胞和毛细胞存活。庆大霉素组在48小时,用树酯包埋半薄切片观察,感觉上皮层的毛细胞溶解破坏,支持细胞存活。结论椭圆囊斑器官体外培养方法可行,并培养成功,为今后进一步研究椭圆囊斑提供了实验模型。     

An electron microscopic study of the otolithic maculae of the lamprey (Entosphenus japonicus).

The sensory epithelium of the otolithic maculae from the inner ear of the lamprey (Entosphenus japonicus) was studied under the scanning and transmission electron microscope. Two different types of sensory hair cells were discerned and each had a patterned distribution over the various maculae. One type of hair cell had 20-30 short stereocilia and one long kinocilium on its surface. The other type exhibited much longer stereocilia which were arranged in graded heights (organ-pipe configuration) from one side of the cell to the other. So-called striated organelles were found in the cytoplasm of both types of sensory cells. These striated organelles exhibited periodic electron-dense and less dense striations of 170 mum extending from the cuticular plate down to the basal portion of the cell. Generally, this organelle was found in profusion in the sensory cells with long hair bundles but rarely found in the other type of cells.     

Actin-associated proteins and fibronectin in the fetal human inner ear

The distribution of alpha-actinin, vinculin, alpha-spectrin, beta-spectrin and fibronectin was analyzed in 14- to 21-week-old fetal human inner ears using immunofluorescence microscopy. Staining for alpha-actinin was fairly evenly distributed at the epithelial surfaces of all five vestibular organs, whereas in the cochlea it was mainly at the surface of the receding greater epithelial ridge and in some foci apically at the lesser epithelial ridge. Fluorescence for vinculin was observed mainly at the surface of vestibular organs, but was lacking in the LER. Intense fluorescence for alpha-spectrin was found at the apical surface of individual cells of the cristae and maculae. Antibodies against beta-spectrin mainly stained the endothelial cells of blood vessels, but faint staining of the epithelial cell surfaces of the vestibular organs was also detected. The fluorescence pattern of the actin-associated proteins is indicative of structural differences between cochlear and vestibular hair cells. Fibronectin was identified only between mesenchymal cells and its functional importance in the mature inner ear epithelia can be discounted.     

Comparison of vulnerability between avian and mammalian inner ears--electrophysiological and morphological studies]

In order to assess the vulnerability of the inner ear, auditory function and morphology of the inner ear were compared between adult budgerigars and adult guinea pigs. Budgerigars have been considered to have an excellent auditory-vocal system. Two experimental conditions were produced in each species; one by acoustic hyperstimulation (1500 Hz, 120 dBSPL) for 96 hours, the other by administration of kanamycin (200 mg/kg) for 7 weeks. Measurement of auditory evoked potentials (AEP) and observation of hair cells by electron microscopy were performed both immediately and 14 days after exposure. In the acoustic hyperstimulation experiment, AEPs of budgerigars showed less damage and better recovery than those of guinea pigs, probably because of morphological differences between the two species in hair and supporting cells. Electron microscopic observation on the budgerigars showed that a small part of the hair cell area was damaged and that regeneration of hair cells had occurred in this area 14 days after exposure. Such observations in guinea pigs revealed that outer hair cells had been damaged and replaced by supporting cells 14 days after exposure. In the kanamycin administration experiment, AEPs showed the same degree of damage and recovery in both species. This suggests that blood supply and drug transport to the inner ear are almost the same although the structure of the inner ear differs markedly between the two species. Electron microscopic observation did not show an apparent regeneration of hair cells 14 days after administration in the budgerigars. Guinea pigs had a tendency to show progressive damage of both auditory function and inner ear morphology even after the cessation of administration. Regeneration of hair cells in the budgerigar differed under both experimental conditions, suggesting that there is a difference in the mode of auditory disturbance between acoustic hyperstimulation and administration of kanamycin. In conclusion, resistance to acoustic hyperstimulation is higher in the avian inner ear than in the mammalian inner ear, while resistance to administration of kanamycin does not differ significantly between the two species.     

Is substance P the neurotransmitter in the vestibular end organs?

The vestibular end organs of the rabbit were fixed intracardially with phosphate buffered 4% formaldehyde, sectioned for 15 micron cryostat sections and incubated with monoclonal substance P (SP) antibody. Specific SP-like immunoreactivity (SPLI) was observed within and below the sensory epithelia of both utricular and saccular maculae and ampullary cristae. Particularly strong SPLI was seen in the maculae which showed many SPLI-positive axons in the subepithelial space and a network of SPLI-positive structures in the basal zones of the sensory epithelium. The calyceal terminals of type I hair cells showed regularly SPLI. The possibility of a transmitter or modulator role for SP in the vestibular function is suggested.     

Expression of cochlin in the vestibular organ of rats

The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Cochlin constitutes 70% of the inner ear protein and cochlin isoforms can be classified into three subgroups, p63s, p44s and p40s. Symptoms of some DFNA9 patients are consistent with those of Ménière's disease. Here, we report the expression of cochlin in the vestibular organ of rats using isoform-specific antibodies that recognize all three isoforms. Cochlin is highly expressed in the stromata of the maculae of otolithic organs and cristae of semicircular canals, and in the channels in the bony labyrinth that transmit the dendritic innervation to the cristae and maculae. Cochlin cannot be detected in the sensory cells, dark cells, nor in the acellular structures, otolithic membrane or in the cupula. These findings support the theory that deposition of acidophilic substance in the inner ear caused by mutation of cochlin can induce a secondary retrograde dendritic degeneration of the vestibular nerves.     

Effects of systemic versus local gentamicin on the inner ear in the Atlantic cod, Gadus morhua (L.), relevance for fish hearing investigations

Fish models are increasingly being used for hearing research investigations. Aminoglycoside antibiotics that are used for damaging the inner ear hair cells can have systemic side effects leading to death of study animals. This study aimed to compare two methods: (i) systemic (intravenous) and (ii) local (intrasaccular) gentamicin administration for induction of inner ear hair cell damage in the Atlantic cod, Gadus morhua (L.). Hair cell damage was assessed using scanning electron microscopy; hair cell density, prevalence of immature hair cells and kinocilia length were measured. Gentamicin-treated fish were compared with control and sham fish. Intravenous gentamicin led to dose-dependent mortality caused by nephrotoxicity. The only visible effect after treatment was more immature hair cells and shorter kinocilia, the effect on hair cell density was equivocal. Following intrasaccular gentamicin treatment, fish mortality was negligible, and hair cells were damaged regardless of dose. Here, we observed decreased hair cell density, high prevalence of immature hair cells, and significantly shortened kinocilia. Conclusion: intrasaccular injection is preferable to intravenous injection of gentamicin for the study of ototoxicity in the Atlantic cod.     

Quantitative study of human Scarpa's ganglion and vestibular sensory epithelia

Methods for counting vestibular ganglion cells and determining the densities of hair cells and intraepithelial basophilic inclusions (IBI) in samples of cross-sectioned vestibular sensory epithelia are described. Data obtained by means of these methods in vestibular sensory epithelia and Scarpa's ganglia in individual temporal bones from subjects at different ages are presented. Both vestibular hair cells and nerve cells in Scarpa's ganglia are found numerically reduced in ears of aged individuals. Changes in the vestibular sensory epithelia appear to precede those seen in Scarpa's ganglion. The incidence of intraepithelial basophilic inclusions correlates with degeneration in the respective vestibular sensory epithelia. There are no striking differences in hair cells densities of the different vestibular sense organs of the same ear and from subjects at about the same age.     

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.