Developmental stage-dependent pattern of inner ear expression of intermediate filaments

The expression of vimentin, cytokeratins (CKs) and neurofilament (NF) proteins was analysed (using monoclonal antibodies) in the mouse inner ear at the otocyst stage (13th gestational day), when organogenesis was largely completed (16th gestational day) and at birth (21st gestational day). Co-expression of vimentin and CKs occurred at the otocyst stage. On the 16th gestational day, most epithelial cells lacked immunoreactivity for vimentin and considerable variation in CK positivity was found between different regions of the epithelial lining. At birth, CK positivity was lacking in the developing organ of Corti but was present in other types of epithelium lining the scala media. In the vestibular half of the labyrinth, positivity for CKs was found at the apical surfaces of both sensory cells and supporting cells and in epithelia lining the membranous labyrinth. Vimentin positivity occurred in the greater epithelial ridge of the differentiating organ of Corti. Even at this stage the statoacoustic ganglion comprised two subpopulations of ganglion cells: those staining for NF proteins and those lacking this immunoreactivity. Thus, as the inner ear matures, a pattern of cytoskeletal reorganization occurs that is dependent on developmental stage.     

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.     

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.     

Direct evidence of nitric oxide production in guinea pig vestibular sensory cells

Production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. The utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, and significantly increased after inoculation with LPS. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role for the vestibular physiology and also be involved in disease of the inner ear.     

Direct Evidence of Nitric Oxide Production in Guinea Pig Vestibular Sensory Cells

Production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. The utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, and significantly increased after inoculation with LPS. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role for the vestibular physiology and also be involved in disease of the inner ear.     

Detection of nitric oxide in the guinea pig inner ear, using a combination of aldehyde fixative and 4,5-diaminofluorescein diacetate

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator. 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.     

Glutamate-induced production of nitric oxide in guinea pig vestibular sensory cells

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.     

Glutamate-induced Production of Nitric Oxide in Guinea Pig Vestibular Sensory Cells

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.     

Detection of Nitric Oxide in the Guinea Pig Inner Ear,Using a Combination of Aldehyde Fixative and 4,5-Diaminofluorescein Diacetate

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator, 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.     

Surgical Management of Head and Neck Cancer: The Next Decade

Sensorineural hearing loss is known to be a significant sequela of otitis media (OM). The pathophysiology of such hearing loss in OM is thought to be due to transmission of toxins and other bacterial products through the round window membrane, damaging the hair cells of the basal turn of the cochlea. Other routes, such as those involving the oval window, blood vessels and lymphatics, may also be involved. The purpose of this study was to elucidate the routes from the middle ear cavity to the inner ear and also the distribution pattern of endotoxin in the inner ear after injection of fluorescence-labelled endotoxin into the tympanic cavity and detection of fluorescence in the cochleae, vestibular end organs and facial nerves. This fluorescence was far more intense in the lower turns of the cochlea. These findings suggest that endotoxin can reach the inner ear by various routes, e.g. the round window, blood vessels or lymphatics, and/or interscala exchange, resulting in a disturbance not only of the cochlea but also of the vestibular end organs.     

Localization of endotoxin in the inner ear following inoculation into the middle ear

Sensorineural hearing loss is known to be a significant sequela of otitis media (OM). The pathophysiology of such hearing loss in OM is thought to be due to transmission of toxins and other bacterial products through the round window membrane, damaging the hair cells of the basal turn of the cochlea. Other routes, such as those involving the oval window, blood vessels and lymphatics, may also be involved. The purpose of this study was to elucidate the routes from the middle ear cavity to the inner ear and also the distribution pattern of endotoxin in the inner ear after injection of fluorescence-labelled endotoxin into the tympanic cavity and detection of fluorescence in the cochleae, vestibular end organs and facial nerves. This fluorescence was far more intense in the lower turns of the cochlea. These findings suggest that endotoxin can reach the inner ear by various routes, e.g. the round window, blood vessels or lymphatics, and/or interscala exchange, resulting in a disturbance not only of the cochlea but also of the vestibular end organs.     

Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells

The distribution of actin, alpha-actinin, fimbrin, tropomyosin and tubulin in the apical region of inner and outer hair cells was studied by immunofluorescent localization of antibodies to these proteins. The macromolecular distribution of actin and alpha-actinin was studied using post-embedding immunoelectron microscopic techniques. Actin is present in the stereocilia and cuticular plate of both inner and outer hair cells. Antibodies to actin were localized with fluorescence and colloidal gold. Colloidal gold particles were distributed uniformly over the stereocilia, stereocilia rootlets and cuticular plate. Fimbrin is present in the stereocilia and the cuticular plate. Immunofluorescent label was more intense over the cuticular plate of outer hair cells than over the cuticular plate of inner hair cells. Alpha-actinin is present in the cuticular plate only. At the ultrastructural level, antibodies to alpha-actinin were labeled throughout the cuticular plate, with larger accumulations of colloidal gold over the electron dense bodies in the cuticular plate, as well as over the electron dense region at the junctional complex. There was no label over the electron dense portion of the stereocilia rootlets. Tropomyosin is observed in the area of the stereocilia rootlets by immunofluorescent techniques, but like fimbrin, the antigenic sites of tropomyosin did not withstand processing for ultrastructural localization. Tubulin is not present in the apical region of inner or outer hair cells, although its presence could be documented in the hair cell body and in the supporting cells.     

Alpha and beta subunits of acetylcholine receptors in the human inner ear

The localization and distribution of nicotinic acetylcholine receptors (n-ACh-r) was characterized by studying alpha and beta subunits in the adult human inner ear by FITC fluorescence technique. In the cochlea, distinct fluorescence staining occurred for beta subunits in outer hair cells (OHCs), but no alpha subunits were identified. Beta subunits differ quantitatively between the three rows of OHCs, decreasing along a base-to-apex gradient in the cochlea. Both alpha and beta subunits were identified on spiral ganglion cells, adjacent nerve fibres and in vestibular hair cells (HCs). It would appear that they form an active complex in n-ACh-r at these locations.     

Inner ear pathology associated with Reye's syndrome

Severe pathological changes were observed in the inner ear tissues of a 2-month-old patient who died of Reye's syndrome after 5 days of hospitalization. In the organ of Corti, the inner hair cells appeared to be more severely damaged than the outer hair cells. Various degrees of degeneration were observed in all non-sensory epithelial cells lining the cochlear duct. In most turns of the cochlear duct, Reissner's membrane was ruptured and/or collapsed onto the organ of Corti. Likewise, both sensory and non-sensory cells of the vestibular end organs were markedly degenerated. These observations suggest that the inner ear tissues are acutely affected in patients with Reye's syndrome, and that the changes may cause impairment of hearing and/or equilibrium in patients who recover.     

Expression patterns of aquaporins in the inner ear: evidence for concerted actions of multiple types of aquaporins to facilitate water transport in the cochlea

Water transport between the perilymph and endolymph is important in regulations of volume and osmotic pressure of the inner ear labyrinth. It is now known that expression of water channels (aquaporins or AQPs) in the cell membrane dramatically increases the ability of water to cross epithelial cells. The aims of the current study were to investigate the cellular localization of AQPs by immunolabeling, and to study the developmental expression and relative abundance of various subtypes of AQPs. We report here that AQP3, AQP7 and AQP9 were expressed in the inner ear. Specific subtypes of AQPs were found in discrete regions expressed by both epithelial cells and fibrocytes in cochlear and vestibular organs. Semi-quantitative measurements showed that AQP4 and AQP1 were the two most abundantly expressed AQP subtypes in the inner ear, and their expressions were dramatically upregulated during development. These data showed a highly localized and largely non-overlapping distribution pattern for different subtypes of AQPs in the inner ear, suggesting the existence of regional subtype-specific water transport pathways, and global regulation of water transport in the inner ear may require concerted actions of multiple types of AQPs.     

Localization of melatonin and its receptors (melatonin 1a and 1b receptors) in the mouse inner ear

Abstract

Background: In the inner ear, evidence has been gathered indicating that melatonin plays important roles in inner ear physiology and pathophysiology. However, no attempt has been made previously to investigate the localization or expression of melatonin and its receptors in the whole inner ear.

Aims/objectives: To analyze the presence of melatonin and its receptors in the normal mouse inner ear.

Material and methods: C57BL6/J mice were used in this study. The localizations of melatonin, MT1a and MT1b in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion and endolymphatic sac (ES), were studied by immunohistochemistry.

Results: The organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory cells, vestibular dark and transitional cells, and ES epithelial cells showed an immunofluorescence reaction to melatonin, MT1a and MT1b.

Conclusion and significance: The present findings show that melatonin and its receptors (MT1a and MT1b) are present in the inner ear, thus supporting the hypothesis that melatonin plays a physiological role in the inner ear.     

前庭器官超微结构研究及结构图构建

目的通过扫描电镜观察小鼠椭圆囊、球囊、壶腹超微结构,研究分析并据此构建前庭器官新的结构示意图。方法选取3个年龄段小白鼠各10只,分别是年轻组≤2个月、中年组2~12个月、老龄组>12个月。分离出椭圆囊、球囊、壶腹,采用扫描电镜技术样品制备方法制备样品,应用扫描电镜进行样品观察。结果扫描电镜下可以得到:①椭圆囊斑及球囊斑不同层面图片:表面为堆积并相互黏附的"表面耳石",表面耳石下是无结构胶状质;底层表面耳石深入到无结构胶质层里;无结构胶质层下面是毛细胞纤毛及"纤毛间耳石"层,不同纤毛束之间均有纤毛间耳石存在,立于支持细胞表面,表面平坦;蜂窝状胶质物质联接无结构胶质层、纤毛间耳石及毛细胞纤毛。②壶腹超微结构的图片:嵴帽是无结构的胶状质与壶腹外侧壁紧贴,但较易分离,嵴帽和壶腹外侧壁之间有纤细的晶状体物质,在壶腹嵴两侧壁上也有纤细晶体物质(壶腹嵴表面耳石);不同的毛细胞纤毛之间有耳石结构的存在(壶腹嵴纤毛间耳石)。结论通过对前庭器官扫描电镜的观察,发现了椭圆、球囊斑及壶腹的新结构成分,由此构建出新的前庭器官超微结构示意图。     

Effect of gentamicin on the carbohydrates of the vestibular end organs: an investigation by the use of FITC-lectins

The effect of gentamicin on the glycoconjugates in the vestibular end organs is demonstrated using FITC-lectins. Five milligrams of gentamicin dissolved in 0.1 ml. saline was injected in a single dose into the middle ear of adult guinea pigs. Seven days after the injection, the fluorescent reactivity of Wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Concanavaline A (Con A), and Latyrus odoratus agglutinin (LOA) was decreased in the apical epithelial cell surface as well as in the gelatinous layer of the otolithic membrane of the maculae. In the cupula of the crista ampullaris, the reactivity for WGA and Con A was reduced. The otoconia, however, showed no detectable changes. This indicates that gentamicin may induce an altered carbohydrate metabolism resulting in a decrease of N-acetyl-glucosamine, mannose, galactose and glucose in the glycocalyx lining the epithelial cells as well as in the gelatinous layer of the otolithic membrane or cupula in the vestibular end organs.