Direct evidence of nitric oxide production in the guinea pig organ of Corti.

Production of nitric oxide (NO) in the organ of Corti of the guinea pig was investigated using the new fluorescence indicator 4,5-diaminofluorescein diacetate for direct detection of NO. The organ of Corti, lateral wall of the cochlea and isolated outer and inner hair cells were examined to locate NO production sites. The fluorescence intensities were augmented by stimulation with L-arginine or glutamate, and significantly increased after inoculation with lipopolysaccharide. This is the first direct evidence of NO production in the cochlea. NO may play an important role in the physiology of the organ of Corti and may also be involved in hearing disorders.     

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.     

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.     

Gentamicin increases nitric oxide production and induces hearing loss in guinea pigs

Objectives/Hypothesis: Gentamicin application is an important therapeutic option for Ménière's disease. However, even if given at intervals, a destruction of the cochlea was often observed in various animal models together with an increased content of nitric oxide (NO) and reactive oxygen species. The present study was undertaken to identify the correlation between hearing threshold alteration and the NO production in the lateral wall and organ of Corti of the guinea pig in response to gentamicin application. Study Design: Prospective animal study in guinea pigs. Methods: A single dose of gentamicin (10 mg/kg body weight) was injected intratympanally into male guinea pigs and the auditory brainstem responses were recorded before treatment and 1, 2, and 7 days after application. The organ of Corti and the lateral wall were removed from the bulla, incubated separately for 6 hours in cell culture medium and the amount of NO production was determined by chemiluminescence. Results: Gentamicin application resulted in a hearing threshold shift beginning on the second day after gentamicin application. This hearing impairment correlates simultaneously with an increased NO₂^? content—the stable oxidation product of NO—in the lateral wall. In the organ of Corti, a slight increase in NO₂^? production was seen as early as on day 1 after gentamicin injection. Conclusions: The correlation between hearing threshold shift and NO production in the cochlea leads to the assumption that increased NO contributes to gentamicin‐induced hearing impairment.     

豚鼠耳蜗外毛细胞和Deiters细胞的同时分离法

目的：探讨豚鼠耳蜗外毛细胞（OHC）和Deiters细胞（DC）同时分离的方法。方法：选健康杂色豚鼠，解剖出耳蜗基底膜，采用酶消化后机械分离。结果：可以同时获得一定数量，活性良好，长短不一的OHC和DC。结论：熟悉耳蜗的解剖特性，保持Corti器的完整及掌握机械吹打的力度是同时获取活性良好的OHC和DC的关键。     

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.     

Potassium induced release of GABA and other substances from the guinea pig cochlea

Gamma-aminobutyric acid (GABA) has been proposed as a neurotransmitter of a subset of efferent nerve fibers in the mammalian cochlea. We tested this hypothesis by examining if GABA was released by high concentrations of K+ from the guinea pig cochlea. Artificial perilymph solutions containing either normal K+ (5 mM) or high K+ (50 mM) were perfused through the perilymphatic compartment of the guinea pig cochlea while collecting the effluent. Nineteen primary amines including GABA were quantified in the effluent by HPLC. This was carried out in normal animals and in animals pretreated with ethacrynic acid and kanamycin to destroy the organ of Corti. Significantly greater levels of GABA, taurine, glutamate, aspartate, glycine and three unidentified substances appeared in effluent collected during exposure of the cochlea to solutions containing higher K+ than normal K+. Compared to normal animals, destruction of the organ of Corti significantly decreased the K(+)-induced release of GABA, taurine, glutamate, aspartate, glycine and one of the unidentified substances; although significant release of glutamate and taurine still occurred in the destroyed ears. The release of GABA is consistent with it being a neurotransmitter in the cochlea. In addition the results: confirm the release of glutamate and taurine from the organ of Corti; suggest that additional substances may be released; and demonstrate the release of glutamate and taurine from tissue other than the organ of Corti.     

The electrochemical and fluorescence detection of nitric oxide in the cochlea and its increase following loud sound

A nitric oxide (NO)-selective sensor (tip diameter 30 microm) was inserted into the perilymph of the basal turn of the guinea pig cochlea. The basal level and stimulation-induced changes of NO were measured. The mean (+/-S.E.M.) basal level of NO was 273+/-42.9 nM. Following perilymphatic perfusion of the artificial perilymph containing NO synthase (NOS) substrate L-arginine (100 microM) combined with cofactor (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride (100 microM), a rapid and significant increase of NO to a mean concentration of 392+/-32.3 nM (P < 0.01, n = 10) was recorded. In contrast, a significant decrease of mean NO concentration to 180+/-32.7 nM (P < 0.01, n = 10) was observed following the perfusion of the NOS-inhibiting agent N(G)-nitro-L-arginine methyl ester (100 microM). No change in the NO concentration was found following the perfusion of either artificial perilymph or N(G)-monomethyl-D-arginine (100 microM) solution employed as controls. Broadband noise exposure (3 h/day at 120 dBA SPL) for three consecutive days produced an increase in NO concentration to 618+/-60.7 nM (P < 0.05, n = 10) in the perilymph. In addition, by using specific dyes for NO, 4,5-diaminofluoresceine diacetate and for the reactive oxygen species (ROS), dihydrorhodamine 1,2,3, the distribution of NO in the whole mounts of the organ of Corti and the production of ROS in vivo in the organ of Corti were investigated in both control (n = 5) and noise-exposed (n = 5) animals. The more intense NO and ROS fluorescence was observed in both the inner and outer hair cells in the noise-exposed groups. It is proposed that both the basal level and the increase in NO concentration following the addition of substrate (L-arginine) are produced by the constitutive NOS while the elevated NO and ROS following noise exposure indicate that NO may be involved in noise-induced hearing loss.     

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.     

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.     

Distribution of pendrin in the organ of Corti of mice observed by electron immunomicroscopy

The distribution of pendrin, which is encoded by the Pendred syndrome gene, has been investigated immunohistochemically in the inner ear. In the cochlea, pendrin has been found in the spiral prominence, external sulcus cells, Hensen’s cells and Claudius cells, but its expression in the organ of Corti remains unclear. We examined whether pendrin localizes in the organ of Corti by postembedding immunogold analysis. In the organ of Corti, gold particles were clearly observed in outer and inner hair cells, including the stereocilia. The density of the particles was especially high in the cuticular plates of the hair cells. Gold particles were also detected in the external sulcus, in part of the spiral ligament adjacent to the external sulcus, in supporting cells, and in the spiral ganglion of the cochlea. Our study revealed that pendrin occurs in the organ of Corti. The role of pendrin in the organ of Corti and its association with the Cl^- or pH regulation of neurotransmission require further study.     

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.     

胚胎小鼠耳蜗感觉上皮的形态发生

目的了解胚胎小鼠耳蜗感觉上皮发育的形态学特征及规律。方法利用光镜、扫描电镜、透射电镜观察从胚胎第8天到刚出生期间C57BL/6小鼠耳蜗感觉上皮发育过程中的形态变化。结果C57BL/6小鼠胚胎第10.5天耳蜗开始发育,感觉上皮由高柱状上皮细胞组成,到胚胎第14天才开始分化为原始的毛细胞;胚胎第16天耳蜗底转Corti器原基形成,毛细胞和支持细胞初具雏形;到出生时,耳蜗形态基本成熟,但底转Corti器仅趋向成熟,蜗尖才形成Corti器原基。结论小鼠耳蜗感觉上皮发育的形态变化在胚胎第13天到出生时最明显,感觉细胞都来源于一种高柱状上皮细胞,到胚胎第14天开始分化为毛细胞;而Corti器的分化成熟是从底转向顶转进行的,出生时尚未完全发育成熟。     

Localization of Nitric Oxide Synthase Isoforms in the Human Cochlea

The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.     

水杨酸钠作用后大鼠耳蜗热休克蛋白27表达的研究

目的　研究水杨酸钠作用后大鼠耳蜗热休克蛋白 2 7(heatshockprotein2 7,HSP2 7)的表达特点 ,探讨HSP2 7在水杨酸钠耳毒性中的作用及意义。方法　用免疫组化SP法结合图像分析技术检测水杨酸钠注射后大鼠耳蜗HSP2 7的表达。结果　HSP2 7在正常及水杨酸钠作用后大鼠耳蜗各回均有不同程度的染色 ,主要阳性部位是Corti器、螺旋神经节、螺旋韧带和血管纹。但水杨酸钠作用后HSP2 7在耳蜗各阳性部位的表达均明显增强 ,其中以Corti器的表达更明显 ,P值均小于 0 .0 1。结论　HSP2 7在大鼠耳蜗组织表达有选择性 ,水杨酸钠能够明显诱导HSP2 7在大鼠耳蜗中的表达 ,HSP2 7可能对耳蜗形态结构损害后的修复起作用 ,且与耳鸣的产生发展可能有关。     

Localization of nitric oxide synthase isoforms in the human cochlea

The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.     

Cation distribution in the organ of Corti of the guinea pig

Cations were precipitated with potassium antimonate in the cochlea of the guinea pig and the distribution of the formed precipitates was studied by electron microscopy. The precipitate density in different cells of the organ of Corti was determined on electron micrographs by counting numbers of precipitates per unit area. The spatial distribution of the precipitates was also determined by electron spectroscopic imaging (ESI). Significant differences were found among the cells of the same tissue being analyzed. These precipitate-rich cells may play a role in a postulated current flow in the organ of Corti.     

Cation distribution in the organ of Corti of the guinea pig

Summary Cations were precipitated with potassium antimonate in the cochlea of the guinea pig and the distribution of the formed precipitates was studied by electron microscopy. The precipitate density in different cells of the organ of Corti was determined on electron micrographs by counting numbers of precipitates per unit area. The spatial distribution of the precipitates was also determined by electron spectroscopic imaging (ESI). Significant differences were found among the cells of the same tissue being analyzed. These precipitate-rich cells may play a role in a postulated current flow in the organ of Corti.     

The ototoxic potential of EMLA. A new local anesthetic for the tympanic membrane

Instillation of EMLA, a new local anesthetic, into the middle ear of the guinea pig caused severe morphological damage to the organ of Corti in the first 4 mm from the round window. Further up the cochlea, only derangements of the stereocilia were found. The extent of morphological damage was the same, whether the agent was administered once or several times. The ototoxic potential of EMLA was obvious and is probably due to direct damage in areas where present in high concentrations. Over a short distance of approximately 0.1 mm there is a transition from a total destruction of the organ of Corti to a completely normal morphology.