Immunoelectron microscopic localization of nitric oxide synthase III in the guinea pig organ of Corti

Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III was also found in the cytoplasm and in the nuclei of inner and outer hair cells. Immunoreactivity was not distributed homogeneously within receptor cells. Numerous gold particles could be identified at the border of the cuticular plates, in the middle parts of the stereocilia and in the cytoplasm. Gold-labeled anti-NOS III antibodies in these sites were seen mostly on the cytoplasmic side of the submembranous cisterns in the vicinity of mitochondria and in the central parts of the hair cells, whereas the cisterns were nearly free from any immunoreactivity. NOS III was also detected in the efferent and afferent nerve endings that were located at the basal and basolateral side of the outer hair cells. Some immunoreactivity was visible in different nerve fibers of the inner and outer spiral tunnels. Besides this, gold-labeled antibodies were also present in the cuticular plate of inner and outer pillar cells, in the cytoskeletal elements located in the apical parts of Deiters cells, forming the lamina reticularis, and in the cytoskeletal-containing region of the cytoplasm of those Deiters cells located at the basal side of the outer hair cells. The role of the NOS III immunoreactivity identified in the organ of Corti was consistent with respect to hair cell and tissue modulation. Received: 24 September 1997 / Accepted: 26 June 1998     

Electron microscopic localization of nitric oxide I synthase in the organ of Corti of the guinea pig

Nitric oxide synthase (NOS) activity has been detected previously in the mammalian cochlea at a light microscopic level. Here we present results of electron microscopic analysis for post-embedding immunoreactivity of neural-type NOS I in the cochlea of the guinea pig. Strong enzyme immunoreactivity was identified in the cytoplasm of inner and outer hair cells. Gold-labeled NOS I antibodies were mainly located in electron-dense areas of the cytoplasm, whereas electron-lucent regions of the receptor cells were nearly free from any immunoreactivity. In both types of hair cells anti-NOS I antibodies were also visible in the cuticular plates, hair bundles and nuclei. Further ultrastructural analysis revealed that the submembranous cisternae of the outer hair cells were nearly free from any reaction product, demonstrating that the whole cytoplasm of this hair cell was not immunoreactive. Other NOS I immunoreactivity was identified in the cuticular plates of the inner and outer pillar cells and in the cytoskeletal elements located in the apical parts of Deiter cells, forming the lamina reticularis or in cytoskeletal-containing regions in basal Deiter cells. Anti-NOS antibodies were visible in the nuclei of various cell types. Our findings suggest that nitric oxide produced by NO I synthase in the organ of Corti may act as a modulator of hair cell physiology during the processes of signal transduction with frequence selectivity.     

Postnatal changes in the reticular lamina of the guinea pig organ of Corti

The dimensions of the apical surfaces of hair cells were measured in guinea pigs, aged from 3 weeks before term to 25 weeks after birth. In the basal two-thirds of the cochlea, the apical surfaces of the outer hair cells and their supporting cells changed with age, shrinking in a direction radial across the cochlear duct. There was an associated widening of the angle of the ‘V’ of the rows of stereocilia. Further apically, between 12 and 16 mm from the base of the cochlea, the outer hair cells and their supporting cells underwent the opposite change, becoming wider in a radial direction with age. The changes were seen before birth and continued for more than 3 weeks after birth. The results suggest that the guinea pig cochlea continues certain developmental processes for a considerable time after birth.     

Immunocytochemical detection of peptides in the guinea pig cochlea

Summary The cochleae of juvenile guinea pigs were investigated for the presence of several neuropeptides. Glucagon, insulin, CCK and -endorphin immunoreactive neurons and nerve fibers as well as hair cells were demonstrated by the peroxidase antiperoxidase technique. Small amounts of substance P were also found in different sites in the inner ear. In contrast, prolactin-like material could not be found at all. These findings have significance with regard to the putative role of neuropeptides in neuromodulation.     

Transplacental kanamycin ototoxicity in the guinea pig

Summary The effect of kanamycin on the cochlear sensory epithelium of albino guinea pig fetuses was studied histologically following the administration of 200 or 400 mg/kg body weight kanamycin sulfate to the pregnant mothers for 8 consecutive days at different stages of gestation. Surface view analysis of Corti's organ revealed slight damage following treatment in the middle trimester of gestation and marked damage due to treatment in the last trimester. The pattern of hair cell loss induced during and after the functional differentiation of the cochlea resembled the pattern of ototoxic lesions in the adult ear. The assumed mechanisms for induction of teratogenesis by kanamycin in the fetal organ of Corti are discussed.Established investigator of the Chief Scientist's Bureau, Ministry of Health, Israel     

Lipopolysaccharide-induced expression of inducible nitric oxide synthase in the guinea pig organ of Corti

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiter’s cells pointing to the cuticular membrane, Hensen’s cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O₂⁻. On the basis of these findings, it can be concluded that NO together with O₂⁻, which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.     

Evidence for a possible NOS back-up system in the organ of Corti of the guinea pig

Recently, the two Ca²⁺/calmodulin-regulated nitric oxide synthase isoforms, nNOS and eNOS, and NO itself have been identified in the cochlea of vertebrates using specific antibodies and a new fluorescence indicator. In order to acquire more information about the quantitative and spatial distribution of these two constitutively expressed NOS isoforms (cNOS) in the organ of Corti at the cellular and subcelluar levels, ultrathin sections of London resin (LR) White-embedded cochleae of the guinea pig were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labeled secondary antibody and the amount of the immunoreactions/m² was quantified for different cell types and subcellular regions. Both NOS isoforms were identified to varying degrees in the same cell types and subcellular regions. A prominent eNOS immunoreactivity was identified in nearly every cell type. In all analyzed animals the highest number of gold-coupled anti-eNOS antibodies was always seen in the cells of the reticular lamina, especially in the cuticular structures of outer and inner hair cells, pillar cells and apical Deiters' cells. Also the microtubuli-containing cytoplasmic regions of Deiters' cells were scattered with gold-coupled anti-eNOS antibodies. A clear eNOS immunoreaction was also found in the remaining cytoplasm of inner and outer hair cells and in the apical Deiters' cells. Numerous anti-nNOS antibodies were located in the outer hair cells and in the cuticular structures of the apical Deiters' cells. The amount of the gold-labeled anti-nNOS antibodies in the cuticular plates of the pillar cells and outer hair cells and in the cytoplasm of inner hair cells and apical Deiters' cells were clearly less but still above unspecific background labeling. The spatial co-localization of the two NOS isotypes in the same cell regions was proven in double-labeling experiments. The spatial distribution of the two cNOS isoforms confirmed recent findings of other authors who localized NO distribution and production sites. The cNOS co-expression with similar function in the same cell type and subcellular regions may represent a functional "back-up system" in which one NOS isoform can replace the other in case of pathophysiological malfunction.     

An anionic charge barrier in the guinea pig cochlea

Summary A charge barrier has been found in the the glomerular basement membrane of the kidney and plays an important role in the filtration of solutes. In the present study, we used electron microscopy to localize anionic sites of a similar charge barrier in the guinea pig cochlea. Polyethyleneimine (PEI) was used as a cationic marker to detect anionic sites. Our results showed a localization of PEI with regular interspaces, indicating the anionic sites to the charge in the capillary basement membrane of the stria vascularis and the spiral ligament, and in the basal lamina of Reissner's membrane and the spiral prominence. This charge barrier, as well as structural size barrier, may play an important role in the maintenance of normal inner ear functions.     

钙蛋白酶在卡那霉素致毒豚鼠耳蜗中的表达

目的探讨钙蛋白酶(calpain)在卡那霉素(kanamycin,KM)致耳中毒豚鼠耳蜗的表达。方法将豚鼠随机分成对照组、KM 3d组、KM 7d组和KM1 4d组,应用免疫组织化学SABC(streptavidin-biotin peroxidae complex,链霉亲合素-生物素过氧化物酶复合物)法和显微图像分析技术检测耳蜗中钙蛋白酶的表达,用药前后给予短纯音刺激检测听性脑干反应阈值,观察豚鼠听力的变化。结果对照组calpain 1阳性免疫反应主要见于耳蜗毛细胞、螺旋神经节、血管纹和螺旋韧带,以螺旋神经节的染色较深,而其它部位均呈阴性。肌肉注射KM后,calpain 1在耳蜗中的阳性反应部位与对照组大致相同,显微图像分析结果表明,随着给药天数的增加,calpain 1在耳蜗上述部位的阳性反应逐渐减弱。Calpain 2在各组豚鼠耳蜗中的表达部位与calpain 1的相同,显微图像分析结果提示,随着给药天数的增加,calpain 2在耳蜗上述部位的阳性反应逐渐增强。结论正常豚鼠耳蜗中有calpain 1和calpain 2的表达。注射KM后,随着给药天数的增加,calpain 1在耳蜗的表达逐渐减弱,而calpain 2的表达则逐渐增强,提示calpain 2可能参与了卡那霉素致耳中毒的过程。     

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.     

谷氨酸调节耳蜗内毛细胞游离钙的实验观察

目的探索谷氨酸(glutamate,Glu)对离体耳蜗内毛细胞(innerhaircells,IHC)内钙信号的调控作用及其生理病理意义。方法在激光共聚焦显微镜下用钙敏荧光探针Fluo-3作为指示剂,观察外源性谷氨酸对分离的10个豚鼠耳蜗IHC胞内游离钙离子浓度(［Ca2+］i)的影响。结果分离好的正常IHC呈烧瓶形状,有明显的颈部,皮板上可观察到静纤毛,大球形的细胞体中间可见圆形的细胞核。形态完好的IHC大约存活2h,Fluo-3钙敏荧光探针染色后IHC胞体、胞核及表皮板有明显的染色梯度,表明游离Ca2+浓度从细胞核向细胞质逐渐减少。终浓度为3.85μmol/L的Glu对游离IHC内［Ca2+］i有增高趋势,而对游离外毛细胞(outerhaircells,OHC)内［Ca2+］i浓度无影响。观察10个IHC,发现9个［Ca2+］i浓度增加,1个无变化;观察10个OHC,发现7个［Ca2+］i无变化,3个略有下降。当Glu浓度增高后,IHC内［Ca2+］i先是迅速升高,继而逐渐下降。IHC外形由烧瓶状逐渐变成球形,提示IHC水肿变性。结论Glu可选择性调控IHC内［Ca2+］i,而对OHC内［Ca2+］i无影响,而过量的Glu刺激,可造成IHC［Ca2+］i的堆积,从而IHC水肿变性。     

豚鼠耳蜗一氧化氮合酶异型体的定位

目的研究一氧化氮合酶(NOS)的异型体在豚鼠耳蜗的定位分布,以探讨一氧化氮(NO)在内耳听觉生理和病理生理机制中的作用。方法使用特异性NOS异型体抗体,采用ABC免疫组化染色法,观察NOS异型体在正常豚鼠耳蜗的定位表达。结果NOS Ⅰ主要分布在内骨膜、螺旋神经节的核周体、螺旋韧带和Corti's器的细胞。NOSⅢ是耳蜗的主要NOS异型体免疫染色,其主要免疫染色分布于耳蜗神经、螺旋神经节核周体、螺旋韧带和耳蜗毛细血管球的内皮细胞,也见于Corti's器的细胞和神经纤维。NOS Ⅱ在正常豚鼠耳蜗内不表达。 结论结构型NOS(cNOS)表达在耳蜗的多个部位,表明NO参与内耳的正常生理功能,包括神经突触的神经传导、耳蜗血流的调节和耳蜗的骨代谢。     

豚鼠耳蜗局灶性微循环障碍模型的建立

目的 :探讨耳蜗局灶性微循环障碍对耳蜗血流、听功能及耳蜗形态学的影响。方法 :采用光化学诱导法 ,经豚鼠颈外静脉注射 2 %四碘四氯荧光素二钠 (RB)后 ,用 (5 4 0± 4 0 )nm的绿色光照射耳蜗第 2转外侧壁(范围约 1.2mm× 1.0mm) ,诱导该部位血管纹微血栓形成。结果 :诱导后观察 5h ,被照射部位的耳蜗血流随时间逐渐下降 ,动作电位阈移值逐渐提高 ,二者的变化呈负相关。耳蜗铺片显示 ,被照射部位诱导后 30min耳蜗外侧壁毛细血管扩张 ,部分毛细血管轮廓不清、血流中断 ;诱导后 90～ 30 0min ,大片的毛细血管萎缩、闭塞、数目减少 ;诱导 3h后 ,光照区内部分外毛细胞、内毛细胞坏死缺失 ,外毛细胞病变较内毛细胞为重 ,毛细胞坏死区的长度为 (115 2 .5 0± 36 3.2 6 ) μm(n =4 )。非光照区CBF及形态学均无明显变化。结论 :光化学法可诱导耳蜗外侧壁局限性微血管损伤 ,造成豚鼠听力下降 ,为耳蜗不同部位微循环障碍导致不同类型听力损伤的研究提供了实验依据 ,并为耳蜗血栓性疾病探索新的治疗方法提供了动物模型。     

Cross-links between stereocilia in the guinea pig organ of Corti, and their possible relation to sensory transduction

Hair cells of the guinea pig cochlea were preserved for electron microscopic examination by fixing in glutaraldehyde without the use of osmium. An extensive array of cross-links was seen between the stereocilia, by both scanning and transmission electron microscopy. The stereocilia were linked together laterally, particularly near their apical ends, by links running approximately at right angles to the long axis of the stereocilia. One set joined stereocilia of the same row, and another set joined stereocilia of the different rows, holding the tips of the shorter stereocilia in towards the longer stereocilia of the next row. In addition, the tip of each shorter stereocilium on the hair cell gave rise to a single, upwards-pointing link, which ran up to join the taller stereocilium of the next row. We suggest that distortion of this link would give rise to sensory transduction. On this basis, we are able to explain the V shape of the rows of stereocilia on outer hair cells. Within the rows, the three-dimensional arrangement of the stereocilia was different from that seen conventionally. Rather than standing parallel, the stereocilia of the different rows tapered in together at the tips, presumably held by the laterally-running cross-links. In addition, a membrane roughness, particularly pronounced in the region of the stereocilium which gives rise to the cross-links, was seen. However, the lateral and basal surface membranes of the hair cell, and the membranes of the internal organelles, had a more conventional appearance.     

Alteration of the calcium content in inner hair cells of the cochlea of the guinea pig after acute noise trauma with and without application of the organic calcium channel blocker diltiazem.

Calcium ions are known to be important to the process of signal transduction across the apical and basal sides of the inner hair cells. Calcium channel antagonists have been demonstrated by light microscopy to provide protection against acoustic trauma. To evaluate the protective effect of calcium channel blocker on the inner ear cells to noise exposure, the amount of the histochemical reaction products formed in the cytoplasm of the inner hair cells of the guinea pig after application of pyroantimonate was measured by an image processing system connected to an energy-filtering transmission electron microscope (EFTEM). Compared to untreated control specimens (experimental animal group I) the amount of precipitable calcium had clearly increased in the inner hair cells of noise-exposed cochleae 60 h after an acute noise trauma (group III). In addition, small electron- lucent areas could be identified in the cytoplasm of the hair cells probably representing damage to the cellular fine structure. When the calcium channel blocker diltiazem was administered without any additional noise exposure the calcium content was drastically reduced in the inner hair cells (group II), but when the antagonist was given before and after acute noise trauma (group IV), the calcium content in the inner hair cells was nearly compared to the amount determined in the untreated group of animals. The role of diltiazem is discussed in respect of tissue protection and to the maintenance of the calcium-dependent physiological processes in the inner hair cells during signal transduction.     

高渗透压对豚鼠耳蜗离体外毛细胞膜电位的影响

为了解高渗透压对豚鼠耳蜗离体活外毛细胞膜电位及细胞长度的影响。我们运用粘附式细胞仪和膜电位敏感针ＤｉＢＡＣ，对豚鼠耳蜗离体外细胞在高渗环境中的膜电位相对值及细胞长度的变化进行了动态测定。     

Comparative oto-vestibular effects in the pigmented guinea pig after dibekacin and netilmicin treatment

Summary We treated groups of pigmented guinea pigs with either intramuscular netilmicin or dibekacin at 100 and 150 mg/kg per day for 3 weeks. Saline was used as the control solution. All animals were tested weekly for both vestibular and auditory functions. The vestibular function was evaluated by the duration of postrotatory nystagmus (PRN) elicited by interrupting the rotation of the animal around the vertical axis; auditory function was evaluated by the threshold response for the Preyer's pinna reflex (PPR). All animals were then sacrificed and either their labyrinths or Corti organs were processed for further investigations using the scanning electron microscope (SEM). The duration of PRN decreased over the treatment period in all of the groups as a result of adaptation. However, 150 mg/kg dibekacin produced a significant decrease of the PRN responses as compared to the control and other groups. This effect also continued during the recovery period. Likewise, the PPR threshold of the animals receiving 150 mg/kg dibekacin showed a significant increase at the end of the treatments and during the recovery period, while the other dibekacin group had no significant auditory impairment. Netilmicin at both doses did not significantly affect responses following either vestibular or auditory stimulations. SEM observations demonstrated that the sensory epithelia of the labyrinths and Corti organs affected by 150 mg/kg dibekacin had great losses of stereocilia, while comparable doses of netilmicin (150 mg/kg) had only very moderate losses of stereocilia in the labyrinths but not in the Corti organs.     

Localization of dynorphin B-like and alpha-neoendorphin-like immunoreactivities in the guinea pig organ of Corti

Antiserum to dynorphin B and antiserum to alpha-neoendorphin were used in an immunocytochemical examination of the guinea pig organ of Corti. Immunoreactive staining for these two proenkephalin B (prodynorphin)-derived peptides was seen in the lateral system of olivocochlear efferents in the organ of Corti: the inner spiral bundle, the tunnel spiral bundle and by the bases of inner hair cells. Immunoreactive staining with both antisera was also seen in efferent terminals on outer hair cells at or above the level of the nucleus, which may represent terminals of either the lateral or the medial system. No immunoreactive staining was seen in tunnel crossing fibers and at bases of outer hair cells corresponding to the medial system of efferents. The staining seen with antiserum to dynorphin B and to alpha-neoendorphin has similar distribution to that seen with antisera to methionine enkephalin; there may be co-localization of these neuropeptides in the lateral system of efferents. Choline acetyltransferase-like immunoreactivity (co-localized with enkephalin-like immunoreactivity in the lateral system in the brainstem) and glutamic acid decarboxylase (GAD)-like immunoreactivity have also been found in olivocochlear efferents. Further studies will be necessary to determine if the dynorphins are co-localized with other neurotransmitter candidates and what their interactions may be.     

豚鼠耳蜗各回外毛细胞L型钙通道的分布

目的探讨耳蜗2～4回外毛细胞(outer hair cell,OHC)L型钙通道分布密度.方法豚鼠耳蜗外毛细胞L型钙通道经DM-BODIPY DHP和RH414标记后用激光扫描共聚焦显微镜观察,以DM-BODIPY DHP / RH414荧光比值大小指示L型钙通道密度的高低.结果①应用硝苯地平阻断后,质膜的DM-BODIPY DHP荧光染色明显减弱,表明其钙通道为特异性L型钙通道;②L型钙通道密度从低频区(第4回)向高频区(第2回)有逐渐增加的趋势,方差分析示第4回和第2回OHC之间L型钙通道的分布差异有显著性意义;③OHC质膜不同区域L型钙通道分布差异无显著性意义.结论耳蜗2～4回OHC之间L型钙通道分布的差异可能是耳蜗音位分布图的分子机制之一.     

Enkephalin-like immunoreactivity in the guinea pig organ of Corti: ultrastructural and lesion studies

Enkephalin-like immunoreactivity (ELI) was examined in a light and electron microscopic study of the normal guinea pig cochlea and of cochlea de-efferented through evulsion of the vestibular nerve. Antiserum to methionine enkephalin, 164, which gives immunoreactive labeling of only the lateral system of efferents, and antiserum 163, which gives immunoreactive labeling of lateral and medial efferents, were used. In de-efferented cochleae no immunoreactive labeling was seen with either antiserum, confirming that in the organ of Corti ELI is confined to efferents. At the ultrastructural level antiserum 163 but not 164 showed ELI in efferent terminals at the base of outer hair cells. ELI with 164 was seen in efferents ending on outer hair cells at the level of the nucleus. Medially, ELI was seen with both antisera in the inner and tunnel spiral bundles. Efferent terminals containing ELI were seen apposing afferent dendrites, other efferents and the inner hair cell. However, only rarely could synaptic contacts be unambiguously identified and then only with afferent dendrites.