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     

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.     

Actin-binding and microtubule-associated proteins in the organ of Corti.

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.     

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.     

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.     

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.     

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.     

Immunohistochemical localization of cyclooxygenase isoforms in the organ of Corti and the spiral ganglion cells of guinea pig cochlea

Prostaglandins have been used in experimental models and clinical studies for the therapy of sudden hearing loss and tinnitus with conflicting results. However, little is known about the rate-limiting enzymes of prostaglandin synthesis in the inner ear, the generally constitutively expressed cyclooxygenase 1 (COX-1) and the distress-inducible cyclooxygenase 2 (COX-2). To extend our knowledge concerning the physiological expression and localization of these two enzymes, immunohistochemical stainings of the guinea pig cochlea were performed. Light microscopical analysis revealed a homogenous distribution of COX-1 within nearly all cell types of the organ of Corti, but no COX-1 expression in the cuticular plates of pillar cells. COX-2 was found to be expressed in all cell types, with much stronger expression in Hensen cells, neighboring Deiters cells and cuticular plates of outer hair cells. Both COX-1 and COX-2 immunoreactions were also found in the spiral ganglion. We conclude that both COX subtypes are expressed in the guinea pig cochlea under physiological conditions. The prominent expression of the distress-inducible COX-2 isoform in cell types under mechanical stress during noise reception might support the hypothesis of a cytoprotective function of COX products in hearing and in cellular stress situations like intense noise exposure.     

Tubulin and microtubules in cochlear hair cells: comparative immunocytochemistry and ultrastructure

The distribution of tubulin has been investigated in surface preparations of the guinea pig organ of Corti using indirect immunofluorescence microscopy. Two different monoclonal antibodies to tubulin produce similar distinct patterns of labelling in hair cells. Labelling is greater in inner hair cells than outer hair cells. It occurs in rings around the cell apex, and in a meshwork below and channels through, the cuticular plate. In outer hair cells from the apical region of the cochlea, labelling occurs around the location of a basalward protrusion of the cuticular plate. These patterns correlate with the location of microtubules observed using transmission electron microscopy. A large patch of labelling occurs on the strial side of the cell corresponding to the largest channel through the cuticular plate and the kinociliary basal body. Strands of labelling are seen running parallel to the long axis of the cell between the subcuticular and synaptic region. Many more of these strands are seen in the inner hair cell than the outer hair cell and may correspond to tracks of microtubules transporting neurotransmitter vesicles or other organelles. In outer hair cells, intense labelling and many microtubules are seen in the subnuclear region. The possible roles of the different microtubule arrangements are discussed.     

Evidence for an Akt-kinase/NO/cGMP pathway in the cochlea of guinea pigs

The protein kinase Akt (protein kinase B) can be activated by numerous growth factors via PI-3 kinase-generated phosphoinositides and is thought to have anti-apoptotic properties. Activated Akt/PKB boosts the activity of endothelial NO synthase (NOS III), which has been found in the key areas of the inner ear (e.g., hair cells and stria vascularis). In order to localize activated Akt/PKB (phospho-Akt) in the cochlea of guinea pigs, sections of ten temporal bones were observed immunohistochemically. The strongest immunoreactivity was found in and underneath inner hair cells (IHC). Within the organ of Corti, reactivity was found in supporting cells, while outer hair cells remained unstained. Spiral ganglion cells, the endothelium of the lateral wall and the vascular area of the modiolus showed moderate staining. The results give evidence that activated Akt/PKB influences the activity of the NO/cGMP pathway in the cochlea. Because of the antiapoptotic properties, activated Akt should now be examined under non-physiological conditions.     

Morphological changes of cochlea in a strain of new-mutant mice

The hearing ability and histological characteristics of the cochlea of a strain of new-mutant mice were analyzed. This new mutant arose as a spontaneous mutation in the C3H/He stock. The genetic mode is autosomal recessive and the animals show abnormal behavior such as circling, head-tossing and hyperactivity. The audiological findings exhibited no recordable auditory brain stem response (ABR) in any homozygotes at ages ranging from 11 days to 117 days. For morphological examination, we used 36 homozygote with ages ranging from 10 days to 18 months. The primary morphological abnormalities were observed in the organ of Corti. The stereocilia of the outer hair cells showed disarray throughout the whole cochlea, although outer hair cell cytoplasm became fully developed, including the nerve terminals. Age-dependent degeneration of the outer hair cells subsequently occurred from the basal to the apical part of the cochlea. The earliest change demonstrated in the outer hair cells was cuticular degeneration. Although the abnormalities of the inner hair cells occurred late, a complete loss of inner and outer hair cells was demonstrated. The stria vascularis was well preserved at a later age as were spiral ganglion cells. These histological findings confirm that this mouse is classified as a neuroepithelial-type mutant. As this animal was expected to have a single gene abnormality, molecular genetic studies on this animal can provide important information on the nature of histological changes of the hair cell from a mode of gene action.     

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.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathological changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.     

Spatiotemporal development of cochlear innervation and hair cell differentiation in the rat

The apical cytoskeleton of cochlear hair cells is largely comprised of actin microfilaments and actin-associated proteins, of which fodrin is one of the most prominent. We studied the development of this mechanosensory apical portion of cochlear hair cells of the rat by fluorescence microscopy using rhodamine conjugated phalloidin to detect F-actin and an antibody against alpha-fodrin. An antibody against the 160 kDa neurofilament polypeptide was used for tracing nerve fibers. The first sign of differentiation of the mechanosensory region, actin-containing stereocilia, was observed on the 19th gestational day in the inner hair cells of the basal coil. The appearance of expression of cytoskeletal actin in the cochlear hair cells proceeded gradientally from basal to apical coil and from inner to outer hair cells. Corresponding maturation sequences were observed in the development of fodrin immunoreactivity in the cuticular plates, but the first evidence of this reactivity was found one day later than the appearance of stereocilia in the hair cells at the same location. Also the penetration of neurofilament-positive neurites into the sensory epithelium followed the same kind of longitudinal and radial maturation gradients throughout the cochlea. Fibers were revealed beneath the sensory cells shortly before the first appearance of differentiation of their mechanosensory region. The results suggest that ingrowing nerve fibers may influence the timing of the apical cytoskeleton differentiation in cochlear hair cells or that both these processes could be controlled by the same external signals that are gradientally expressed throughout the cochlea.     

An immunohistochemical method for the study of aminoglycoside ototoxicity in the guinea pig cochlea using decalcified frozen sections

Summary An immunohistochemical technique with decalcified frozen sections was used to study aminoglycoside ototoxicity. Decalcified guinea pig cochleas were cut with a fine blade parallel to the plane of the modiolus to facilitate the penetration of inclusion material and the manipulation of frozen sections. Light microscopy was carried out and additional frozen sections were employed for an immuno-electron microscopic study. Twenty-four hours after a single transtympanic injection of 10 mg gentamicin, there was a definite distribution of the drug in only type I hair cells of the ampullae as well as in both inner and outer hair cells along the length of the cochlea. In those animals treated intraperitoneally with 200 mg/ kg amikacin for 8 days, the drug was located in the outer hair cells of the cochlea, with a tendency to decrease from base to apex and in the inner hair cells towards the apex.Read at the XVIIIth International Congress of Audiology, Prague, Czechoslovakia, 26 August 1986     

Immunocytochemical localization of glutamate immunoreactivity in the guinea pig cochlea

The localization of glutamate immunoreactivity was examined in the guinea pig cochlea using affinity purified polyclonal antibodies to glutamate and immunoperoxidase post-embedding staining techniques on one micron plastic sections. Glutamate immunoreactive staining was seen in both inner and outer hair cells and in spiral ganglion cells and auditory nerve fibers. These results support the hypothesis that glutamate may function as the hair cell transmitter or as a precursor to the transmitter and add further support for an excitatory amino acid as the transmitter of the auditory nerve.     

豚鼠耳蜗一氧化氮合酶的分布

目的 用组织化学法,通过观察NADPH-黄递酶的活性了解一氧化氮合酶在豚鼠耳蜗内分布。方法 经4％多聚甲醛心脏灌注固定后,取出耳蜗,经3％依地酸脱钙后,作厚10μm冰冻切片,用辅酶Ⅱ孵育液在37℃条件下孵育l小时。结果 发现在耳蜗内、外毛细胞底部与耳蜗神经末梢接头处及毛细血管球内皮细胞有明显NADPH-黄递酶活性,此外在内、外柱细胞、支持细胞、血管纹及螺旋神经节细胞也有NADPH-黄递酶活性反应。结论 NO在维持耳蜗正常神经传导及毛细血管张力中起着重要作用。     

Ryanodine receptor localisation in the mammalian cochlea: an ultrastructural study

Calcium-induced calcium release (CICR) in the mammalian cochlea has been suggested to enhance neurotransmitter release from inner hair cells and facilitate the efferent response in outer hair cells. Light microscopic evidence exists for the presence of ryanodine receptors in the organ of Corti but there is so far no information about their ultrastructural localisation. We have therefore used post-embedding immunogold labeling with antibodies that predominantly recognise ryanodine receptor isoforms 1 (RyR1) and 2 (RyR2) to investigate their distribution in rat cochleae. In inner hair cells, the highest levels of labeling were observed over an area of rough endoplasmic reticulum that lies in the cytoplasmic region beneath the nucleus; in outer hair cells, the cytoplasmic region above the nucleus displayed most labeling. Labeling was also associated with the subsurface cisternae adjacent to the lateral membranes of both types of hair cell, with the efferent terminals on the outer hair cells and was observed in adjacent supporting cells. Labeling in outer hair cells was significantly higher than that in inner hair cells or in the supporting cells. Our results support the presence of RyR1 in the cochlea but do not rule out the presence of other isoforms. CICR may be involved in the control of calcium levels in the base of the inner hair cells and supporting cells, and in the cholinergic efferent response and motile behaviour of the outer hair cells.     

小鸡和豚鼠耳蜗毛细胞β-肌动蛋白分布比较

目的 :比较小鸡基底乳头和豚鼠耳蜗毛细胞 β-肌动蛋白 (β- actin)分布的特点。方法 :应用免疫组织化学方法观察小鸡和豚鼠耳蜗中β- actin免疫反应活性。结果 :鸡基底乳头高、矮毛细胞的静纤毛 ,盖膜根部附着处缘上皮细胞胞浆 ,豚鼠耳蜗三排外毛细胞胞浆 ,内、外支柱细胞胞浆和指状突β- actin免疫反应阳性。结论 :小鸡和豚鼠耳蜗毛细胞具有相同结构蛋白β- actin,但两种动物之间存在明显的分布差异。     

Nonerythroid spectrin (fodrin) is a prominent component of the cochlear hair cells

We studied the distribution of nonerythroid spectrin, fodrin, in surface preparations and cryosections of the cochlear hair cells as well as isolated hair cells of the guinea pig by using a monoclonal antibody (Mab) reacting with Mr 240 kD alpha-fodrin polypeptide. The Mab gave a strong reaction with the cuticular plate of both the inner and outer hair cells (IHCs and OHCs). Stereocilia were nonreactive and only a weak cell surface reaction was seen in the supporting cells. In the outer hair cells the upper turns of the cochlea, fodrin was observed in a cytoplasmic spiralling structure extending from the cuticular plate towards the cell nucleus. Some labelling was also seen along the cell surface membrane and in the synaptic region. The results suggest that fodrin may be an important constituent in the active processes of hair cells such as cell motility and/or signal transduction.