Spiral Prominence Research Articles

Monoclonal antibodies to inner ear antigens: I. Antigens expressed by supporting cells of the guinea pig cochlea

Murine monoclonal antibodies against guinea pig cochlear epithelium were generated with the goal of identifying cochlea-specific antigens and elucidating their function. To compensate for the limited amount of cochlear tissue, intrasplenic immunization was used. Hybridoma supernatants were screened by ELISA for antibody production and for binding to homogenates from cochlea, liver, lung, kidney and brain. Hybrids producing antibody to cochlea were subcloned and tested immunocytochemically against frozen sections and surface preparations of paraformaldehyde-fixed cochlear tissue. KHRI-1, a low titer IgM antibody stained only Hensen cells. KHRI-2, also an IgM antibody, stained tectorial membrane, cells of the spiral limbus, cells bordering the space of Nuel, Hensen cells and the root cells of the spiral prominence. KHRI-3, an IgG1 antibody, stained the phalangeal processes of outer pillar cells and the apical portion of phalangeal processes of Deiters' cells in a distinctive wine goblet pattern on surface preparations. KHRI-3 antibody also reacted with peripheral nerves and pia mater of brain in unfixed frozen sections but the antigenic site was not stable to fixation in contrast to the epitope detected in the cochlea. In Western blots of detergent extracts from cochlea KHRI-3 stained a broad tissue-specific band of M r 70–75 kDa; a narrower band of M r 68–70 kDa was identified by KHRI-3 in extracts of tongue and brain. KHRI-1 and KHRI-2 did not detect any proteins in Western blots. The monoclonal antibodies KHRI-1, -2, and -3 which define epitopes expressed by discrete populations of supporting cells in the inner ear should be useful in characterizing the nature and function of cellular structures in the cochlea.

Expression of Intermediate Filament Proteins in the Adult Human Cochlea

The immunohistochemical localization of intermediate filament proteins was studied in frozen sections of chemically fixed, nondecalcified adult human cochleas. Cytokeratins were found in all epithelial cells lining the cochlear duct (including most supporting cells of the organ of Corti) but were absent in the hair cells. Neurofilament proteins were present in the nerve endings at the hair cells, in the neural bundles, and in the ganglion cells. Vimentin staining occurred in most of the supporting structures and was roughly complementary to the regions showing cytokeratin staining and neurofilament staining. However, the region of the spiral prominence and outer sulcus, as well as the pillar cells and Deiters' cells in the organ of Corti, showed coexpression of vimentin and cytokeratins. No definite immunostaining was observed with antibodies to desmin and glial fibrillary acidic protein.

Ultrastructural Study of Cytochemical Localization of Carbonic Anhydrase in the Inner Ear

Vibratome sections were stained for cytochemical localization of carbonic anhydrase (CA) activity in vestibular neuro-epithelium, spiral ligament and spiral limbus. The new finding is the localization of reaction products in the interdental cells of the spiral limbus, Claudius' cells, mesothelial cells of the lower border of spiral ligament, vestibular sensory cells and perilymphatic cells, which have not earlier been proved to have CA activity. The interdental cells showed the products only on the basolateral infoldings. Claudius' cells showed prominent products in the microvilli. In the vestibular sensory cells, the products were present only in the stereocilia and cuticular areas. The perilymphatic fibrocytes under the vestibular sensory epithelium, like the fibrocytes of the spiral ligament, revealed diffuse products throughout the whole cell. In the vestibular supporting cells and transitional cells, the reaction products were localized diffusely in the cytosol, but not in the secretory granules. In the long cell projections of the transitional cells, type II fibrocytes at spiral prominence, mesothelial cells at the uppermost region of the spiral ligament and Borghesan's zone, the localization of the reaction products was the same as that of the basolateral infoldings of the vestibular dark cells and marginal cells of stria vascularis shown previously.

Cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase in the guinea pig inner ear. Evaluation of the cerium-based method vs. the lead-based method.

The cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase in the guinea pig inner ear was studied using both the lead-based method and the cerium-based method. The influence of primary fixation and incubation time was investigated under both incubation conditions. A high p-NPPase activity was observed in the basolateral infoldings of the stria vascularis in the cochlea and the dark cells in the vestibular labyrinth. The stromal cells of the spiral prominence only showed a weak reaction with the lead-based method. No specific enzyme activity was found in the endolymphatic sac. The results of the present study are discussed with emphasis on the cation transport mechanisms of the inner ear.

Immuno-Electron Microscopic Study of Keratin Distribution in the Cochlea Using Monoclonal Antibody

Keratin distribution in the cochlea has been studied immuno-electron microscopically by both pre-embedding and postembedding methods. Keratin immunoreactivity in the guinea pig cochlea was clearly demonstrated in Hensen's cells, the reticular lamina surrounding both outer and inner hair cells, outer and inner pillar cells, Claudius' cells, inner and external sulcus cells, interdental cells of the spiral limbus, Reissner's membrane, border cells, inner phalangeal cells, Deiters' cells, and spiral prominence cells. Keratin expression at the ultrastructural level showed a nonfilamentous keratin system in the cytoplasm of cochlear supporting cells.

Regional variations in the expression of cytokeratin proteins in the adult human cochlea

In the adult human cochlea, a cytokeratin (Ck) network exists along the entire surface of the organ of Corti, enclosing it like a shell. Only the surfaces of the outer and inner hair cells are not integrated in this network. In temporal bone specimens, Ck filaments in Hensen's cells were found to be arranged parallel with and closely apposed to the plasma membrane. In the stria vascularis, Cks were identified only in the marginal cells. Cells in Reissner's membrane and spiral prominence showed varying degrees of immunoreactivity to different monoclonal antibodies directed against Cks. A distinct positivity for Cks was found in most spiral ganglion cells, indicating their presence in all cells. The principal pattern of immunoreactivity was the same in the organ of Corti of the entire cochlea. However, a quantitative gradient in the expression of Cks was observed, with more Cks at the apex than at the base. This was correlated to a difference in the number of Hensen's cells between the two regions. The distinct shell configuration of the Ck network in Corti's organ gives it a tonotopically related difference in rigidity which must be of considerable importance for the perception of sound in the cochlea. The absence of Cks in inner and outer sulcus cells gives them cytoskeletal characteristics of mesenchymal cells with a possible regenerative potential.

Spiral prominence epithelium.

Recent studies have shown neuroendocrine-type cells in human spiral prominence epithelium. A light microscopic and ultrastructural study was made on the guinea pig spiral prominence epithelium to try and identify neuroendocrine cells. The study showed that such cells were not present in the guinea pig spiral prominence epithelium. The spiral prominence epithelium was characterized by uniformity of cellular staining with Toluidine blue and the ultrastructural appearance. The morphology of the spiral prominence epithelium was similar to that previously described. The absence of neuroendocrine cells in spiral prominence epithelium in the guinea pig is discussed in relation to species differences.

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissner's membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.

The vessels of the stria vascularis: quantitative comparison of three rodent species

The stria vascularis (SV) was quantitatively compared in three species commonly used in auditory research: guinea pig, mouse and gerbil. Measurements were obtained for surface area, cross-sectional area, length, width and thickness of SV. Surface area and length were proportional to the overall size of the cochlea in each species, but there was no significant difference between species in mean cross-sectional area. In guinea pig and mouse, there was no significant difference in thickness (endolymphatic surface to spiral ligament) and a similar pattern was observed for width (Reissner's membrane to spiral prominence): the width of SV increased from the apical end to a point 80% of the distance from the apex, then decreased to the basal end of SV. The thickness of gerbil SV was significantly less ( P < 0.001) and there was less of a gradient in width as compared to guinea pig and mouse. The vessels of SV were compared in terms of vascular density (vessels per unit area), rbc density (red blood cells per unit area), R/V (rbc density/vascular density), inter-vessel spacing and vessel diameter. Highly significant ( P < 0.001) differences between species were found in vascular density, RBC density and vessel diameter, but there were no differences between species for R/V or inter-vessel spacing. The results of this study may reflect differences in the metabolic requirements of SV among different species.

Potentiation of inner ear damage following electron beam irradiation with CDDP administration

The study was designed to examine the combined ototoxic effect of CDDP (Cisplatin, Cis-diammine dichloroplatinum) and electron beam irradiation, using guinea pigs. One group received physiological saline solution of 4 ml/kg/day, and another group received CDDP of 2 mg/kg/day for five days. And following the injection of saline or CDDP, the electron beam of 14Gy/day was applied to the both groups to the right ear for five days. Animals were sacrificed after 21 days, and temporal bones of these animals were removed for the inner ear histopathology. Temporal bones were classified into four groups (control, electron beam irradiation, CDDP administration, and combined administration group), and the inner ears were observed by the surface preparation technique with a phase contrast microscope, and by scanning and transmission electron microscopy, and by temporal bone study of serial sectioned slides. The main pathologic findings of the inner ear are as follows: Electron beam irradiation group showed no hair cell damage. CDDP group induced slight damage to the outer hair cells. Combined administration group (CDDP + electron beam irradiation) showed severe outer hair cell damage. Stria vascularis was degenerated moderately in the combined administration group and slightly in some animals of electron beam irradiation group. And Reissner's membrane and Hensen's cells, were damaged in the basal turn of cochlea of the combined administration group. All other cochlear structures (spiral ganglion, spiral lamina, basilar membrane, spiral prominence, tectorial membrane, blood vessels of the cochlea) and vestibular organs were lack of significant changes in all groups by a light microscopic observation. This study was clarified that combined administration of CDDP and electron beam irradiation showed severe ototoxic potentiation. Therefore, it is important that we must pay attention to the inner ear damage caused by combined therapy of CDDP and electron beam irradiation involving inner ear for the head and neck tumor.

An anionic charge barrier in the guinea pig cochlea

A charge barrier has been found in 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.

Demonstration of adenylate cyclase activity in stria vascularis of guinea pig cochlea

Cholera toxin-stimulated adenylate cyclase activity was demonstrated cytochemically in the stria vascularis of the guinea pig inner ear by the cerium method of Rechardt and Hervonen, and it was compared with ALPase, Mg-ATPase, Ca-ATPase and Na, K-ATPase activity. Adenylate cyclase activity was observed in the marginal and basal cells. The reaction products were fine granular particles on the plasma membrane of the basal infoldings of the marginal cells, but not on that of the free surface facing the endolymphatic space. Enzyme activity was also observed in the large mitochondria;the reaction was intense in the outer chamber and weak in the cristae, was inhibited by 5 mM alloxan. The plasma membranes of adjacent basal cells showed a positive rea-ction. No enzyme activity was detected in the intermediate cells, the endothelial cells of the capillaries in the stria vascularis, fibrocytes in the spiral ligament, epithelial cells of the spiral prominence, Corti's organ or Reissner's membrane. In the marginal cells, the location of adenylate cyclase activity corresponded with that of Na, K-ATPase activity. Therefore, the adenylate cyclase activity of the stria vascularis may play a role in the regulation of the membrane permea-bility to water and electrolytes while Na, K-ATP-ase acts as an ion pump to maintain high potassium and low sodium ion concentrations in the endolymph.

Analysis of structural changes in the stria vascularis following chronic gentamicin treatment.

Changes in the stria vascularis following chronic gentamicin treatment have been examined using quantitative methods. Albino guinea pigs were given gentamicin at 100 mg.kg-1.day-1 subcutaneously for 10 days. Comparisons were made of strial tissue from the treated animals sacrificed either 1 h or 4 weeks following the last injection with that from saline-injected controls. Strial width (spiral prominence to Reissner's membrane) and marginal cell (MC) number across the stria were determined from scanning electron micrographs. Strial thickness (endolymphatic surface to spiral ligament) and the volume fractions of the strial components (MCs, intermediate cells (ICs), basal cells (BCs) and capillaries) were derived from thin sections. Qualitative changes to both MCs and ICs were apparent 1 h after the last injection. At four weeks post-treatment, there was a small, but statistically significant, decrease in the number of marginal cells and a highly significant decrease in strial thickness. This was almost entirely due to a highly significant decrease in the volume fraction of MCs (i.e. shrinkage). The volume fraction of ICs was increased but this could be accounted for by MC shrinkage; after allowing for the reduction in strial thickness, the volume occupied by ICs was unchanged. Thus, following chronic gentamicin treatment, the stria is affected but significant progressive and permanent structural effects are confined to the marginal cells.

Ultracytochemical Study of Ouabain-sensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in the Inner Ear of the Squirrel Monkey

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na(+),K(+)-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K(+)-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K(+)-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.

Cytochemical localization of Ca++-ATPase activity in the lateral cochlear wall of the guinea pig

Ca++-ATPase activity was examined cytochemically in the lateral cochlear wall of the guinea pig. The reaction products showing Ca++-ATPase activity were found along the folded plasma membrane of the strial marginal cells. In contrast, little or no reaction was seen on the apical surfaces of these cells. There were also marked reaction products on the microvilli and the endolymphatic cell surface of Reissner's membrane, and the apical and lateral plasma membranes of the spiral prominence and the external sulcus cells. These reactions completely disappeared when Ca++ or ATP was removed from the incubation medium. Our results strongly suggest that Ca++-ATPase plays an important role in Ca++ transport system for the regulation of Ca++ concentration in the cochlear endolymph.

Ultracytochemical Study of Ouabain-sensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in the Inner Ear of the Squirrel Monkey

The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of the Na+, K+-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K+-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K+-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.

Somatostatin (somatostatinlike) immunoreactive cells in the human inner ear.

Certain epithelia of the human inner ear and human endolymphatic sac display somatostatin and/or somatostatin-like immunoreactivity. Histologic sections from 13 human temporal bones and from 15 endolymphatic sacs were studied using the unlabeled antibody peroxidase-antiperoxidase technique. The somatostatin and/or somatostatin-like immunoreactive cells were located exclusively in the covering epithelium of the spiral prominence and in the epithelium of the intermediate and rugosal part of the endolymphatic sac. In the epithelium of the spiral prominence and endolymphatic sac, secretory granules of the same size and appearance as those of intestinal or pancreatic somatostatin-producing cells were demonstrated ultrastructurally. The findings are consistent with a local exocrine, paracrine, and/or endocrine system of the inner ear.

Temporal bone showing polyarteritis nodosa, otosclerosis, and occult neuroma

The clinical and general histopathologic manifestations of systemic vasculitis of the polyarteritis nodosa type are well known. Although hearing loss associated with polyarteritis nodosa has been reported, only two temporal bone studies are available. The findings in a 60-year-old man with well-documented hearing loss who had rheumatoid arthritis, polyarteritis nodosa, and otosclerosis are presented. Polyarteritis nodosa extensively involved the subarcuate arteries and arteries of the facial canal. There were decreased nerve fibers to and sensory cells in the crista of the semicircular canals and macula of the utricle and saccule. Focal and diffuse atrophy of the stria vascularis and decreased cellularity in the spiral prominence and ligament were present. There was a loss of outer hair cells. Otosclerosis involved the left and right oval window niches (bilateral stapedectomy had been performed). There was a small Antoni type A neuroma of the superior division of the vestibular nerve on the left.

Incorporation of inositol in the cochlear tissues as determined by autoradiography.

The sites of incorporation of [3H]inositol perfused perilymphatically in the guinea pig cochlea were localized autoradiographically. In the organ of Corti, active incorporation occurred in the synapses, nerve fibers, pillars and nuclei of various cells. Both hair cells and supporting cells moderately incorporated inositol. In the lateral wall of the cochlear duct marked incorporation was observed in the epithelia of the vascular stria and spiral prominence. The possible involvement of inositol phospholipids in auditory transduction at the organ of Corti and in ionic transport in the lateral wall of the cochlea is briefly discussed.

Hormonproduzierende (parakrine) Zellen im menschlichen Innenohr

Presence of immunoreactive somatostatin in the covering epithelial cells of the spiral prominence was demonstrated by immunohistochemical methods. The concentration of somatostatin increases from the basal turn to the apical turn of the cochlea. Ultrastructural investigations of these cells reveal secretory granules with a diameter of 250-350 nm. The findings promote the existence of a local paracrine system of the inner ear.