Cytokeratin expression in the epithelia of the adult human cochlea

Summary Epithelia can be characterized by the specific expression pattern of their cytokeratin components. Therefore, we investigated the immunohistochemical expression of different cytokeratin subunits in frozen sections of chemically fixed, non-decalcified, adult human cochleas. The organ of Corti and the marginal cells of the stria vascularis showed reactivity for cytokeratin subunits 8, 18 and 19, whereas the other cochlear epithelia in addition expressed cytokeratin 7. The expression of cytokeratins 7, 8, 18 and 19 by the epithelia of the adult human cochlea is typical of simple epithelia. The deviant cytokeratin pattern of the organ of Corti and marginal cells of the stria vascularis may well reflect their differences in functional state and/or differentiation as compared to the other cochlear epithelia.This work was presented in part at the 25th Workshop on Inner Ear Biology, 4–7 September 1988, London, UK     

Differential immunohistochemical detection of cytokeratins and vimentin in the surgically removed human endolymphatic duct and sac

Summary Immunohistochemical detection of intermediate filament proteins and different subgroups of cytokeratins (Cks) was used to characterize the epithelium of the surgically removed adult human endolymphatic duct (ED) and sac (ES). The epithelium of the ED and ES demonstrated immunostaining for Cks 7, 8, 14, 17, 18 and 19, a pattern typical of so-called complex or mixed epithelia. This is a remarkable finding, since this pattern differs strikingly from previously reported data on the adult human cochlea and vestibular labyrinth that demonstrated a Ck pattern typical of simple (or single-layered) epithelia. Furthermore, the epithelium of the ED and ES demonstrated co-expression of Cks and vimentin. The present data indicate that the epithelium of the ED and ES exhibits another type of epithelial differentiation and demonstrates a higher degree of complexity than the other epithelia in the adult human inner ear.     

An immunocytochemical study of cytokeratin expression in human middle ear cholesteatoma

Summary An indirect immunofluorescent method with monoclonal anti-cytokeratin antibodies was used to localize various cytokeratins in human middle ear cholesteatoma. The 50 K/58 K and 56.5 K/65–67 K paired cytokeratins are markers of skin type and were found in the specimens of human middle ear cholesteatomas studied. In contrast, the 40 K and 45 K cytokeratins (markers of simple epithelia), the 48 K cytokeratin (marker of hyperproliferative epidermal disease) and the 51K cytokeratin (marker of internal organ epithelia) were absent in human middle ear cholesteatoma. These findings indicate that the pattern of cytokeratins in human middle ear cholesteatoma is similar to that of skin but is different from those of simple epithelium, internal organ epithelia, and hyperproliferative epidermal disease. These findings also support the skin type epithelial origin of cholesteatoma and strongly favor the migration theory in the genesis of cholesteatomas.     

角蛋白、波形蛋白、神经细丝蛋白在人胎内耳的定位表达

本文对合法引产9～32周胎龄的人内耳中角蛋白、波形蛋白、神经细丝蛋白的表达进行了免疫组织化学定位研究。结果发现增殖期的前庭器和Corti器始基细胞均有这三种中间纤维的表达。随着内耳分化的进行，用蛋白表达部位限于表皮板、网板、支持细胞、血管纹和前庭壁细胞。波形蛋白的表达部位分布在Corti器始基细胞、一些支持细胞、环绕的结缔组织、软骨细胞、骨组织、血管纹缘细胞、螺旋神经节细胞和神经纤维，前庭感觉上度表达反应弱。神经细丝蛋白除了在神经细胞和神经纤维表达阳性外，在Corti器站基细胞上部、血管纹原始细胞表达明显，软骨细胞和骨组织表达弱。内耳发育成熟期，这三种中间纤维蛋白在感觉毛细胞和听毛细胞的内部都无阳性表达。研究结果提示：这三种中间纤维参与人内耳的发育，可能与人内耳功能的发生有关。     

Unique expression of connexins in the human cochlea

Mutations in the genes GJB2 and GJB6, which encode the proteins Connexin 26 (Cx26) and Connexin 30 (Cx30), have been linked to nonsyndromic prelingual deafness in humans. These proteins may form so-called gap junctions (GJ) or transcellular pathways between cells. The pathogenesis of deafness due to GJ Connexin mutations remains unclear partly because examinations performed in the human ear are infrequent. Here we analysed the expression and distribution of Cx26 and Cx30 in five fresh normal human cochleae taken out at occasional surgery. Immunohistochemistry including confocal microscopy in decalcified specimen showed that these proteins are widely expressed in the human cochlea. In the lateral wall there was strong antibody co-labeling for Cx26 and Cx30 that support the existence of channels comprising heteromeric Cx26/Cx30 connexons. In the organ of Corti there were some co-labeling in the supporting cell area including mainly the Claudius cells and Deiter cells of these two Cxs, apart from isolated Cx26 and Cx30 labeling in the same area, suggestive of both homomeric/homotypic pattern and hybrid pattern (heteromeric or heterotypic). Cx30, Cx26 and Connexin 36 (Cx36) immunoreactivity was also associated with spiral ganglion type I neurons, the latter being a gap junction protein specific to neurons. Gap–junction-based electrical synapses are not known to occur in mammalian auditory system other than in bats where they may play a role for fast electrical nerve transmission useful for echolocation. Their potential role in the processing of human auditory nerve signaling as well as non-GJ roles of the connexins in human cochlea is discussed.     

Nerve supply to the inner sensory cells in a human cochlea

Summary A cochlear apical turn taken from a 66-year-old woman showed an area with marked decrease in number of the inner sensory cells. After observation by scanning electron microscope, the same specimen was thin-sectioned tangentially to the osseous spiral lamina. Numbers of myelinated nerve fibers were counted and the thickness of the fiber was measured by computed measuring equipment. Compared to the area with inner sensory cells intact, the area without inner sensory cells showed a 70% decrease in number of myelinated fibers. These degenerated fibers might have had intimate relationship with the inner sensory cells, probably being the afferent nerve supply to the inner sensory cells.This study was supported by the Japanese National Science Research Fund (No. 753-6016-257432)     

Immunohistochemical demonstration of the co-expression of vimentin and cytokeratin(s) in the guinea pig cochlea

Summary Using a decalcification procedure for guinea pig petrous bones, we were able to prepare cryostat sections for immunohistochemical studies. A panel of monoclonal antibodies against intermediate filament proteins was then used to show a co-expression of cytokeratin and vimentin in the epithelial cells of the stria vascularis.     

Deep-water waves in the cochlea

In order to obtain physical insight from a mathematical solution of the cochlear mechanics problem, a delicate balance is required between and refinements in modelling. For the study of the transition of long to short waves (deep-water waves) a closed-form solution is advantageous; this can, however, only be obtained at the cost of further simplification. In previous work the course of the impedance function z(x) was, therefore, reduced to the extreme; in the neighbourhood of the resonance location z(x) was assumed to be a linear function of x - the so-called ‘straight-line approximation’.This restriction is removed in the present paper. A ‘hyperbolical approximation’ of the impedance function z(x) is introduced and it is shown that with this function the two-dimensional cochlea model can be solved in closed form. The computation results show that, for not too large values of the damping parameters δ, the response in the neighbourhood of the resonance location is almost as well represented by the formerly used ‘straight-line approximation’ as by the ‘hyperbolic approximation’. Hence the principal aspects of cochlear resonance are well brought to light with the ‘straight-line approximation’. This implies that in the case under consideration the dominant part played by short waves is confirmed.When a larger range of x values is to be considered, the hyperbolic approximation is advantageous. The computed response functions agree better with experimental data from the literature. However, it is clear that really satisfactory agreement seems not possible with a two-dimensional model.     

High-resolution magnetic resonance imaging of human cochlea

OBJECTIVES: High-resolution MRI (MRI) of human inner ear structures provides several advantages over other imaging modalities. High-resolution visualization of inner ear ultrastructure in a noninvasive manner may provide important information about inner ear disease that is not obtainable in other ways. The study was performed to demonstrate the capabilities of MRI at high resolution on the human cochlea, vestibular structures, and facial nerve. Comparative analyses of MRI anatomy with that seen on histological dissection were made. The aim of the study was to define the anatomy of human cadaveric cochlea using a 9.4-Tesla magnetic resonance scanner, currently the most powerful magnetic resonance magnet available. STUDY DESIGN: Experimental pilot study of cadaveric human cochleae. METHODS: Serial scanning using a 9.4-Tesla magnetic resonance imager on normal preserved and fresh cadaveric inner ears was performed in different planes. RESULTS: The images revealed detailed anatomy of the modiolus, utricle, saccule, semicircular canals, and facial nerve. Specifically, identifiable structures within the cochlea included the osseous spiral lamina, Reissner's membrane, membranous spiral lamina, spiral ligament, and others. CONCLUSIONS: Data established through the acquisition of images from cadaver cochlea, facial nerve, and vestibular complex provide a foundation for developing steps for testing temporal bones and, eventually, patients with Meniere's disease and other inner ear disease. The present ongoing project will provide information on baseline images of the inner ear using high-resolution MRI.     

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.     

Immunocytochemical localization of contractile and contraction associated proteins in the spiral ligament of the cochlea

Most of the extracellular fibers of the spiral ligament are associated with a distinct band of ‘anchoring’ cells which occur at the boundary between the spiral ligament and the otic capsule. These cells are characterized by parallel arrays of intracellular filaments which, along with the extracellular fibers, insert into electron dense, conical adhesion plaques. The intracellular filaments show a close morphological resemblance to the ‘stress fibers’ of cultured fibroblasts (Henson et al., 1984). In the present study we have demonstrated by immunofluorescence techniques that the anchoring cells, unlike adjacent cells of the spiral ligament, contain a complement of proteins that is typically associated with stress fibers and with contractile systems. In addition to actin, the cells contain myosin, tropomyosin, -actinin and talin. These results lend further support to the hypothesis that the anchoring cells have the capacity to create and/or maintain tension on the spiral ligament-basilar membrane complex and to influence the mechanical properties of the basilar membrane.     

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCII and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy (SR-SIM) were used in the study. CD4⁺ and CD8⁺ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons, CD4⁺ and CD8⁺ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus, macrophages, CD4⁺ and CD8⁺ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4⁺ and CD8⁺ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.     

Immunohistological analysis of neurturin and its receptors in human cochlea

ObjectiveDifficulties in obtaining properly preserved human cochlea have been a major obstacle to in vitro study of this deeply located and hard bone-fortressed hearing organ. Our study aimed at investigating GDNF family ligands (GFLs) and their receptors in the human cochleae that were surgically obtained during a transcochlear approach dealing with life-threatening, intra-cranial meningiomas.MethodsThe specimens were properly fixed with 4% paraformaldehyde in the operating room. By using immunohistochemical techniques, distribution of GDNF, Neurturin (NTN, one member of GFLs), as well as cRet, GFRα-1 and GFRα-2 receptors in the human cochleae was investigated. Five cochleae from five adult patients were processed for the study. The patients had normal hearing threshold before operation.ResultscRet receptor immunoreactivity was seen in the spiral ganglion neurons, mainly inside the cell bodies but rarely in the nerve fibers and not in the organ of Corti. Immunolabeling for GFRα-1 and GFRα-2 receptors was identified mainly in the cell bodies of the spiral neurons than in the nerve fibers. In the organ of Corti, GFRα-1 immunostaining could be demonstrated in the Deiters’ cells, Hensen cells, inner pillar cells, and weakly in the inner hair cells but not in the outer hair cells; no structures in the organ of Corti were labeled with GFRα-2 receptor antibody. NTN immunostaining was found in the supporting cells of organ of Corti, including Deiters’ cells, Hensen cells as well as Claudius’ cells. In the spiral ganglia, NTN immunostaining was seen in both the cell bodies and the nerve fibers of neurons. GDNF immunoreactivity was not revealed in human cochlea.ConclusionSurgically obtained human cochleae were properly fixed and underwent immunohistochemical investigation of neurotrophic elements. NTN and its receptors discovered in current study can be responsible for the unique neuronal survival properties in human spiral ganglion (hSG); a prerequisite for the function of cochlear implants.     

Changes in the gene expression pattern of cytokeratins in human respiratory epithelial cells during culture

The replacement of extensive tracheal defects resulting from intensive care medicine, trauma or large resections is still challenged by the re-epithelialization of an autologous or alloplastic trachea replacement. Therefore, this study was performed to investigate the potential of culture-expanded human respiratory epithelial cells (hREC) to regenerate a functional epithelium for tracheal tissue engineering. hREC from seven male nasal turbinates were freshly isolated, expanded on a collagenous matrix and subsequently cultured in high-density multi-layers to allow epithelial differentiation. The composition of epithelial cells in native respiratory epithelial tissue and culture-expanded hREC was analyzed by histological staining with Alcian blue and by immunohistochemical staining of cytokeratin pairs CK1/10 and CK5/14 with the antibodies 34E12 and CD44v6. Differentiation of culture-expanded hREC was further characterized by gene expression analysis of cytokeratins CK5, CK13, CK14 and CK18 using semi-quantitative real-time RT-PCR technique. Histological and immunohistochemical staining of culture-expanded hREC demonstrated basal cells covering the collagenous matrix. These cells formed a cellular multi-layer, which was composed of a basal layer of undifferentiated basal cells and an upper layer of cells differentiating along the squamous metaplasia and ciliated cell lineage. Lineage development of culture-expanded hREC was further documented by the induction of cytokeratins CK13 and CK18. Our results suggest that culture-expanded hREC have the potential to colonize collagen-coated biomaterials and to regenerate epithelial cell types for tracheal tissue engineering.     

γ─氨基丁酸在人体耳蜗的定位

应用免疫细胞化学方法，研究γ－氨基丁酸（ＧＡＢＡ）在人体耳蜗的定位。结果表明：ＧＡ－ＢＡ定位于人体耳蜗传出神经系统；是人体耳蜗传出神经递质。     

鸟纲蜗管微血管的比较解剖学观察

目的：了解鸡内耳蜗管微血管的超微结构，为相关研究提供资料。方法：分别应用透射电镜和扫描电镜对10只成年鸡内耳蜗管微血管的超微结构进行观察。结果：鸡内耳蜗管组织中末见典型的薄壁毛细血管，基底膜中末见微血管分布；鸡耳蜗组织中的微血管管壁较厚，从内皮细胞至周边基膜问有呈交替排列的多层纤维、周细胞及其胞突；血管盖的每条皱签中都含有1条微血管，螺旋神经节内微血管与蜗管内的微血管结构相似；扫描电镜下，微血管表面周细胞胞突交织成网状。结论：鸡内耳蜗管微血管与哺乳纲动物内耳蜗管微血管的超微结构及分布有较大差别，其意义尚待进一步探讨。     

噪声暴露对大鼠耳蜗碳酸酐酶活性及mRNA表达的影响

目的：探讨噪声暴露后耳蜗碳酸酐酶（CA）活性及其mRNA表达的变化。方法：选用健康白色大鼠24只,随机分为4组,1组为正常对照组,另外3组为实验组。实验组暴露在4kHz、110dBSPL窄带白噪声环境中4h／d,分别测试噪声暴露1d、1周、3周和对照组大鼠脑干反应（ABR）。采用免疫组织化学法观察耳蜗外侧壁CA的活性,RT-PCR检测大鼠耳蜗CAIImRNA的表达。结果：噪声暴露后大鼠ABR阈值较对照组显著增加;而耳CA活性及CAIImRNA的表达较对照组显著下降;随着噪声暴露时间的延长,ABR阈值增加而cA活性及CAIImRNA的表达呈下降趋势。结论：噪声刺激能降低耳蜗CA的活性及CAIImRNA的表达,CA可能参与了噪声性聋的发病机制。     

Wave propagation and dispersion in the cochlea

Insight into cochlear mechanics can be obtained from semi-analytical and asymptotic solution methods of which the Liouville-Green (LG) method — in another context known as the WKB method — is the most important one. This paper describes the dispersion properties of fluid waves in general terms and develops the LG formulation on that basis. The eikonal equation of the LG method is shown to be identical to the dispersion relation in dispersive-wave theory. Consideration of the group velocity then leads to the derivation of the central LG formula as it has been used in an earlier paper on the LG method (Boer E. and Viergever M.A. (1982): Hearing Res. 8, 131–155). The formulation appears to apply as well to dissipative and active (i.e., energy-producing) systems. Of the many possible collateral subjects two are selected for a deeper discussion: amplification, concentration and expansion of energy, and the problem of reflection of cochlear waves. In the latter context, it is shown why — and under which conditions — cochlear waves are not reflected, despite the large degree of dispersion that they show. The analysis brings to light a fundamental asymmetry of the model regarding the direction of wave travel: waves travelling in the direction opposite to the normal one are likely to undergo reflection, while waves in the normal direction are not reflected.     

Electron microscopic atlas of the simian cochlea

An electron microscopic atlas of simian cochlear duct structures is presented with the hope that it may help to clarify certain questions of structural-functional relationships. Emphasis is placed on structures directly involved in sensory transduction and in synaptic transmission, at afferent and efferent nerve fiber terminals. A brief discussion is included as an introduction to the possibilities of interpretation of electron microscopic data. Orientation of electron micrographs is assisted by means of numerical coding on light microscopic photographs, and by means of a diagram which combines electron microscopic and light microscopic data.