Intermediate filaments in the inner ear of normal and experimentally damaged guinea pigs

The hypothesis that proteins known to occur in glial cells in the central nervous system may be present in inner-ear supporting cells was investigated. Immunocytochemical techniques were used to look for the existence of two classes of intermediate filaments, vimentin and glial fibrillary acidic protein (GFAP), in cellular elements of the inner-ear epithelium in normal and experimentally damaged guinea-pig cochleas. Vimentin is present in two types of supporting cells in the normal organ of Corti: Deiters' cells and inner pillar cells. Differences in intensity and distribution of vimentin immunostaining are observed across the three rows of Deiters' cells. GFAP immunoreactivity was not detected in any supporting-cell type in the organ. Cochlear hair cells were not labeled for either GFAP or vimentin. Following hair-cell destruction by exposure to noise or the administration of aminoglycosides, GFAP and vimentin are not present in phalangeal scars replacing lost hair cells.     

Cytoskeletal identification of intermediate filaments in the inner ear of the jerker mouse mutant

The expression of the five main groups of intermediate filaments (IF) and their subgroups, especially cytokeratins, was analysed in cryosections of the labyrinth of the jerker mouse mutant and compared with normal CBA/CBA controls. Fourteen different well characterized monoclonal antibodies were used. In principle the same pattern of IF was found in the two mouse strains. IF were not found in hair cells of the mouse inner ear, which may reflect a disparity as compared with human fetal hair cells. Chain-specific (epitope-specific) differences were visualized in cytokeratin no. 8 with regard to cells in epithelia involved in the homeostasis of inner ear fluids. Differences in immuno-staining occurred between adjacent cells in the same epithelium, for instance in the semicircular canals.     

Developmental stage-dependent pattern of inner ear expression of intermediate filaments

The expression of vimentin, cytokeratins (CKs) and neurofilament (NF) proteins was analysed (using monoclonal antibodies) in the mouse inner ear at the otocyst stage (13th gestational day), when organogenesis was largely completed (16th gestational day) and at birth (21st gestational day). Co-expression of vimentin and CKs occurred at the otocyst stage. On the 16th gestational day, most epithelial cells lacked immunoreactivity for vimentin and considerable variation in CK positivity was found between different regions of the epithelial lining. At birth, CK positivity was lacking in the developing organ of Corti but was present in other types of epithelium lining the scala media. In the vestibular half of the labyrinth, positivity for CKs was found at the apical surfaces of both sensory cells and supporting cells and in epithelia lining the membranous labyrinth. Vimentin positivity occurred in the greater epithelial ridge of the differentiating organ of Corti. Even at this stage the statoacoustic ganglion comprised two subpopulations of ganglion cells: those staining for NF proteins and those lacking this immunoreactivity. Thus, as the inner ear matures, a pattern of cytoskeletal reorganization occurs that is dependent on developmental stage.     

The immunohistochemical analysis of pendrin in the mouse inner ear

Pendred's syndrome (PS) is an autosomal recessive disorder characterized by deafness and goiter, which are caused by mutations in the Pendred's syndrome gene (PDS). PDS encodes a membrane protein named pendrin that is considered to act as an anion transporter. An expression pattern of the PDS ortholog (Pds) mRNA in the auditory and vestibular systems has been reported in mice, and the localization of pendrin has been reported recently. We generated antipeptide antibodies against human pendrin, and performed immunohistochemical analysis of mouse inner ears. We detected pendrin in the endolymphatic duct and sac, and the utricle, saccule, and external sulcus. In the endolymphatic duct and sac, the expression of pendrin was apparent at the apical membrane. In addition, we detected pendrin in the spiral ligament, Claudius cells, Deiter's cells, and the spiral ganglion of the cochlea. Our results are key to defining the role of pendrin in inner ear development and elucidating the pathogenic mechanisms underlying deafness in PS.     

Developmental expression of aquaporin 2 in the mouse inner ear

OBJECTIVES: The maintenance of endolymph homeostasis is critical for the inner ear to perform its functions of hearing and maintaining balance. The identification and cloning of aquaporins (a family of water channel proteins) has allowed the study of a novel cellular mechanism potentially involved in endolymph homeostasis. The objective of the present study was to define the developmental temporal and spatial expression pattern of aquaporin 2 (Aqp2) in the developing mouse inner ear. STUDY DESIGN: A systematic immunohistochemical study of Aqp2 protein expression was performed on embryonic mouse inner ears ranging from embryonic day 10 (otocyst stage) to embryonic day 18 (just before birth). METHODS: Serial cryosections of embryonic mouse inner ears were used for immunohistochemical experiments. A rabbit polyclonal antisera raised against a synthetic Aqp2 peptide was used with a standard nickel intensified 3,3-diaminobenzidine reaction protocol for immunolocalization of Aqp2 in tissue sections. RESULTS: Aquaporin 2 is expressed diffusely in the early otocyst, then becomes progressively restricted as the inner ear matures. During early cochlear duct formation (embryonic days 12 and 13), expression of Aqp2 is homogeneous; later, it becomes restricted to specific regions of the endolymphatic compartment (embryonic days 15 and 18). Similar restriction of expression patterns could be noted for the vestibular structures. Endolymphatic duct and sac and stria vascularis expression of Aqp2 was noted to occur fairly late during development but demonstrated a distinct pattern of immunolabeling. CONCLUSIONS: Aquaporin 2 shows an early and specific pattern of expression in the developing mouse inner ear, suggesting a significant role for this water channel protein in the development of endolymph homeostasis and meriting further functional studies of Aqp2 in the inner ear.     

Noise-induced inner ear damage in newborn and adult guinea pigs

Two-day (Group A), eight-day (Group B), and eight-month (Group C) old guinea pigs were exposed to 30 continuous hours of white noise at 119–120 db SPL. One month later pathology of the organ of Corti was evaluated and quantitated by use of the surface preparation technique. Percent cell damage was determined for outer hair, inner hair, outer pillar, and inner pillar cells at each of the four turns of the cochlea and for the cochlea as a whole. Comparisons of pathology of each cell type were made between groups. Mean percent outer hair cell damage per cochlea (± 1 S.E.) was 23.72 ± 3.69 for Group A, 36.98 ± 5.76 for Group B, and 7.24 ± 1.75 for Group C. There was no significant difference in outer hair cell damage between Groups A and B. Outer hair cells of Group A were significantly more damaged than those of Group C when damage in the cochlea as a whole was considered due to significantly greater damage in Group A at three and one half turns; likewise, outer hair cells of Group B were significantly more damaged than those of Group C when damage in the cochlea as a whole was considered due to significantly greater damage in Group B at two and one half and at three and one half turns. A similar effect was observed in terms of pathology of inner hair cells and pillar cells: there was a trend toward increased damage in animals of Groups A and B compared with C. Group C showed no outer or inner pillar cell damage, and only one of six animals had alterations in inner hair cells. In contrast, outer and inner pillar cells were damaged in Groups A and B, and four of six animals of Group A and six of eight of Group B showed inner hair cell damage. Recent electrophysiological and audiometric studies are discussed which, with the results of the present study, indicate greater susceptibility of young cochleas when compared with older cochleas, to noise-induced physiological and pathological alterations. It would seem medically prudent to take special precautions to avoid exposing newborns to excessive noise.     

X-ray microanalysis of inner ear fluids in the embryonic and newborn guinea pig

Summary In energy-dispersive histograms, changes in the relative peak intensities were followed, especially Cl and K, which indicate the maturation of endolymph. The maturation of endolymph in the guinea pig occurs prior to birth. In X-ray histograms, distinct peaks for Cl and K, but also for Na, were observed approximately 20 days before birth (DBB). The lesser relative peak intensities for Cl and K as compared with mature endolymph indicate an immature endolymph composition at this stage of development. The relative peak intensities of Cl and K increased at approximately the 10-DBB stage and showed similar values as at birth.Supported by grants from the Swedish Medical Research Council (project 12X-720) and the Foundation Tysta SkolanPresented at the 18th Workshop on Inner Ear Biology in Montpellier/La Grande Motte, September 14–16, 1981     

Expression of glycoconjugates in the mouse inner ear after prenatal irradiation

Summary Irradiation of the murine fetal inner ear is known to produce damage both to the vestibular and cochlear parts in the adult mouse. Fluorescein-labelled lectins were used to reveal possible differences in the glycoconjugate content between normal and irradiated inner ears. In the vestibular part, the otoconia showed the highest uptake of labelled sugars. This uptake was weaker after irradiation when compared to non-irradiated specimens. The type I hair cells in the ampulla and in the utricle showed a weaker uptake, but no labelling was demonstrated in the type II hair cells compared to the non-irradiated controls. In the cochlear part of the inner ear almost no uptake of fluorescent-binding lectins could be demonstrated in the irradiated groups except for in the tectorial membrane. In the endolymphatic sac no uptake was shown after prenatal irradiation. These findings are discussed and correlated to the already known damage of the inner ear following prenatal irradiation.     

A morphometric study of the pallid mutant mouse inner ear

Mice homozygous for the mutant gene pallid (pa/pa) often lack otoconia in some or all of their maculae and are used to study the influences of gravity receptor hypostimulation on vestibular-related behaviors. Since the value of this animal model is based on the assumption that the vestibular sensorineural elements are normal, a morphometric analysis was done on the inner ear of these otoconia-deficient mice to see whether sensori-neural structures are also affected by the pallid gene. In pallid mice lacking all otoconia, the sensory epithelia of the utricle, saccule, and semicircular canal cristae were the same size as in their heterozygous (pa/+) controls. Although the superior and inferior divisions of the vestibular ganglion of the pallid mice were smaller than normal, the first-order neurons within these divisions were normal in size, number, and density. However, the superior divisions in both groups had larger neurons than did the inferior divisions. Within the pallid cochlea, first-order auditory neurons within the spiral ganglion were smaller than normal, but the scala media was larger. Since the significant vestibular influences of the pallid gene are limited primarily to the otoconia, behavioral abnormalities reported for these otoconia-deficient mice are apparently due only to gravity receptor hypostimulation.     

Prenatal low-dose gamma irradiation of the inner ear induces changes in the expression of intermediate filaments

The expression of intermediate filaments (1F) was analysed in the inner ear in normally developed adult CBA/CBA mice and in mice of the same age which had been gamma irradiated in utero with a low dose 1-2 Gy single exposure. Well characterized monoclonal antibodies (mAbs) against all classes of intermediate filament proteins (cytokeratins-Cks, vimentin, neurofilaments, desmin and glial fibrillar acidic protein) were used. With the exception of neurofilament proteins, the expression of intermediate filament proteins was the same in adult normal and irradiated inner ears, irrespective of gestational age at exposure. A complex Ck pattern occurred in the various cell types comprising the membranous labyrinth. In spite of the differences in cell shape and internal organization of organelles, epithelia actively involved in inner ear fluid homeostasis (stria vascularis, dark cell epithelium, endolymphatic duct and sac) revealed, according to our mAbs, the same expression of Cks, except for the mouse counterpart of human Ck 7, which was found exclusively in the stria vascularis and the endolymphatic duct and sac. The pattern of intermediate filament composition in the labyrinth was the same in the mouse as in man. Irradiation on gestational days 12 or 13 (the otocyst stage)--but not at more advanced embryonic age--induced immunoreactivity for neurofilament proteins in vestibular hair cells (HC) and to a minor extent also in cochlear HC. No such positivity was found in the control material.     

Spatiotemporal expression of otogelin in the developing and adult mouse inner ear

Using a PCR-based subtractive method on cDNA from 2-day-old mouse cochlea, we identified a gene encoding otogelin, Otog, an inner ear specific glycoprotein expressed in all acellular structures. Here, we provide evidence that otogelin is detected as early as embryonic day 10 in the otic vesicle. At this stage, otogelin is detected in the epithelial cells which do not overlap with the myosin VIIA-expressing cells, namely the precursors of the hair cells, thus arguing for an early commitment of the two cell populations. Analysis of otogelin spatiotemporal cell distribution allows a molecular tracing for the contribution of the cochlear and vestibular inner ear supporting cells to the formation of the acellular structures. Throughout embryonic and adult life, the expression of the otogelin gene as monitored by LacZ inserted into Otog, and the abundance of the protein are greater in the vestibule than in the cochlea. In adult, otogelin is still produced by the vestibular supporting cells, which argues for a continuous process of otogelin renewal in the otoconial membranes and cupulae. In contrast, in the tectorial membrane, otogelin should be a long-lasting protein since both the otogelin gene and protein were almost undetectable in adult cochlear cells. The data are consistent with the requirement for otogelin in the attachment of the otoconial membranes and cupulae to their corresponding sensory epithelia as revealed in Otog -/- mice.     

小鼠耳蜗胚胎发育过程中Smad4基因的表达

目的探讨小鼠内耳胚胎发育演变过程，检测Smad4基凶在小鼠耳蜗中的表达情况。方法选用耳廓反应灵敏、健康的C57BL／6小鼠作为种鼠交配，用观察阴栓方法获得适龄胎鼠，≤17天取胚胎头，≥18天在显微镜下取耳蜗，胚胎头水平冰冻切片，耳蜗平行于蜗轴冰冻切片，HE染色方法观察小鼠耳蜗发育形态演变过程，免疫组织化学方法检测Smad4蛋白在小鼠胚胎10～20天耳蜗巾的表达情况。结果胚胎10天，听泡发育．胚胎12天听泡下部有蜗管始基形成并开始发育．胚胎18天，蜗管发育2圈，形成了可以辨认的内、外毛细胞．血管纹开始分化。Smad4从胚胎15天才开始表达，最初表达部位为耳蜗底转将发育成蜗轴以及柱状上皮分化为感觉细胞及支持细胞的区域，但在胚胎早期表达较弱，后期在耳蜗内广泛表达，在螺旋器、血管纹、前庭膜均有表达，且表达逐渐增强，而在蜗轴部位的表达逐渐减弱。结论Smad4在小鼠内耳分化期有阳性表达，且表达具有明显的阶段性和区域性，说明其参与内耳听觉器官的发育过程并且可能存耳蜗的形成过程中起着重要的作用。     

A ferritin-containing cell type in the stria vascularis of the mouse inner ear

This report describes a new cell type within the stria vascularis of the mouse inner ear. The cell is similar ultrastructurally to the classically described intermediate cell. However, it can be distinguished by the presence of dense vacuoles, presumably lysosomes, within which can be visualized electron dense particles resembling ferritin molecules. In addition, the ferritin-like particles are present throughout the cytoplasm and occasionally within the endoplasmic reticulum. These cells characteristically abut capillary basal lamina. Electron probe analysis of the dense vacuoles revealed the presence of iron. It is suggested that these cells may sequester iron released from dying erythrocytes in the strial capillary system, whereupon the iron is conserved through ferritin synthesis.     

Pharmacodynamics of adenovector distribution within the inner ear tissues of the mouse

Recent studies have demonstrated that delivery of genes to the inner ear can achieve a variety of effects ranging from support of auditory neuron survival to protection and restoration of hair cells, demonstrating the utility of vector based gene delivery. Translation of these findings to useful experimental systems or even clinical applications requires a detailed understanding of the pharmacokinetics of gene delivery in the inner ear. Ideal gene delivery systems will employ a well tolerated vector which efficiently transduces the appropriate target cells within a tissue, but spare non-target structures. Adenovectors based on serotype 5 (Ad 5) are commonly used vectors, are easy to construct and have a long track record of efficacious gene transfer in the inner ear. In this study we demonstrate that distribution of Ad5 vector occurs in a basal to apical gradient with rapid distribution of vector to the vestibule after delivery via a round window cochleostomy. Transduction of the vector and expression of the delivered transgene occurs by 10 min post vector delivery. At 24 h post delivery only 16% of vector that was initially detectable within the inner ear by quantitative PCR remained. Perilymph sampling was used to determine that vector concentrations in perilymph peaked at 30 min post delivery and then declined rapidly. Understanding these basic distribution patterns and parameters for delivery are important for the design of gene delivery vectors and vital for modeling dose responses to achieve safe efficacious delivery of a therapeutic agent.     

Melanin in the inner ear

Summary Following several studies on the effects of kanamycin toxicity on the inner ears of guinea pigs, we have studied the importance of melanin in this phenomenon. Transmission electron microscopy showed that, under the influence of kanamycin, the intermediate strial cells developed a secretory aspect similar to that seen in skin melanocytes. This aspect as yet has never been described for the inner ear cells. A planimetric, morphometric method was also used to determine the strial cell melanin status in control animals. Additional findings in the study confirmed an increase in the number of melanosomes during kanamycin poisoning. Statistical data are discussed.Presented at the First European Congress of Oto-Rhino-Laryngology and Cervico-Facial Surgery, Paris, 26–29 September 1988