Scanning electron microscopy of the normal human cochlea

Preservation of the fine structures of the human cochlea has been achieved by perfusing the cochlea with fixative shortly after death. Following the dissection of the temporal bone the surface of the organ of Corti and stria vascularis has been examined in the scanning electron microscope. The surfaces of the inner and outer hair cells can be seen and the stereocilia projecting from their surfaces closely examined. The number and length of the stereocilia of the outer hair cells changes linearly with distance along the cochlear duct. The surface of the stria vascularis is similar to that seen in other animals.     

Electron microscopic study on inner ear barotrauma: with emphasis on the cochlear damage

In order to investigate inner ear barotrauma, guinea pigs were subjected to rapid decompression between 2 absolute pressure (ATA) and 1 ATA in a chamber. After pressure loading and observation for absence of Preyer's reflex, they were sacrificed immediately, 1 day, 1 week and 1 month later, respectively. Then, morphological changes of the organ of Corti and stria vascularis were studied under TEM and SEM. The immediate features noted were fracture of stereocilia with minimal intracellular changes. One day later, there was marked degeneration of outer hair cells and expansion of supporting cells. The damage to stereocilia clearly preceded morphological alterations within hair cell bodies and cannot be interpreted as arising secondary to hair cell degeneration. Most of outer hair cells eventually disappeared and were replaced by supporting cells. Inner hair cells degenerated slowly; 1 month later, some of them remained almost intact, despite disappearance of stereocilia. The continuity of reticular lamina was maintained not only immediately but also through the period of hair cell degeneration, thus preventing any leakage of endolymph into the organ of Corti. There was reversible dendritic swelling of inner hair cells immediately following the trauma. No changes of stria vascularis were observed over passage of time. The mechanism of hair cell damage due to inner ear barotrauma is presumed to be a deformity of the organ of Corti caused by pressure discrepancy between perilymph and endolymph resulting in an injury to stereocilia.     

Microprobe analysis of cryofixed, chemically untreated freeze-dried cochlear hair cells

Elemental (energy-dispersive X-ray) microanalysis was performed of cryofixed, chemically untreated freeze-dried cochlear hair cells of 1-month-old CBA/CBA mice. This technique is very difficult to carry out and time consuming, requiring a large number of specimens to be sectioned, quite at random, in order to obtain absolutely perpendicular section planes. Microprobe determination was performed in different parts of outer and inner hair cells. In addition to the normal intracellular elemental content a high calcium concentration was found at the base of all hair cells. Lower calcium levels were evident in the entire cytoplasm, including the cuticular plate and stereocilia. Technically it was feasible to analyze individual stereocilia.     

Ultrastructural localization of calmodulin in gerbil cochlea by immunogold electron microscopy

Localization of calmodulin, a calcium binding protein, was identified in adult gerbil cochleas using paraffin section immunohistochemistry and immunogold electron microscopy with monoclonal antibody against bovine calmodulin. Immunoreactive calmodulin was abundant in inner hair cells (IHCs), outer hair cells (OHCs) and Boettcher cells of the cochleas. Other cell types containing calmodulin were marginal cells and basal cells of the stria vascularis, fibrocytes in the spiral ligament, spiral ganglion neurons and vascular smooth muscle cells. Immunogold labeling for calmodulin was observed in cuticular plate, stereocilia, and within cytoplasm of IHCs and OHCs. In OHCs the labeling was mostly observed in the region underlying lateral wall corresponding to subsurface cisterna. In IHCs the staining was diffuse in the cytoplasm and denser than that in OHCs. Boettcher cells showed dense staining along the microvillous projections facing to the intercellular spaces between Boettcher cells and Claudius cells and between the neighboring Boettcher cells. These distributions of calmodulin in the hair cells consist with the assumption that IHCs act as a true neurotransducer and OHCs as an active bi-directional mechanotransducer. The rich presence of calmodulin in Boettcher cells suggests that the cells may involve in mediating Ca(2+) regulation and play a distinctive active role in ion transport.     

Stereociliary bundle morphology in organotypic cultures of the mouse cochlea

Organotypic cultures of the neonatal mouse cochlea have a band of hair cells consisting of 3-5 rows of outer hair cells and a single row of inner hair cells. The outer hair cell stereociliary bundles show progressive differentiation from the apical to the basal ends of the band. Undifferentiated apical bundles have a disk-like array of short stereocilia resembling microvilli. Partially differentiated bundles are hemispherical with poorly organized rows of thickly clustered stereocilia, which gradually increase in height in the direction of the kinocilium. More differentiated bundles remain hemispherical with many microvilli-like stereocilia, but have highly organized rows of sterocilia along the side nearest to the kinocilium, and well-defined height increments between the rows. Highly-differentiated, basal bundles usually have a 'V' or 'W' shape, although some can be almost polygonal. The basal bundles have 4-5 regular rows of stereocilia with a well-defined gradient in height across the rows, and very few microvilli-like stereocilia. Cross-links are only consistently observed in more differentiated bundles, where the rows of stereocilia are regular and have significant height increments across them. The links show a wide variety of forms and orientations not previously observed in other preparations. Spoke-like arrays of links project from the upper regions of many stereocilia and other stereocilia appear to bear distinct tip-to-side links, although with a variety of orientations. A similar variety of cross-links is observed in early postnatal cochleae in vivo, but not in the cochleae of adult mice, indicating that this variety may be a transient feature of sterociliary bundle development. In vitro, inner hair cell stereociliary bundles are often covered by overlying material from the developing tectorial membrane. The variations in morphology of inner hair cell bundles and their cross-links are similar to those of the outer hair cell bundles.     

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.     

Rotated stereociliary bundles and their relationship with the tectorial membrane in the guinea pig cochlea

Hair cells with rotated stereociliary bundles have been observed in the cochleae of control and kanamycin-treated guinea pigs. The affected outer hair cell bundles have a variable degree of rotation, with some being completely reversed. The inner hair cells are more rarely affected, and only small areas of an individual inner hair cell bundle are abnormal. In counts from ten cochleae, the number of outer hair cells with rotated bundles was most commonly between 10% and 20%, with almost 27% of all outer hair cells affected in the most extreme case. The rotated outer hair cell bundles often have distorted outlines but in other respects closely resemble normal bundles. In particular, they have the usual gradation in stereociliary height, intracellular cross-links and intercellular links to adjacent normally-orientated bundles. There are also corresponding imprints in the tectorial membrane which match the pattern of the stereocilia. In kanamycin-treated guinea pigs, imprints of both normal and rotated hair bundles are present, even when the corresponding bundle is absent, and there are frequently remnants of stereocilia inserted in the imprints. These observations suggest that, apart from their abnormal orientation, the rotated bundles are similar to normal bundles in both their organization and association with the tectorial membrane. The implications of this with respect to transduction and cochlear mechanics are discussed.     

High resolution scanning electron microscopy of the human organ of Corti. A study using freshly fixed surgical specimens

Scanning electron microscopy on immediately fixed human cochleae obtained during surgery for life-threatening petro-clival meningioma showed excellently preserved morphology. We compared the morphological findings with those from transmission electron microscopic sections of well preserved human and animal tissue. The characteristics of neural innervation, the pathways of the nerves through the organ of Corti and the intimate relation of nerves to supporting cells along their route could be studied in detail. The lateral membranes of Hensen and Claudius cells were folded creating a surface enlargement. Marginal pillars extended the distal end of the tectorial membrane and correspond to the marginal net or "randfasernetz" described earlier. Stereocilia imprints at the undersurface of the tectorial membrane go as far as to the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cell, attached to the marginal pillars, raises questions about differences in the excitation of the last row of outer hair cells. The complex nature of many supporting cells, stria vascularis and Reissner's membrane, intracellular complexities as well as surface features are described. Supernumerary inner hair cells were observed and the different arrangement of outer spiral fibres in contrast to findings in animals and variations of nerve fibres within the organ of Corti between apex and base are discussed.     

Expression of heregulin and ErbB/Her receptors in adult chinchilla cochlear and vestibular sensory epithelium

Immunolabeling of heregulin, a growth factor that enhances cell proliferation in damaged utricles, and one of its binding receptors, ErbB-2, has been briefly described in the P3 rat cochlea and utricle [Zheng et al. (1999) J. Neurocytol. 28, 901-912]. However, little is known about the distribution of heregulin and its three binding receptors in adult animals. Here we describe the immunolabeling patterns for heregulin, ErbB-2, ErbB-3 and ErB-4 in the cochlea, spiral ganglion, utricle and saccule of the adult chinchilla using confocal microscopy. Heregulin immunolabeling was intense along the apical pole of Deiters cells and Hensen cells and along the membrane of supporting cells of the utricle and saccule; light immunolabeling was present in the outer layer of the spiral prominence and cytoplasm of spiral ganglion neurons. In the cochlea, intense to moderate ErbB-2 immunolabeling was evident in the cytoplasm of pillar cells, outer hair cells (OHCs), border cells, stria vascularis and spiral ligament; moderate ErbB-2 immunolabeling was present in the cytoplasm of the hair cell and supporting cell layers of the utricle and saccule. In the cochlea, light ErbB-3 immunolabeling was present in the inner hair cells, OHCs, marginal and intermediate cell layers of the stria vascularis and spiral ganglion neurons; moderate ErbB-3 immunolabeling was present in the cytoplasm of hair cells and supporting cells of the utricle and saccule. In the cochlea, utricle and saccule, ErbB-4 immunolabeling was intense in the nuclei and light to moderate in the cytoplasm and membrane of sensory cells and supporting cells. These results suggest that heregulin acting through ErbB receptors and various receptor complexes may play an important role in cell proliferation and survival in the cochlea and vestibular system.     

Deterioration of the elemental composition of endolymph in genetic inner ear disease

Summary A normal elemental composition of the endolymph in the scala media of the Shaker-1 and the Shaker-2 mouse does not occur even when there is a normal cell morphology of the stria vascularis. In addition to distinct peaks for potassium and chlorine — but with a decreased relative peak intensity as compared with normals — sodium, phosphorus, sulphur, and calcium were detected. In normal cochlear endolymph only potassium and chlorine were found. At a point of time when the dark cells of the stria vascularis showed severe atrophy, the deterioration of the elemental content of endolymph in the cochlea had further increased showing low concentrations of potassium and chlorine.The dark cell region around vestibular organs remained ultrastructurally rather intact in several months old animals but could reveal an extensive vacuolization of cell cytoplasm and occasionally a reduced number of intercellular extensions towards the basal membrane. The elemental analysis of vestibular endolymph revealed reduced but still high peaks for chlorine and potassium and a distinct peak for sodium thus having an endolymph-like elemental composition. The secretory/reabsorptive epithelia in the vestibular part of the inner ear thus are either not sufficient alone to maintain the specific ionic composition of endolymph or have an inborn type of metabolic disorder in ultrastructurally rather normal cells and therefore are unable to produce a normal composition of endolymph.The fluid filled spaces of the homozygotic Kreisler mouse inner ear did not reveal an elemental composition similar to endolymph or even to a modified type of endolymph.Supported by grants from the Foundation Tysta Skolan, the Torsten and Ragnar Söderberg's Foundation and the Swedish Medical Research Council (12X-720)     

Ultrastructure of the inner ear of NKCC1-deficient mice

The transduction of the auditory signal is dependent on the flow of ions within the inner ear. We have generated mice deficient in NKCC1, an ion cotransporter that is thought to be involved in the secretion of K+ by the strial marginal cells. Inner ear histology revealed partial to almost total absence of the scala media and collapse of Reissner's membrane. Ultrastructural analysis showed that Reissner's membrane consists of 3-4 cell layers instead of the usual two, and a substance of unknown composition is present between Reissner's membrane and underlying structures. Within the tunnel of Corti, hair cells and supporting cells were difficult to identify. The location of the tectorial membrane was altered, and a precipitate was observed surrounding it. Severe structural defects were noted in the interdental cells and Boettcher cells, and mild defects were observed in the stria vascularis and in type II and type IV fibrocytes. The finding that major defects occur predominantly in cells that are not known to express NKCC1 suggests that loss of NKCC1 results in functional defects in cells expressing NKCC1 and a morphological effect on cell populations downstream in the proposed K+ recycling pathway.     

Mechanical transduction in outer hair cells

The outer hair cells are responsible for the exquisite sensitivity, frequency selectivity and dynamic range of the cochlea. These cells are part of a mechanical feedback system involving the basilar membrane and tectorial membrane. Transverse displacement of the basilar membrane results in relative motion between the tectorial membrane and the reticular lamina, causing deflection of the stereocilia and modulation of the open probability of their transduction channels. The resulting current causes a change of membrane potential, which in turn produces mechanical force, that is fed back into the motion of the basilar membrane. Experiments were conducted to address mechanical transduction mechanisms in both the stereocilia and the basolateral cell membrane, as well as modes of coupling of the outer hair cell force to the organ of Corti.     

Tectorial membrane. II: Stiffness measurements in vivo

The tectorial membrane is assumed to play a crucial role in the stimulation of the cochlear hair cells and was thought for decades to serve as a stiff anchor for the tips of the hair-cell stereocilia, particularly those belonging to the OHCs. Yet, its stiffness has never been measured under conditions approximating its normal environment in live animals. We have developed a method for doing this. The tectorial membrane is approached through the lateral wall of scala media. The bony cochlear capsule is removed along scala media over somewhat less than 1/4 turn, and the underlying spiral ligament and stria vascularis are carefully reflected. With the help of a three axial hydraulic manipulator, a flexible micropipette filled with isotonic KCl is inserted into the tectorial membrane at one of two different angles and moved either transversally, away from the basilar membrane, or radially, toward or away from the modiolus. This causes the tectorial membrane to be deformed and the micropipette to bend. The micropipette stiffness is calibrated on an instrument of a new kind, so as to convert the bend into force. The calibration allows us to determine the point stiffness of the tectorial membrane from the amount of micropipette bend. The stiffness of the tectorial membrane per unit length has been calculated from the point stiffness with the help of the deformation pattern. Transversal and radial stiffness magnitudes have been determined in the second cochlear turn in Mongolian gerbils. Both are smaller by almost an order of magnitude than the corresponding aggregate stiffness of the OHC stereocilia. As a consequence, the tectorial membrane cannot act as a stiff anchor for the stereocilia but only as a mass load, except at relatively low sound frequencies where mass effects are negligible. This means that the classical model of shear motion between the tectorial membrane and the reticular lamina must be replaced.     

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。     

Presbycusis: a human temporal bone study of individuals with flat audiometric patterns of hearing loss using a new method to quantify stria vascularis volume

OBJECTIVE: The purpose of this study was to determine the prevalence of stria vascularis atrophy in individuals with presbycusis and flat audiometric patterns of hearing loss. Individuals with presbycusis have historically been categorized by the shape of their audiograms, and flat audiometric thresholds have been reported to be associated with atrophy of the stria vascularis. Stria vascularis volume was not measured in these studies. STUDY DESIGN: Retrospective case review. METHODS: Archival human temporal bones from individuals with presbycusis were selected on the basis of strict audiometric criteria for flat audiometric thresholds. Six temporal bones that met these criteria were identified and compared with 10 temporal bones in individuals with normal hearing. A unique quantitative method was developed to measure the stria vascularis volume in these temporal bones. The hair cell and spiral ganglion cell populations also were quantitatively evaluated. RESULTS: Only one of the six individuals with presbycusis and flat audiometric thresholds had significant atrophy of the stria vascularis. This individual with stria vascularis atrophy also had reduced inner hair cell, outer hair cell, and ganglion cell populations. Three of the individuals with presbycusis had spiral ganglion cell loss, three individuals had inner hair cell loss, and all six individuals had outer hair cell loss. CONCLUSIONS: The results of this investigation suggest that individuals with presbycusis and flat audiometric patterns of hearing loss infrequently have stria vascularis atrophy. Outer hair cell loss alone or in combination with inner hair cell or ganglion cell loss may be the cause of flat audiometric thresholds in individuals with presbycusis.     

Early development of cochlear hair cell stereociliary surface morphology

The early development of the surface structures of differentiating cochlear hair cells (guinea-pig) was analysed by scanning electron microscopy (SEM). A basal-to-apical gradient was evident in hair cell maturation. Inner hair cells developed before outer hair cells at the same level in the cochlea. The first sign of the onset of hair cell differentiation was a regularization of the pattern of microvilli on the future hair cell. Later, the cluster of regularized microvilli was rebuilt to form the stereociliary bundle, with a stepwise increase in the length of those stereocilia facing the stria vascularis.     

Localization of the integral membrane glycoprotein synaptophysin and the surface glycoprotein Egp-34 in the embryonic and adult human inner ear

By using well-defined monoclonal antibodies, the distribution of synaptophysin and the glycoprotein Egp-34, the first of its kind, was analyzed immunohistochemically in the fetal and the adult human inner ear. In fetal labyrinths, a distinct immunoreactivity for synaptophysin occurred in the apical region of both outer and inner hair cells as well as in nerve terminals adjacent to both cochlear and vestibular hair cells. In adult hair cells, immunoreactivity was found throughout the cytoplasm. Synaptophysin may act as an important calcium binding protein in the sensory transduction of hair cells. A selective expression of Egp-34 was found in the area of marginal and intermediate cell infoldings in the stria vascularis. A similar localization pattern was found for a number of membrane transport enzymes. The glycoprotein Egp-34 is probably of importance for the active mechanisms regulating the homeostasis of endolymph.     

Effects of type 2 diabetes mellitus on cochlear structure in humans

OBJECTIVE: To evaluate the effects of type 2 diabetes mellitus on cochlear elements in humans. DESIGN: Comparative study of the histopathologic characteristics of human temporal bones. SETTING: Otopathology laboratory in a tertiary academic medical center. PATIENTS: Temporal bones from 18 patients with type 2 diabetes mellitus were divided into 2 groups according to the method of management of diabetes: insulin in 11 patients (mean age, 51.9 years; age range, 44-65 years) and oral hypoglycemic agents in 7 patients (mean age, 54.4 years; age range, 45-64 years). The diabetic groups and 26 age-matched controls (mean age, 52.9 years) were examined using light microscopy, and the cochlear changes were compared between groups. MAIN OUTCOME MEASURES: Morphometric measurements of vessel wall thickness in the basilar membrane and stria vascularis were made in all turns of the cochlea at the midmodiolar level. Area measurements of the stria vascularis were made in all turns of the cochlea at the midmodiolar level. Cochlear reconstructions and standard cytocochleograms were prepared using an oil immersion objective. The number of spiral ganglion cells was determined for each segment of the cochlea. Comparisons were made in each segment between diabetic and control groups. RESULTS: In the insulin group, walls of the vessels of the basilar membrane and stria vascularis in all turns were significantly thicker than those of controls. Walls of the vessels of the stria vascularis in the basal turn were also significantly thicker in the oral hypoglycemic group than in controls. Atrophy of the stria vascularis in most turns of the insulin group and the lower middle turn of the oral hypoglycemic group was significantly greater than in the controls. Loss of cochlear outer hair cells was significantly greater in the lower and upper basal turns in both diabetic groups. No significant difference was found in the number of spiral ganglion cells or inner hair cells between groups. CONCLUSION: This study demonstrates that cochlear microangiopathy and degeneration of the stria vascularis and cochlear outer hair cells are found in patients with type 2 diabetes mellitus.     

Effect of furosemide upon morphology of hair bundles in guinea pig cochlear hair cells

Furosemide was administered by intraperitoneal injection and intracochlear perfusion. The peak-to-peak amplitude of the cochlear microphonic at high sound intensities was reduced however the drug was administered. After a period of time ranging from 1-4 h following drug application, cochleae were fixed and prepared for scanning electron microscopy to examine the hair cells. Changes in the hair bundles from basal turns tended to be more extensive than those of more apical turns. Initially there was an increase in the granularity of the surface of the stereociliary membrane and a tendency for cross-links to swell, stretch and break. Later, the surface texture of stereocilia became smoother than normal, and hair cells in the basal turns showed extensive erosion and fracture of cross-links. Tip links could survive even when extensively stretched. It is possible that the morphological changes in stereocilia reported here do not arise from direct actions of furosemide, but indirectly, from perturbations of the ionic composition of cochlear fluids induced by effects of the drug upon the stria vascularis.     

Experimental mumps labyrinthitis in monkeys (Macaca irus)--immunohistochemical and ultrastructural studies

Three monkeys (Macaca irus) were inoculated with mumps virus into unilateral cochleas and their inner ear were examined by immunofluorescent microscopy and transmission electronmicroscopy. The temporal bones were removed after survival period of 14 days when serological tests disclosed elevation of anti-mumps antibody titers. Immunofluorescent microscopy revealed that the viral antigen was positive in the stria vascularis. The ultrastructural study revealed that the pathologic changes in the cochleas were marked in the organ of Corti and stria vascularis. The outer hair cells were more susceptible to the infection than the inner hair cells. In the stria vascularis, both marginal and intermediate cells were affected. It was possible to find some of marginal cells in the basal turn shedding a large number of mature virions into the endolymph. These pathologic changes observed in the cochleas of the monkeys were similar to those previously revealed in the guinea pig cochleas and thus were considered as the specific features of acute mumps labyrinthitis.