Ultrastructure of the normal human organ of Corti. New anatomical findings in surgical specimens

Conclusion A thorough scanning electron microscopy (SEM) investigation of immediately fixed human adult cochleae obtained during surgery for petro-clival meningiomas conveyed new information about morphology.

Objective To investigate the ultrastructure of human adult cochleae using SEM.

Material and methods Two human cochleae were decalcified, fixed with glutaraldehyde and osmium and prepared for SEM.

Results The excellently preserved morphology showed the pathways of nerve fibres through the organ of Corti. Undulating lateral cell membranes of Hensen and Claudius cells created an enlarged surface that may be important for homoeostasis. The distal attachment of the tectorial membrane to the reticular lamina was present in the shape of a marginal net, which was extended through marginal pillars. Stereocilia imprints extended as far as the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cells attached to the marginal pillars raises questions about differences in the excitation of the last row of outer hair cells as a result of differences in the composition of the tectorial membrane.     

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.     

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。     

豚鼠耳蜗盖膜下面的超微结构

目的 利用扫描电镜技术详细地观察豚鼠盖膜下的超微结构,为耳蜗的感音机制提供新的认识.方法 用S-4800型超高分辨率扫描电子显微镜观察了6只豚鼠12只耳蜗的盖膜.结果 (1)耳蜗各圈盖膜下面均可观察到外毛细胞静纤毛压迹,这种压迹多半与静纤毛最高排的形态一致,第一圈呈“W”型,由基底圈到顶圈,逐渐由“W”型渐变为“V”型及不规则的簇状.压迹为一排圆形的小凹,将盖膜下表面表层辐射状纤维断开,只有最高排的静纤毛才与盖膜接触形成压迹,压迹排列呈串珠一样.(2)盖膜下内毛细胞相应的位置有一条较深的波浪状沟槽,呈线性结构,沟槽比外毛细胞静纤毛的压迹浅而宽,由耳蜗的基底圈到顶圈这种压迹逐渐呈带状.(3)盖膜下的纤维由盖膜的外缘到盖膜与内毛细胞静纤毛压迹之间以蜗轴呈辐射状排列,但在外毛细胞静纤毛压迹的位置被这些压迹所阻断,而总的辐射方向和纤维走行并没有改变,纤维非常的精细,每根纤维之间又相互交错相连.内、外毛细胞压迹之间,柱细胞顶部的盖膜纤维很细,均匀呈细丝状.而内毛细胞与螺旋缘之间,即内沟上方的盖膜下方的纤维更加细长.结论 通过对盖膜超微结构的观察,盖膜下内外毛细胞静纤毛的压迹充分说明毛细胞静纤毛与盖膜的接触.盖膜下的纤维由盖膜的外缘到柱细胞和柱细胞到盖膜下内毛细胞静纤毛带状压迹之间也就是内、外毛细胞压迹之间,柱细胞顶部的盖膜的纤维与内毛细胞顶部盖膜带状结构到螺旋缘之间的纤维的粗细都不一样.这种纤维的分布特点,可能与盖膜在声音调谐方面所起的作用有一定的关系.     

Three-dimensional structure of outer hair cell pillars

Conclusions. Electron tomography was used to generate three-dimensional reconstructions of the pillars that connect the cell membrane with the cytoskeleton of the outer hair cell. Results are consistent with the hypothesis that pillars are important for mechanically linking the membrane with the cytoskeleton. Objective: To make a qualitative assessment of the morphology of the sub-membrane pillars of cochlear outer hair cells. Materials and methods. Guinea pig cochleae were fixed and prepared for electron microscopy using protocols described previously. Sections were imaged on an electron microscope equipped with a goniometer. The specimens were tilted through a range of 120°, and an image was acquired at each tilt angle. Filtered back-projection was used to generate three-dimensional reconstructions. Results. Twelve individual pillars were successfully reconstructed. Pillars often connect to the cell membrane through a thin segment, and to the cytoskeleton through a forking structure that may form a central cavity.     

Elemental composition of individual cells and tissues in the cochlea

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.     

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.     

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.     

Stereociliary bundles reorient during hair cell development and regeneration in the chick cochlea

We have examined changes in the orientation of stereociliary bundles of hair cells in the cochlear sensory epithelium that occur during normal embryonic development and during the regeneration of hair cells that follows acoustic trauma. At the time when hair cell surfaces become recognizable in the embryonic cochlea, the bundles of stereocilia exhibit a range of orientations, as indicated by the position of the kinocilium and later, by the location of the tallest row of stereocilia. With time, the orientations of bundles on neighboring hair cells become more uniform, a condition that is maintained in the adult. Changes in stereocilia orientation are also observed during the regeneration of hair cells after acoustic trauma. When new hair cells first differentiate at sites of trauma in the recovering sensory epithelium, their stereociliary bundles are not uniformly oriented. Then as the cells mature over a period of days, the bundles become aligned both with the neighboring bundles in the region of the previous lesion and with the pre-existing bundles that surround the site of regeneration. We conclude that the stereociliary bundles of hair cells are reorienting as the cells differentiate. A common mechanism may guide reorientation both during embryonic development and during regeneration. Observations in living cochleae indicate that differentiating stereociliary bundles establish asymmetric linkages to the extracellular matrix of the developing tectorial membrane. During the growth of the tectorial membrane, its progressive extension across the surface of the sensory epithelium may exert traction forces through those asymmetric linkages that pull the bundles of the hair cells into uniform alignment.     

Smad5 基因敲除小鼠耳蜗扫描电镜观察

目的观察Smad5基因敲除杂合（3月组2只小鼠4只耳蜗）子小鼠内耳形态学超微结构变化。方法野生型动物组：1个月组4只小鼠5只耳蜗，2个月组及3个月组各有2只小鼠4只耳蜗：杂合子动物组1个月组3只小鼠3只耳蜗，2个月组2只小鼠4只耳蜗，3个月组3只小鼠6只耳蜗。按常规方法制成标本作扫描电镜观察。结果Smad5基因敲除杂合子小鼠扫描电镜观察：1月组耳蜗中均观察到耳蜗第一和第二圈外毛细胞有不同程度的静纤毛融合，未见外毛细胞缺失，内毛细胞未见明显地病理变化。2月组耳蜗中观察到第一和第二圈均可见内外毛细胞静纤毛不同程度缺失，有的部位以内毛细胞静纤毛缺失为主，而有的部位以外毛细胞静纤毛缺失为主，有的以片状缺失为主，有的则以散在性缺失为主。3个月组3只小鼠6只耳蜗中均观察到不同程度第一和第二圈内外毛细胞静纤毛广泛性缺失，有外毛细胞缺失的部位多，以第一排外毛细胞的静纤毛缺失为主．第二和第三排外毛细胞静纤毛多以散在性缺失为主，有内毛细胞缺失的部位多伴有外毛细胞静纤毛的大片状缺失。野生型动物组扫描电镜观察：1个月组4只小鼠5只耳蜗，2个月组2只小鼠4只耳蜗均未观察到内外毛细胞缺失和静纤毛的变化，3月组有少量的外毛细胞缺失。结论野生型小鼠2个月以内耳蜗毛细胞无明显变化．杂合子小鼠随着月龄的增加内外毛细胞缺失的程度逐渐加重。     

Acoustic overstimulation alters the morphology of the tectorial membrane

After a permanent threshold shift was induced by exposing guinea pigs to a 1 kHz pure tone at 105 dB(A) for 72 h, light microscopic observations of freshly dissected and stained tectorial membranes showed an increased waviness and clumping of the fibers of the middle zone. Hensen's stripe was not seen as a continuous dense structure running through the middle zone but was at times discontinuous and curved. As measured from cross-sections of the cochlea, the thickness of the tectorial membrane was decreased after acoustic overstimulation. The stereocilia of the inner and outer hair cells lie directly under the middle zone. Visual detection levels of threshold of tectorial membrane movement was determined by stimulating the marginal zone of the tectorial membrane of isolated cochlear coils by an oscillating water jet. After acoustic overstimulation the tectorial membrane became more compliant. The tectorial membrane abnormalities were restricted to the regions of the cochlea that demonstrated a 40–50 dB hearing loss.     

Repair pattern of the reticular lamina of the organ of corti in human ears

The repair pattern of the reticular lamina of the organ of Corti was studied in the cochleae of human ears. The inner ears were obtained at autopsy from the individuals who had no evidence of auditory or vestibular disorders or therapy with ototoxic drugs:

• After a loss of outer or inner hair cells, the framework of the reticular lamina was distorted but no gaps were detected. The supporting cells were hypertrophied but no reparative proliferation of the supporting cells was found in the organ of Corti.
• Defects due to the collapse of outer hair cells of the first row were filled in mainly by the hypertrophied heads of the outer pillar cells.
• Defects due to the collapse of outer hair cells of the second, third and fourth row were filled in mainly by the hypertrophied phalanges of Deiters' cells of the first, second and third row respectively.
• The space due to inner hair cell loss was filled in by the nearest inner supporting cells which were hypertrophied and extended toward the space.
• The distortion of the reticular lamina and the aging degeneration were regarded as two possible causes for the total loss of the organ of Corti near the end of the first turn.

     

Ultrastructural changes in the outer hair cells of the guinea pig cochlea after exposure to quinine

The outer hair cells have been shown to have motile properties which are likely to participate in the cochlear performance. Quinine is known to induce hearing loss as well as contraction of skeletal muscles. Isolated outer hair cells and isolated cochleae from guinea pigs have been exposed to quinine, which was also injected into living guinea pigs. When a physiological response was registered, the cells and cochleae were fixed and examined by transmission electron microscopy. In the isolated cells the formation of a central microtubule core occurred and in the cochleae a swelling of the subsurface cisternae in the outer hair cells was observed. The results are discussed in the context of a proposed effect of quinine on the contractile processes of the outer hair cells.     

Neural tuning in the granite spiny lizard

Scanning electron microscopy was used to examine the basilar papilla of the granite spiny lizard. The papilla contains three distinct hair cell populations: an apical and a basal population with free-standing cilia, and a central population with a tectorial membrane. In the free-standing populations, stereocilium length decreases towards the ends of the papilla. Ciliary tuft morphology differs in the free-standing and the tectorial membrane populations, except that several of the free-standing hair cells with the shortest stereocilia have a tuft morphology like the hair cells in the tectorial membrane population. On the basis of single-fiber physiology, auditory nerve fibers can be divided into a low characteristic frequency (CF) and a high CF population. Mappings of the tonotopic organization of the nerve demonstrated two groups of high CF fibers that correspond to the two free-standing hair cell populations. The low CF fibers are associated with the tectorial membrane hair cell population. Fiber CF correlated with hair cell cilium length, not position on basilar membrane, for hair cells with free-standing cilia. Tonotopic organization of high CF fibers could be predicted reasonably well from the histogram of fiber CFs.     

人Corti‘s器扫描电镜观察

目的 了解正常人Corti’s器的超微结构特征。方法 用扫描电镜观察成年人和胎儿的Corti’s器。结果成人和胎儿Corti’s器基底圈内、外毛细胞排列规则,中圈和顶圈毛细胞排列不规则,内毛细胞以一排,外毛细胞以三排排列。可观察到列外的内、外毛细胞,四排排列和自然缺失的外毛细胞。外毛细胞粗大的静纤毛以及由于固定不及时引起的外毛细胞静纤毛气球样改变。结论 用扫描电镜观察成人和胎儿Corti’s器的精细结构,发现了毛细胞排列的基本规律和一些异常的形态。及时固定是保存Corti’s器结构完好的必要条件。     

Morphological changes in the cochlea of the mouse after the onset of hearing

The cochleae of 5, 10, 12 and 15 day old mice and of adults (Mus musculus, strain NMRI) were studied by light and electron microscopy. In each case the same part of the organ of Corti (2.8–3.3 mm from the helicotrema) was examined. The results were correlated with the development of auditory thresholds (in mice of the same strain) obtained by Ehret (Ehret G. (1971) J. Am. Audiol. Soc. 1, 179–184). It was demonstrated that morphological development of the organ of Corti is by no means complete at the onset of behavioural responses to acoustic stimuli. After this event the following morphological changes occur: (1) The basilar membrane filaments stain more intensly and the tympanic cover layer is greatly reduced in thickness and almost completely disappears. (2) The filaments of the pillar cells stain more intensly and apparently increase in number, and the angle between the outer and inner pillar cells increases so that the cross-sectional area of the tunnel of Corti expands. (3) Hook-shaped connections (marginal pillars) between the reticular membrane and the tectorial membrane disappear. The consequences of these morphological changes for the cochlear mechanics are discussed, especially in respect to the increase of sensitivity of hearing.     

Functional morphology of the outer hair cells of the human: new aspects]

Serial sections through the outer hair cells of the human organ of Corti were investigated using high-resolution electron microscopy. Delicate monostratified tubular structures of differing lengths are located on the cytoplasmic side of the hair-cell membrane. They are connected to the cell membrane via short fibers (pillars) arranged in pairs. The tubular structures, which are also termed subsurface cisterns (SSC), constitute an extensive network. This network lines the entire inner surface of the outer hair-cell membrane, and in contrast to the mammals investigated so far, also lines the base of the cell. The postsynaptic cisterns of the efferent synapses are integrated into this system. At the surface of the inner lamina of the cisterns, pores 8.5 nm in diameter are to be found. These are surrounded by protein complexes with a stellate arrangement. Similar protein complexes are located on the inner lamina of the postsynaptic cisterns. Pores 8.5 nm in diameter are also present in the outer lamina of the SSC. These constitute the base for the pillar filaments. At high magnification, the pillars are seen to have a lumen about 6 nm in diameter. Pores about 8 nm in diameter are again to be found where the pillars are anchored in the outer cell membrane. The pillars, consisting of actin, are probably ionic channels, and the pores of the inner lamina of the SSC surrounded by protein complexes are thought to be acetylcholine receptors. Since the postsynaptic cisterns of the efferent innervation of the outer hair cells are part of the overall system of the SSC, and acetylcholine is regarded as the neurotransmitter of efferent innervation, it would seem possible to regulate the subsurface cisterns, as a wall-stabilizing, contractile system, via the efferent innervation. On the basis of serial sections, a three-dimensional, true-to-scale model of the contractile wall system of the human outer hair cell was reconstructed.     

Guinea pig tectorial membrane profile in an in vitro cochlear preparation

The guinea pig cochlea was examined under high-magnification light microscopy in an in vitro preparation. After extraction of the otic capsule, the bulla was opened widely and a small hole made into the fourth turn of the scala vestibuli. The organ of Corti was visualized under artificial endolymph at 600 X magnification. Added 1-micron titanium dioxide particles settled on the upper surface of the transparent tectorial membrane. Particle positions showed that much of this upper surface lay in a flat sheet that extended centrifugally almost to the Hensen's cells, giving the impression it was attached there. The sheet extended at least to the level of the inner hair cells, where a tectorial membrane thickness of about 40 micron was reached. Titanium dioxide particles were seen regularly in immediate proximity to the hair cell cilia, indicating that scala media is continuous with the subtectorial space. Upon mechanical manipulation, Hensen's cells proved to be extremely cohesive and elastic. It is suggested that hair cell stereocilia provide major mechanical connections for the tectorial membrane.     

Scanning electron microscopy of intracellular structures in the hair cells of the guinea pig cochlea using backscatter electrons

Guinea pig cochleae were prepared for scanning electron microscopy (SEM). The lateral surface of the outer hair cells was studied first with secondary electrons. It was possible to find a network of tubular cisterns and long mitochondria stained by osmium immediately under the plasma membrane detected by backscattered electrons. The lateral arrangement of the mitochondria and the tubular cisterns was confirmed by observing the embedded SEM tissues by transmission electron microscopy.     

Displacement of the reticular lamina with and without the tectorial membrane in the guinea pig cochlea

OBJECTIVE: In an attempt to establish the mechanical relationships between the reticular lamina and tectorial membrane, we studied the morphological changes of the reticular lamina on a micrometer scale when an in vitro preparation of guinea pig cochlea with and without tectorial membrane was exposed to a potassium-rich medium. MATERIAL AND METHODS: Using video-enhanced differential interference contrast microscopy, the radial displacement of the inner hair cells (IHCs) and outer hair cells (OHCs) in the reticular lamina was measured in real time after exposure to the potassium-rich medium for 3 min. RESULTS: The amplitude of the displacement of the OHCs in preparations with an intact tectorial membrane was half of that observed in those in which the tectorial membrane had been removed. A similar displacement response was also observed for the IHCs, although it was smaller than that for the OHCs. There was no significant difference in the amplitude of the displacement among the three rows of OHCs. CONCLUSIONS: These results suggest that the structure linking the OHCs to the pillar cells is very elastic and that the movement of the OHCs in situ is weakly mechanically coupled to the IHCs. The tectorial membrane provides increased compliance in the motion of the IHCs and OHCs.