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.     

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.     

Assessment criteria for rotated stereociliary bundles in the guinea pig cochlea.

OBJECTIVE: Our study examined the relationship between variant stereociliary bundles of cochlear outer hair cells (OHCs) and auditory function to analyze assessment criteria for rotated stereociliary bundles in the guinea pig cochlea. METHODS: Auditory brainstem response and distortion product otoacoustic emission (DPOAE) were recorded on 100 guinea pigs. Variant hair cells were identified and counted by scanning electron and light microscopy. RESULTS: The most common variation observed was rotation of stereociliary bundles in the first-row OHCs (OHC1), with most 13.3% variant OHC1 rotated 90 degrees and a few 2.5% rotated 180 degrees. Occasionally, the length and angle of the 2 arms of an OHC deviated from the norm. The auditory brainstem response threshold of affected animals increased only slightly, 20- to 30-dB sound pressure level. More importantly, amplitude of DPOAE increased significantly (40.5 dB sound pressure level). CONCLUSION: Our study suggests that rotation of stereociliary bundles in the cochlear OHC was found to be prevalent in 28% of the animals. We established the assessment criteria for rotated stereociliary bundles that were more than 10% OHC1 rotated. This hair bundle seemed to be rotated by 90 degrees from the normal orientation and was accompanied with changes of auditory function. Increased amplitude of DPOAE is associated with the variation of rotated OHC that might result in hearing loss.     

Morphological changes to the stereociliary bundles in the guinea pig cochlea after kanamycin treatment

Pigmented guinea pigs were treated with 400 mg/kg of kanamycin for 8 days and sacrificed 13 days later. The upper surface structures of the organ of Corti were studied using high resolution scanning electron microscopy to reveal fine details of stereociliary fusion and damage to the reticular lamina. Outer hair cell stereocilia showed from partial to complete fusion and loss whilst the inner hair cells showed very little evidence of any damage. In particular, the possible effects of kanamycin on the cross-links between the stereocilia have been investigated. The cross-links are found to be present on all outer and inner hair cell bundles which show no fusion of stereocilia. Partially-fused stereocilia which have free tips still possess the upward-pointing links, and the remaining undamaged stereocilia in these bundles possess the normal cross-links. Where fusion occurs along the sides of the stereocilia, the side-to-side links are missing although whether their absence either results in, or else is caused by, the fusion is not known.     

Tip-link organization in anomalously-oriented hair cells of the guinea pig cochlea

Stereocilia are described in a group of guinea pigs with abnormal conformation of the outer hair cell stereociliary bundles. Rows of Stereocilia were found which were circular instead of V- or W-shaped, and rotated or even reversed in orientation with respect to normal. The Stereocilia have however a normal gradation in heights of the rows of Stereocilia, and have tip links running in the direction of gradation in height.     

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.     

Structure and composition of stereocilia cross-links in normal and hydropic cochleas of the guinea pig

Summary Structure and composition of stereocilia cross-links were investigated cytochemically in normal and hydropic cochleas of the guinea pig. The electron-dense markers colloidal thorium and cationized ferritin were used for visualization. Side links as well as tip links were visualized using both markers. Cationized ferritin allowed a better visualization of the delicate cross-link substructure than did colloidal thorium. Following digestion with neuraminidase, cross-link reactivity for colloidal thorium was virtually abolished. However, the basic structure of the cross-links could still be observed as a result of routine post-fixation and contrast staining. In both 3- and 6-month hydropic cochleas glycocalyx reactivity of the stereocilia appeared to be unaltered, provided that stereocilia were still present. However, loss of cross-links of the outer hair cells — resulting in disarrangement of the stereociliary bundles — was observed in hydropic cochleas. Our results suggest that cross-links are a separate morphological and cytochemical entity, which is different from the glycocalyx. Furthermore, loss of stereocilia cross-links, with concomitant disarrangement of the outer hair cell stereociliary bundle, appears to be one of the early pathological features of surgically induced endolymphatic hydrops, which might be responsible for permanent sensorineural hearing loss.     

豚鼠椭圆囊斑静纤毛间的相互连接形式

目的：探讨豚鼠椭圆囊斑毛细胞静纤毛束间的相互连接形式及其生理意义。方法：用扫描电镜和透射电镜观察豚鼠椭圆囊斑毛细胞静纤毛束间相互连接。结果：豚鼠椭圆囊斑毛细胞静纤毛束间存在３种相互连接形式：①顶部连接：自短纤毛顶至长纤毛的上行连接;②侧连接：同行静纤毛间的云雾状连接;③行间连接：邻近行间的连接。结论：３种连接的存在可将纤毛束连为一体。推测当纤毛偏转时,所有静纤毛可能形成一个整体依次移动,牵拉或放松     

Vulnerability of tip links between stereocilia to acoustic trauma in the guinea pig

The cochleae of anaesthetized guinea pigs were prepared for scanning electron microscopy, immediately after exposure to an intense tone. Stereocilia on hair cells showing relatively small degrees of disruption were analyzed. If the bundles of stereocilia showed no or only a very slight degree of disorganization, the fine links emerging from the tips of the shorter stereocilia remained intact. If the stereocilia were separated more than a very little, the tip links between stereocilia were no longer visible. However, it was possible for tip links to remain intact in some parts of the hair bundle, while tip links in other, more disrupted parts, were lost. In outer hair cells, tip links did not seem any more vulnerable in one position than in another. In inner hair cells, it was commonly found that the tip links running between the tallest stereocilia and the next row of shorter stereocilia had broken, while the tip links running between the other shorter rows of stereocilia remained intact. The results suggest that tip links between stereocilia are preserved as long as the other links between the stereocilia and the cytoskeleton of the stereocilium remain intact. When the latter are damaged the tip links fracture. The results also suggest that, if the tip links are indeed involved in transduction, some degree of stimulus transduction can continue in damaged inner hair cells, albeit with a reduced sensitivity.     

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。     

Stereociliary anomaly in the guinea pig: effects of hair bundle rotation on cochlear sensitivity.

Histological analysis of cochleas from 100 albino guinea pigs (Hartley strain) obtained from Charles River Laboratories revealed an apparently congenital anomaly in 24% of animals, with roughly equal prevalence in males and females. In affected animals, 15-50% of the first-row outer hair cells (OHCs) showed distinctly abnormal orientation of the W-shaped stereociliary array. These abnormal hair bundles could be rotated by up to 180 degrees from the normal quasi-radial orientation. Second- and third-row OHCs appeared normal in all cases. Cochlear sensitivity was assayed in a subset of animals via compound action potentials (CAPs): CAP thresholds in affected animals were, on average, elevated by 5-10 dB with respect to normal controls. If the contributions of individual OHCs to cochlear 'amplification' add linearly, and if the total OHC contribution corresponds to approximately 45 dB of 'gain', a quantitative correlation of the degree of stereociliary rotation and the degree of threshold shift in these ears suggests that first-row OHCs make a larger contribution to the cochlear amplifier than either of the other OHC rows.     

Development of hair cell stereocilia in the avian cochlea

The establishment of an embryonic hair cell's stereociliary bundle involves the coordinated regulation of several morphogenetic events. The developing hair cell organizes the assembly of individual stereocilia, regulates the growth of the stereociliary bundle, and aligns the orientation of the bundle. During development, individual stereocilia exhibit three phases of growth: (1) an initial assembly and elongation of a small number of actin filaments; (2) the development of the stereocilia rootlet and the addition of more filaments to each stereocilium; (3) a second growth phase where elongation of the actin filaments resumes. These three phases involve different biochemical conditions for actin assembly and, thus, are temporally separated during development. Each hair cell also regulates the size of its stereociliary bundle so that it fits into the precise basal-to-apical gradient in stereocilia length and width seen in the mature cochlea. Orientation of the stereociliary bundles also changes during development. Very young hair cells exhibit a non-uniform orientation. Early in development, neighboring groups of cells rapidly acquire a uniform orientation. A more gradual shift in orientation continues throughout development, so that by maturity most of the hair cells are oriented toward the abneural edge of the sensory epithelium.     

Atrophy of outer hair cell stereocilia and hearing loss in hydropic cochleae

We have earlier described selective atrophy of short and middle stereocilia on outer hair cells of the three upper cochlear turns in hydropic cochleae of guinea pigs. The present study describes sequential early stages of stereocilia degeneration leading to this specific atrophy. Comparison of the morpho-pathology with the ultimate CAP audiograms taken before sacrifice indicated a close association between the low frequency hearing loss and this atrophy of stereocilia. The atrophy appeared to be associated first with the short and then the middle stereocilia of the 2nd and 3rd rows of outer hair cells between 0.5 kHz and 2 kHz and with time included the 1st row of all outer hair cells of the upper cochlear turns down to the 8 kHz region.     

An inner ear anomaly in golden hamsters

A new mutation of the inner ear was discovered in golden hamsters raised in our laboratories. Although scanning electron microscopy showed a normal arrangement of individual stereocilia on the first row of outer hair cells, the entire bundle of stereocilia were irregular in orientation and scattered in several directions. Seventy per cent to 85% of the stereociliary bundles were found to be abnormal throughout the cochlea, with no apparent difference between the right and left sides. Transmission electron microscopy showed that the cuticles of the first row of the outer hair cells were dislocated, but no dislocation due to this mutation was apparent in the lower portions. This mutation of the inner ear was already present in the basal turn four days after birth. The kinocilium was located outside of the stereocilia in the first row of outer hair cells, but sensory hairs were scattered in every direction, as in the adult animals. A comparison of auditory brainstem response tests revealed no difference between the abnormal and normal hamsters.     

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.     

Differences in hair bundles associated with type I and type II vestibular hair cells of the guinea pig saccule.

Several studies have reported variations in shape and size of stereociliary bundles and in a limited number of observations have associated them to type I and type II hair cells. A systematic study has been undertaken for which a technique was developed in order to identify both cell types and their corresponding hair bundles. Numerous fissures were obtained in saccular epithelia and observed in scanning electron microscopy. Saccular type I and type II hair cells in the guinea pig were found to have distinctive hair bundles. The tallest stereocilia of almost all type I cells were longer than 6 microns, and were shorter in the striola compared to the periphery. In contrast, the tallest stereocilia of almost all type II cells were shorter than 6 microns and were not found to vary notably in size from the striola to the periphery. Hair bundles with stereocilia organized in straight or in staggered rows were found for both types of cells across the whole saccular epithelium, with no apparent particular distribution. Possible physiological significance of differences in hair bundles is discussed.     

Development of the tectorial membrane

During development, the organization of the stereociliary bundles undergoes drastic changes from the microvilli-like nascent stereocilia to the 'W' formation of the step-like arrangement of the adult form. During this period the developing tectorial membrane (TM) establishes prescribed attachments with various substructures of the developing sensory ciliary bundles and supporting cells. The TM detaches from the supporting cells and inner hair cell stereociliary bundles as K?lliker's organ matures. The inter-connecting linkage system develops postnatally, and the 'tip-linkages' are already found in one-week-old mice, suggesting that the critical organization of the micromechanics of the stereocilia matures rapidly during the postnatal period. The TM develops in stages, and its development parallels that of the organ of Corti. The major TM is initially secreted by the greater epithelial ridge cells, and the minor TM is produced by the lesser epithelial ridge cells. The substructures of the TM are formed by the participation of a number of different supporting cells. During the active stage of production of the substructures by the supporting cells, these cells are intensely Alcian blue-PAS stained, indicating that the glycoconjugates are locally produced by these cells.     

Regeneration of hair cell stereociliary bundles in the chick cochlea following severe acoustic trauma

Examination of pure-tone acoustic damage in the chick cochlea revealed a significant amount of hair cell recovery over a 10 day period following the exposure. The recovery included both a regeneration of stereociliary bundles to replace those that were lost and a reshuffling of the mosaic pattern of the hair cell surfaces that survived. Ten-day-old chicks were exposed to a 1500 Hz pure tone at 120 dB SPL for 48 h and their cochleae were processed for scanning, transmission and light microscopy at 0 h, 24 h. 48 h, 4 d, 6 d and 10 d after exposure. Immediately after exposure the damaged region exhibited two types of hair cell trauma. The first was a defined area of complete hair cell loss and the second was an area where the hair cells survived but exhibited varying amounts of stereocilia injury. After 48 h of recovery, new hair cells were identifiable in the region of hair cell loss and with time they underwent a progressive maturation of their stereociliary bundles. The surviving hair cells showed a dramatic rearrangement and expansion of their surfaces but exhibited no repair of the damaged stereociliary bundles. These results suggest that the chick cochlea is capable of a significant amount of recovery and regeneration following acoustic trauma.     

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.