鸡内耳盖膜的超微结构观察

为了解鸟纲内耳盖膜的超微结构及其与毛细胞、支持细胞的关系，应用扫描电镜和透射电镜对鸡内耳盖膜的超微结构进行观察。发现：扫描电镜下盖膜横断面呈三角形，内有许多空洞，内淋巴面呈网状；透射电镜下见盖膜由微丝及肢状基质构成，听毛细胞的最高排静纤毛及动纤毛插入盖腹中，支持细胞的微绒毛通过絮状细丝与盖膜呈锚状连接。这种连接既有弹性又十分牢固，可在一定范围内限制盖膜运动。由此推测鸟纲内耳与哺乳纲内耳在感受声刺激的方式上有所不同。     

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.     

Three-dimensional observation of the cochlea. Intracellular structure of the hair cell and the supporting cell

Intracellular structures of the guinea pig cochlea were observed by scanning electron-microscopy. The cochlea was freeze-fractured and then macerated with 0.1% OsO4 solution (aldehyde-osmium-DMSO-osmium (AODO) method). Intracellular organelles, such as mitochondria, endoplasmic reticulum (ER), and Golgi apparatus, were demonstrated stereoscopically. The ER of the outer hair cell showed the most interesting features, such as subsurface cisternae and lamellar body. The subsurface cisternae which formed a stratiform network covered the inner surface of the cell membrane of the supranuclear part. Variously shaped mitochondria were found on the innermost layer of the subsurface cisternae. The lamellar body consisted of dilated cisternae and tubules of ER. The tubular ER of the lamellar body were contiguous with the subsurface cisternae. The pillar cells, Deiters' cells and Hensen's cells had well-developed tubular ER, while Claudius' cells had poorly developed tubular ER.     

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.     

Atomic force microscope (AFM). A nanomanipulator for biophysical studies of stereocilia of the cochlear hair cells

OBJECTIVES: Studies of the mechanoelectrical sensor system of the hair cell bundle in the cochlea require a manipulation device that enables controlled force application and movement of individual stereocilia in the nanometer range. METHODS: In our atomic force microscope (AFM) setup, the scan is directly controlled in an upright differential interference contrast (DIC) infrared video microscope with a water immersion objective and in the measured AFM image. Here we present studies on hair cells of the mammalian cochlea. RESULTS AND CONCLUSIONS: The resulting images revealed the tips of individual stereocilia of living sensory cells of the organ of Corti and the typical shape of the ciliary bundle. Scanning electron-microscopic (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that up to four AFM manipulations on the same cell did not cause obvious damage to the surface morphology of the stereocilia.     

Effects of submarine engine room steady noise on the compound action potential tuning curves and its relation to cochlear pathology in guinea pigs

Compound Action Potential Tuning Curves (CAP-TC) for tone pip of 2k, 4 kHz were examined in 8 guinea pigs before and after exposure to noise with main energy centered in the range of 0.25-4.0 kHz. CAP-TC was measured with the pure tone simultaneous masking profiles. AP was evoked by tone pip with an intensity of 10 dB above threshold. Masker level producing 40% reduction in AP amplitude was used. Relations between changes in CAP-TC and AP threshold shifts and the pathology of the stereocilia of hair cells were evaluated by surface preparation and SEM observation in 13 ears. After noise exposure, animals with damaged stereocilia showed AP threshold shift of 20-50 dB, deteriorations of CAP-TC, decrease of Q10 dB value, threshold shift of characteristic frequencies (CF) and displacement of CF towards higher frequencies. It showed that stereocilia damage may affect the susceptibility and frequency selectivity of the cochlea. We consider the CAP-TC may be an useful and sensitive index for detecting physiological and pathological conditions of the cochlea.     

Early development of cochlear hair cell stereociliary surface morphology

Summary 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.Supported by grants from Karolinska Institute, the Swedish Medical Research Council (12X-720), the Swedish Society for Medical Sciences and the Foundation Tysta Skolan     

Post-mortem changes in hair bundles of the guinea pig and human cochlea studied by high-resolution scanning microscopy

High-resolution scanning electron microscopic studies have been made on the guinea pig cochlea on material fixed from 15 min to 4 h post-mortem. Changes in the surface texture and cross-links of stereocilia were detected after only 15 min. Such changes included an increase in granularity of the surface membrane, thickening, stretching and fracturing of all types of cross-links accompanied by splaying apart and loss of rigidity of stereocilia. These changes were more pronounced in the basal turns of the cochlea and in general increased in severity and spread more apically with increasing times post-mortem. By 4 h, many hair bundles consisted of a fused amorphous mass in which individual stereocilia were not discernible. Remarkably at this time, some hair bundles appeared to have suffered little damage. These results will facilitate better discrimination between effects solely due to post-mortem necrotic changes and those due to specific actions of ototoxic drugs and other insults.     

How are inner hair cells stimulated? Evidence for multiple mechanical drives

Recent studies indicate that the gap over outer hair cells (OHCs) between the reticular lamina (RL) and the tectorial membrane (TM) varies cyclically during low-frequency sounds. Variation in the RL-TM gap produces radial fluid flow in the gap that can drive inner hair cell (IHC) stereocilia. Analysis of RL-TM gap changes reveals three IHC drives in addition to classic SHEAR. For upward basilar-membrane (BM) motion, IHC stereocilia are deflected in the excitatory direction by SHEAR and OHC-MOTILITY, but in the inhibitory direction by TM-PUSH and CILIA-SLANT. Upward BM motion causes OHC somatic contraction which tilts the RL, compresses the RL-TM gap over IHCs and expands the RL-TM gap over OHCs, thereby producing an outward (away from the IHCs) radial fluid flow which is the OHC-MOTILITY drive. For upward BM motion, the force that moves the TM upward also compresses the RL-TM gap over OHCs causing inward radial flow past IHCs which is the TM-PUSH drive. Motions that produce large tilting of OHC stereocilia squeeze the supra-OHC RL-TM gap and caused inward radial flow past IHCs which is the CILIA-SLANT drive. Combinations of these drives explain: (1) the reversal at high sound levels of auditory nerve (AN) initial peak (ANIP) responses to clicks, and medial olivocochlear (MOC) inhibition of ANIP responses below, but not above, the ANIP reversal, (2) dips and phase reversals in AN responses to tones in cats and chinchillas, (3) hypersensitivity and phase reversals in tuning-curve tails after OHC ablation, and (4) MOC inhibition of tail-frequency AN responses. The OHC-MOTILITY drive provides another mechanism, in addition to BM motion amplification, that uses active processes to enhance the output of the cochlea. The ability of these IHC drives to explain previously anomalous data provides strong, although indirect, evidence that these drives are significant and presents a new view of how the cochlea works at frequencies below 3?kHz.     

Scanning electron microscopy of hair cells,stereocilia and cross-linkage systems in experimentally induced endolymphatic hydrops

Summary In this study scanning electron microscopy was used to examine the stereocilia and their cross-linkages in guinea pig cochleas 1, 2, 4 and 8 months after endolymphatic sac obliteration. Initial changes were restricted to the stereocilia of the outer hair cells and consisted of a disarrangement and bulging of the stereocilia with an interruption of their cross-linkage systems. Subsequently, the stereocilia became fused and atrophied. Cross-linkages of inner hair cells remained intact. Loss of both outer and inner hair cells started in the apex and progressed towards the base of the cochlea. These findings indicate that early changes in the micro-architecture of the stereocilia may have a mechanical origin, with pressure fluctuations in the scala media possibly playing an important role. Offprint requests to: J. E. Veldman     

SEM analysis of the developing tectorial membrane in the chick cochlea

The development of the tectorial membrane in the embryonic chick cochlea was studied using scanning electron microscopy. Chick embryos ranged in age from embryonic day 7 (E7) to post-hatching day 15. Our studies revealed that a fine filamentous matrix arose on the apical surface of the basilar papilla at approximately E7. This matrix was secreted by the supporting cells which encircled the hair cells. By E9, the early matrix had increased in volume but remained filamentous in structure, except at the inferior edge of the basilar papilla where it was condensed into a layer of laterally-oriented columns. At E9 the TM exhibited an additional layer of matrix, called the amorphous component. It appeared to originate from the homogene cell population, and attached to the early columnar matrix at the inferior edge of the basilar papilla. The two components of the TM were separated by a longitudinal ridge, called the 'track', which marked the inferior edge of the amorphous component. As the cochlea developed, the basilar papilla increased in width, the columnar component elongated and the track appeared to recede. These morphological findings point to separate developmental origins for the two components of the tectorial membrane.     

强噪声暴露后大鼠听觉电生理及形态学改变

目的:观察强噪声暴露后大鼠听觉电生理及形态学的改变,为探讨噪声性聋的发病机制提供实验依据.方法:大鼠随机分为对照组和实验组,实验组暴露于中心频率为4 kHz的窄带噪声中,给声强度为120 dBSPL,暴露时间为4 h.观察2组听觉脑干诱发电位(ABR)及耳蜗形态学的变化.结果:实验组出现ABR反应阈上升,与对照组比较差异有统计学意义(P<0.01);暴露后第1天,基底膜铺片经DNA荧光染料碘化丙锭染色后示;实验组3排外耳毛细胞(OHC)中可出现不同的毛细胞细胞核形态变化(正常、凋亡、坏死和缺失),而第21天未见明显的细胞凋亡;OHC的缺失2组差异有统计学意义(P<0.01),螺旋神经节细胞计数2组差异无统计学意义(P>0.05).扫描电镜示;实验组OHC纤毛异常(散乱、倒伏)及OHC的缺失,以第3排OHC最严重.结论;所应用的强噪声能引起大鼠听觉系统的损害,产生永久性阈移(PTS).该噪声条件下,耳蜗毛细胞的死亡模式在暴露后早期包括凋亡和坏死,而晚期则主要是坏死;PTS的产生可能和OHC纤毛异常及OHC的缺失有关.     

Helical Structure of Hair Cell Stereocilia Tip Links In the Chinchilla Cochlea

Outer-hair-cell stereocilia tip-link structure in the chinchilla cochlea was studied by transmission electron microscopy using tannic acid and Ruthenium red/Alcian blue histochemical procedures. Tannic acid and Ruthenium red/Alcian blue treatments showed the tip link as a compact strand of filaments 9–12 nm thick. Fourier analysis of tip-link images showed that the strand is a three-start helical bundle of fine, coiled filaments which had an axial period of 22.5 ± 1.5 nm. Each of three coiled filaments in the strand showed globular structures, 4.3 ± 0.3 nm in diameter. The globular structures may correspond to individual protein subunits or they may be repeating identical domains of one polypeptide. The three filaments of the helical array may provide a rigidity to the tip link during stereocilia deflections. Alternatively, changes in the subunit or domain structure of each filament may result in a lengthening or shortening of the tip-link strand.     

Secretion of a new basal layer of tectorial membrane following gentamicin-induced hair cell loss

Scanning electron microscopy (SEM) and video-enhanced DIC light microscopy were used to assess morphological changes in the chick tectorial membrane (TM) following gentamicin-induced hair cell loss. Gentamicin was administered (100 mg/kg/day for 3 days) and isolated and in-situ TMs were examined in both fixed and unfixed preparations at days 5 and 10 after the initial injection. Although this protocol induced hair cell damage extending up to 75% of the length of the basilar papilla, there was no apparent damage to the TM itself. However, the ejection of damaged hair cells appeared to sever the filamentous attachments between the TM and the apical surface of the basilar papilla. In SEM preparations this detachment caused the TM to shrink back toward the superior edge. Interestingly, despite the lack of TM damage, gentamicin treatment did reveal the secretion of a new basal layer of TM. Secretion of this new basal layer had begun by day 5 and it was well organized by day 10. This new layer formed attachments to both the recovering basilar papilla and the overlying original TM, a step thought to be necessary for the restoration of auditory function in the regenerating cochlea.     

Sustained cadherin 23 expression in young and adult cochlea of normal and hearing-impaired mice

Cadherin 23 encodes a single-pass transmembrane protein with 27 extracellular cadherin-domains and localizes to stereocilia where it functions as an inter-stereocilia link. Cadherin 23-deficient mice show congenital deafness in combination with circling behavior as a result of organizational defects in the stereocilia hair bundle; common inbred mouse strains carrying the hypomorphic Cdh23(753A) allele are highly susceptible to sensorineural hearing loss. Here, we show that an antibody (N1086) directed against the intracellular carboxyterminus reacts specifically with cadherin 23 and detects with high sensitivity the isoform devoid of the peptide encoded by exon 68 (CDH23Delta68). Cochlea, vestibule, eye, brain and testis produce the CDH23Delta68 isoform in abundance and form moieties with different molecular weight due to variations in glycosylation content. In the cochlea, CDH23Delta68 expression is highest at postnatal day 1 (P1) and P7; expression is down regulated through P14 and P21 and persists at a low steady-state level throughout adulthood (P160). Furthermore, CDH23Delta68 expression levels in young and adult cochlea are similar among normal and hearing deficient strains (C3HeB/FeJ, C57BL/6J and BUB/BnJ). Finally, by immunofluorescence using an antibody (Pb240) specific for ectodomain 14, we show that cadherin 23 localizes to stereocilia during hair bundle development in late gestation and early postnatal days. Cadherin 23-specific labeling becomes weaker as the hair bundle matures but faint labeling concentrated near the top of stereocilia is still detectable at P35. No labeling of cochlea stereocilia was observed with N1086. In conclusion, our data describe a cadherin 23-specific antibody with high affinity to the CDH23Delta68 isoform, reveal a dynamic cochlea expression and localization profile and show sustained cadherin 23 levels in adult cochlea of normal and hearing-impaired mice.     

纯中药制剂复聪汤对庆大霉素致聋豚鼠耳蜗毛细胞修复再生的实验研究

目的观察纯中药制剂对庆大霉素致聋动物和耳蜗毛细胞的修复再生作用。方法选用73只健康青年豚鼠,行听力学检查后,53只动物连续肌肉注射庆大霉素(80mg.kg-1.d-1)24～30天致聋(GM组),其间死亡8只,剩余45只随机分成中药治疗组(25只)和致聋后对照组(20只),中药治疗组给予纯中药制剂"复聪汤Ⅰ号"口服液和"复聪汤Ⅱ号"滴耳液治疗,致聋后对照组同法给予生理盐水。另外20只豚鼠作为正常对照组,每天肌肉注射等量生理盐水。在治疗30、60和90天后对GM组两组动物分别行ABR、DPOAE检测,同时,每一时间点处死6只动物,左侧耳蜗行扫描电镜观察,右侧耳蜗铺片行光镜观察和毛细胞计数。结果实验前所有豚鼠的听功能正常;连续注射庆大霉素30天后,GM组豚鼠ABR反应阈值上升到50～80dB SPL,DPOAE幅值降低,光镜和电镜下表现为耳蜗毛细胞纤毛断碎、倒伏、融合和消失,多处毛细胞表皮板肿胀、突起和疱疹样变性,或毛细胞被完全挤出网状板,基底回和第三回耳蜗毛细胞严重消失;中药治疗30天后,80%的耳聋豚鼠的ABR反应阈恢复到30～50dB SPL,DPOAE幅值明显提高;耳蜗铺片和电镜观察显示耳蜗毛细胞数目有一定恢复,组织结构有修复现象,扫描电镜可见再生毛细胞仅出现少量纤毛,而致聋后对照组未发现此种现象;治疗60天后,耳蜗毛细胞数目明显增多,Corti器的毛细胞区有大量的增殖细胞出现,扫描电镜可见成小束的新生纤毛出现在毛细胞缺失部位,同时支持细胞大量增殖;治疗90天后,再生的静纤毛束已基本形成,尤其是耳蜗基底部位的毛细胞明显增多,听功能基本正常。结论纯中药制剂复聪汤对庆大霉素致聋豚鼠耳蜗毛细胞有一定的修复和再生作用。     

A scanning electron microscopic study of cochlear changes in iron-deficient rats

The effects of iron deficiency on the cochlea were studied in growing rats fed with a basic iron-deficient diet for 80 days. The electrophysiological changes (auditory thresholds raised more than 15 dB) were observed in 47% of the cochleas of iron-deficient rats. When these organs of Corti were examined by scanning electron microscopy, abnormalities of outer or inner hair cells were found, as follows: (1) fusion and torsion of the stereocilia, (2) coalescence of adjacent stereocilia in the same row, (3) loss of sensory hair stiffness, and (4) loss of stereocilia. Within each lesion, the neighbouring supporting cells and their microvilli showed no damage. The findings indicate that cochlear impairment can be induced by iron deficiency. The peroxidative mechanisms responsible for the lesions of stereocilia in iron deficiency are discussed.     

Organ of Corti surface preparations for computer-assisted morphometry

This paper describes a relatively rapid method (67 h) for producing surface preparations of the organ of Corti that are suitable for a computer-assisted morphometric analysis of cochlear hair cells. On day 1, a cochlea was fixed in OsO4, dehydrated in ethanol and infiltrated with an Epon-like plastic (Medcast). On day 2, the Medcast within the cochlea was polymerized at 60 degrees C in an oven. On day 3, the complete organ of Corti was dissected into approximately 20 segments which were trimmed and mounted on a slide in Medcast. Two 400 mesh transmission electron microscope grids are mounted at the apical and basal ends of the organ of Corti. These grids served as reference points for the establishment of an independent X, Y coordinate system on the slide by a computer-assisted light microscope. The segments were permanently attached to the slide by polymerization of the Medcast at 60 degrees C for approximately 15 h. At the beginning of day 4, the organ of Corti surface preparation was ready for examination by light microscopy or could be further sectioned and examined by transmission electron microscopy.