Development of the embryonic chick's tectorial membrane

The nascent tectorial membrane (TM) is identifiable as early as stage 33 (7th day) as thin, wispy material. By stage 37 (11th day), the dense mesh of the immature TM and fibrous webs (subtectorial threads) that attach the TM to the basilar papilla are distinct but scanty. The TM condenses slightly in its upper face. The growth of the columnar cells and basilar papilla during the following days pulls the TM, lifting it upward, and resembling the cables on a suspension bridge in cross-section. As a result, a large hollow wedge forms. During stages 40-44 (14th-18th days), the columnar cells secrete large amounts of fibrous material, which fills the hollow wedge and condenses into the dense meshes. The honeycombed patterns appear at this time. The supporting cells secrete the fibrous webs. Their secretory activity closely corresponds to that of the columnar cells. The secretory material from both cell types remains attached to the apical ends of their respective cells after secretory activity ends. By hatching (stage 46-21 days), the columnar cells have filled with fibrous material and their cytoplasmic organelles are restricted to the apices. The cytoplasm of supporting cells is relatively clear, with few cytoplasmic remnants of their intense secretory activity earlier.     

中耳胆脂瘤肥大细胞形态学观察

对３０例中耳胆脂瘤肥大细胞进行组织化学与透射电镜观察，并与健康外耳道皮肤对照．结果发现中耳胆脂瘤肥大细胞明显多于正常对照组（Ｐ＜０．０１），主要分布范围包括上皮层、下皮下特别是近骨吸收面的肉芽组织内。电镜下大多数肥大细胞呈活化状态，与ＴＣ肥大细胞类似，呈无定型均质细颗粒状或毛玻璃样改变，肥大细胞贴壁的小静脉内见许多多形核白细胞溢出；活化肥大细胞主要呈“缓慢脱颗粒”状态；少数肥大细胞与成纤维细胞及浆细胞密切接触。讨论了肥大细胞在中耳胆脂瘤发病和发展中的可能作用。     

Neurogenin 1 Null Mutant Ears Develop Fewer, Morphologically Normal Hair Cells in Smaller Sensory Epithelia Devoid of Innervation

The proneuronal gene neurogenin 1 (ngn1) is essential for development of the inner-ear sensory neurons that are completely absent in ngn1 null mutants. Neither afferent, efferent, nor autonomic nerve fibers were detected in the ears of ngn1 null mutants. We suggest that efferent and autonomic fibers are lost secondarily to the absence of afferents. In this article we show that ngn1 null mutants develop smaller sensory epithelia with morphologically normal hair cells. In particular, the saccule is reduced dramatically and forms only a small recess with few hair cells along a duct connecting the utricle with the cochlea. Hair cells of newborn ngn1 null mutants show no structural abnormalities, suggesting that embryonic development of hair cells is independent of innervation. However, the less regular pattern of dispersal within sensory epithelia may be caused by some effects of afferents or to the stunted growth of the sensory epithelia. Tracing of facial and stato-acoustic nerves in control and ngn1 null mutants showed that only the distal, epibranchial, placode-derived sensory neurons of the geniculate ganglion exist in mutants. Tracing further showed that these geniculate ganglion neurons project exclusively to the solitary tract. In addition to the normal complement of facial branchial and visceral motoneurons, ngn1 null mutants have some trigeminal motoneurons and contralateral inner-ear efferents projecting, at least temporarily, through the facial nerve. These data suggest that some neurons in the brainstem (e.g., inner-ear efferents, trigeminal motoneurons) require afferents to grow along and redirect to ectopic cranial nerve roots in the absence of their corresponding sensory roots.     

Inflammatory pseudotumor in the middle ear cavity

Inflammatory pseudotumor includes a diverse group of lesions characterized by inflammatory cell infiltration and variable fibrotic responses. It is extremely rare in the middle ear alone. A 7-year-old girl presented right hearing impairment. Because an otitis media with effusion was first suspected, a myringotomy was performed, but it found a mass that was different from a congenital cholesteatoma. Canal wall-down tympanomastoidectomy removed the mass successfully. The pathologic study of the specimen confirmed an inflammatory pseudotumor. We report an extremely rare case of the inflammatory pseudotumor in the middle ear with a review of the poor literature about this subject.     

Cell potential and motility of isolated mammalian vestibular sensory cells

Vestibular hair cells (VHCs) were isolated from the guinea pig inner ear. Using the whole cell variant of the patch clamp recording technique a zero current cell potential of −63.1 ± 9.9 mV was measured in macular hair cells. Depolarization and repolarization were accompanied by mechanical responses of the solitary VHCs. In addition to the evoked motile events spontaneous shape changes of VHCs were observed. Implications for vestibular micromechanics of the observed evoked force generation in VHCs are discussed.     

279例耳闷胀感的病因分析

目的 探讨产生耳闷胀感的部位和原因。方法 收集耳闷胀感为主诉的279例362耳,对患耳的临床表现、鼓室导抗图、纯音测听和咽鼓管功能进行分析。结果 362耳中鼓室导抗图A型203耳、B型111耳、C型33耳以及鼓膜穿孔15耳。纯音测听表现为传导性聋、感音神经性聋、混合性聋以及正常者分别是83耳、8l耳、105耳和93耳。在362耳中,咽鼓管功能异常208耳,在A型鼓室导抗图203耳中,咽鼓管功能异常67耳。结论 产生耳闷胀感的病变部位包括外、中、内耳和面神经的病变,其中以分泌性中耳炎、各种原因引起的感音神经性聋和“咽鼓管狭窄症”为多。     

The progenitors of inner ear hair cells and their regulating genes

Hair cells in the mammalian inner ear are very fragile and are often injured as a result of acoustic trauma or exposure to ototoxic drugs (cisplatin, aminoglycosides, etc)[1]. In amphibians and birds, spontaneous post-injury regeneration of all inner ear sensory hair cell occurs, while in the mammalian cochlea, such hearing loss is usually permanent as there are currently no treatments that can lead to post-injury hair cell regeneration.     

经鼓阶胚胎干细胞内耳导入的初步观察

目的观察未经体外诱导的胚胎十细胞（embyonic stem cells,ESC）导入听力正常大鼠内耳的可行性以及导入后的存活和分布情况,为ESC内耳移植治疗由毛细胞缺失导致的感音神经性耳聋提供实验基础和理论依据。方法5—6周龄Wistar大鼠,10只,右耳为实验耳：经鼓阶打孔法植入带有绿色荧光蛋白（EGFP）的ESCs;左耳为对照组,不实施手术。术前1周与术后即刻行听性脑干反应（ABR）检查,取双侧耳蜗做冰冻切片,观察ESCs植入内耳后存活和分布情况。结果术后动物存活8只,麻醉效果好无干扰完整测完ABR动物5只。鼓阶打孔途径导入耳蜗的ESCs大部分于鼓阶聚集悬浮,少数可在鼓阶基底膜嵴和鼓阶外侧壁处贴壁;未在柯替器等中阶部位巾发现有ESCs的分布。ABR检测结果显示鼓阶打孔途径导入方法对大鼠听力影响较小。结论胚胎干细胞可经耳蜗底转鼓阶打孔途径导入耳蜗。干细胞在内耳成功存活,并且对内耳损伤小,因此。它可以作为内耳细胞移植的重要方式。     

Mechanisms of movement in outer hair cells and a possible structural basis

Summary Isolated outer hair cells were found to slowly shorten when subjected to a solution that would induce contraction in a muscle fibre. Two possible mechanisms underlying this behaviour emerge from ultrastructural and immunocytochemical investigations. Antibody labelling at the electron microscopic level demonstrates that actin is present not only in the stereocilia and in the cuticular plate but also along the wall of outer hair cells, between the plasma membrane and the subsurface fenestrated cisternae. The latter are interconnected by regularly spaced pillars, resembling those seen between the T-tubules and sarcoplasmic reticulum in muscle fibres. Contraction also results from the application of positively charged macromolecules to the bathing solution. This implies sensitivity of the membrane-associated complex (the cortex system) to an electrical current. A second contractile system may reside in the cytoplasm, where calmodulin is present in contracted hair cells. This protein is a calcium-binding control protein for contraction-like events in smooth muscle and non-muscle cells. The unique presence of the cortex system in outer hair cells, and its absence in inner hair cells, indicates a functional significance that relates to a motor function of outer hair cells in hearing.     

Growth of a fish ear: 1. Quantitative analysis of hair cell and ganglion cell proliferation

Proliferation (or addition) of inner ear sensory hair cells continues for a long time postembryonically in cartilaginous and bony fishes, and in amphibians. In contrast, proliferation only occurs during embryonic development in birds and mammals. However, detailed quantitative data on hair cell addition are not available for bony fishes. In order to quantify the extent of proliferation, we determined the number of sensory hair cells on the saccular sensory epithelium in specimens of the cichlid fish Astronotus ocellatus (the oscar) ranging from 2.0 to 19.0 cm in standard length (0.9-343 g). Ganglion cells were counted using serial sections of the saccular branch of the eighth nerve in animals of the same size range. The saccular macula of a 2.0 cm long (0.9 g) Astronotus contains approximately 5500 sensory hair cells; fish from 16 to 19 cm long have over 170 000 hair cells. The increase in number of sensory cells and the increase in both length and weight of the animals studied were statistically correlated (r2 = 0.8). The relative densities of saccular sensory cells in different epithelial regions remained constant in animals from 2.0 to 17 cm; in larger animals the cell density decreased somewhat. Based upon very conservative estimates of the rate of growth of Astronotus, we calculate that an average of 167 hair cells/day are added during the time when the cell population of the saccule increases. Ganglion cell number also increased approximately 4.8 times in the range of fish studied. The smallest animals in our study had about 150 ganglion cells per saccular epithelium, while the largest fish had over 600 ganglion cells. We estimate that the average ratio of hair cells to afferent fibers increases from about 30:1 in the smallest fish to over 300:1 in the largest animals.     

Stem/Progenitor Cells Derived from the Cochlear Sensory Epithelium Give Rise to Spheres with Distinct Morphologies and Features

Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair cell-like cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.     

人类β防御素2在中耳胆脂瘤上皮中的表达及意义

目的:研究中耳胆脂瘤上皮中人类β防御素2(hBD2)的表达及与上皮内朗汉斯巨细胞(LC)密度的相关性,探讨hBD2在胆脂瘤病理过程中的作用。方法:采用免疫组织化学计算机图像分析方法观察21例中耳胆脂瘤上皮和10例外耳道正常皮肤表皮中hBD2的表达及LC密度。结果:胆脂瘤上皮中hBD2的表达水平及LC密度较外耳道皮肤表皮增高,差异具有统计学意义(均P<0.01)。胆脂瘤上皮中hBD2表达与上皮内LC的密度存在正相关(P<0.05)。结论:胆脂瘤上皮中hBD2表达较外耳道表皮显著增高,表明其与胆脂瘤的慢性炎症病理状态关系密切。胆脂瘤上皮中hBD2可能是LC重要的趋化因子,在连结特异与非特异性免疫中扮演一定角色。     

A model for signal transmission in an ear having hair cells with free-standing stereocilia. I. Empirical basis for model structure

No adequate theory for the signal-transmission properties of the peripheral auditory system exists for any vertebrate ear. Because the mammalian ear seems to pose conceptual and technical problems that complicate the development of an adequate theory, it is worthwhile to investigate simpler ears. The ear of the alligator lizard is simpler than mammalian ears in several respects: the motion of the basilar membrane is approximately independent of longitudinal position and is approximately linearly related to the sound pressure at the tympanic membrane; in a large region of the cochlea the hair cells have free-standing stereocilia that are not in contact with a tectorial membrane; the receptor potential of these hair cells is related to the sound pressure at the tympanic membrane in a relatively simple manner; the cochlear-nerve fiber responses from this region do not exhibit two-tone rate suppression. Also, the relative accessibility of this ear has enabled measurement of several response variables: tympanic-membrane volume velocity, extracolumella velocity, basilar-membrane velocity, hair-cell stereociliary displacement, hair-cell receptor potentials, and cochlear-nerve-fiber discharges. A model is developed to represent these results in terms of underlying anatomical structures and physiological mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

A threshold decrease for electrically stimulated motor responses of isolated aging outer hair cells from the pigmented guinea pig

When outer hair cells are isolated from guinea pig cochleas and are placed in normal Hank's medium, they exhibit aging as a slow tonic reduction in length and increase in diameter. During this time the lateral subsurface cisternae become progressively vesiculated and the optical density of the border seen under phase-contrast microscopy decreases. A study of 65 outer hair cells was carried out using video imaging of this process. The base of each cell bonded to the Petri dish and the motility of the cuticular plate was recorded in two ways. To quantify the slow contraction of each preparation, the dimensions of the cell were measured from video replay. Displacements of the cuticular plate in response to an alternating electric field in line with the cell axis were also monitored using a video tracking technique. The amplitude of a 1 Hz stimulus required to cause a visually detectable motor response above baseline noise decreased as the cell degraded. Typically, fresh cylindrical cells exhibiting high optical contrast showed relatively small movements for field strengths up to 2 kVm^–1. However, as the cell depolarized, the rigidity initially decreased and the cell could respond to field strengths down to 0.1 kVm 1 before cell death ultimately occurred. Such a threshold phenomenon in the isolated OHC has not been demonstrated directly until now. This result explains the variability of electromotility in aging in vitro preparations from the cochlea.     

In vitro growth and collagen synthesis in fibroblasts from the rabbit middle ear mucosa

The present methodological study was undertaken to introduce a model system in which individual cells of the middle ear mucosa could be studied under controlled conditions allowing standardized sampling and different manipulations using quantitative methods. The method is based upon isolation and culture of fibroblasts from normal rabbit middle ear mucosae. The growth pattern of the cells was determined by measurement of the total content of cell protein, DNA content and cell division activity. Collagen synthesis was also estimated and results compared with normal skin fibroblasts. Finally, fibroblasts derived from rabbit middle ear mucosae with otitis media were cultured under similar conditions. Results demonstrated the method to be valid and reproducible. Evaluated by any of the parameters applied, growth initially increased exponentially, followed by a stationary phase with a constant cell mass. The growth potentials in middle ear fibroblasts appeared to differ significantly from skin fibroblasts. Except for a decreased cell proliferation, fibroblasts from diseased mucosae did not demonstrate any major differences from the normal fibroblasts probably because the original inflammatory stimuli in vivo were lost in vitro. These findings suggest that future investigations of a model system of otitis media in vitro require the addition of inflammatory mediators.     

Over Half the Hair Cells in the Mouse Utricle First Appear After Birth, with Significant Numbers Originating from Early Postnatal Mitotic Production in Peripheral and Striolar Growth Zones

Many non-mammalian vertebrates produce hair cells throughout life and recover from hearing and balance deficits through regeneration. In contrast, embryonic production of hair cells declines sharply in mammals where deficits from hair cell losses are typically permanent. Hair cell density estimates recently suggested that the vestibular organs of mice continue to add hair cells after birth, so we undertook comprehensive counting in murine utricles at different ages. The counts show that 51 % of the hair cells in adults arise during the 2 weeks after birth. Immature hair cells are most common near the neonatal macula’s peripheral edge and striola, where anti-Ki-67 labels cycling nuclei in zones that appear to contain niches for supporting-cell-like stem cells. In vivo lineage tracing in a novel reporter mouse where tamoxifen-inducible supporting cell-specific Cre expression switched tdTomato fluorescence to eGFP fluorescence showed that proteolipid-protein-1-expressing supporting cells are an important source of the new hair cells. To assess the contributions of postnatal cell divisions, we gave mice an injection of BrdU or EdU on the day of birth. The labels were restricted to supporting cells 1 day later, but by 12 days, 31 % of the labeled nuclei were in myosin-VIIA-positive hair cells. Thus, hair cell populations in neonatal mouse utricles grow appreciably through two processes: the progressive differentiation of cells generated before birth and the differentiation of new cells arising from divisions of progenitors that progress through S phase soon after birth. Subsequent declines in these processes coincide with maturational changes that appear unique to mammalian supporting cells.     

L型电压门控钙通道α1D亚基在内耳毛细胞的分布及在听力中的作用

目的探讨L型电压门控钙通道α1D亚基在内耳毛细胞的分布及在听力中的作用。方法应用免疫细胞化学荧光标记观察L型电压门控钙通道α1D亚基在牛蛙毛细胞上的分布;利用听性脑干反应(ABR)检测携带不同L型电压门控钙通道α1D基因型(α1D / 、α1D /-和α1D-/-)小鼠的听力。结果L型电压门控钙通道α1D亚基主要密集分布于毛细胞侧膜和顶膜,在毛细胞的胞核区域和纤毛丛处没有阳性荧光染色。三种α1D基因型小鼠的ABR检测结果显示,α1D / 野生型小鼠的短声反应阈正常,为34.8±5.7dBSPL;α1D /-杂合子小鼠反应阈高于同窝生α1D / 野生型小鼠,为54.4±12.4dBSPL;α1D-/-小鼠呈现全聋,ABR在100dBSPL时仍无反应。结论L型电压门控钙通道的α1D亚基分布在牛蛙毛细胞的侧膜和顶膜,在内耳的听觉生理中具有重要作用。L型电压门控钙通道α1D亚基缺失或是数量的减少均可以影响小鼠听力。     

鸡内耳毛细胞标志物的比较研究

目的：为研究内耳毛细胞发育和再生提供有价值的标志物。方法：新生鸡内耳冷冻切片，间接免疫荧光细胞化学技术染色毛细胞抗原（HCA）、Espin和肌浆球蛋白7a基因，观察上述基因在毛细胞中的表达部位和强度。结果：HCA、Espin和肌浆球蛋白7a在听毛细胞和前庭毛细胞均表达；HCA染色毛细胞顶部和纤毛，Espin染色毛细胞纤毛和皮板，肌浆球蛋白7a为典型的细胞质染色。结论：HCA、Espin和肌浆球蛋白7a基因是研究毛细胞发育和再生的理想标志物，结合肌动蛋白-F染色毛细胞纤毛可以更好地定性和定量研究内耳毛细胞发育和再生。     

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