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.     

光学相差显微镜下前庭囊斑两型毛细胞的形态学鉴别

目的:探讨电生理实验中,光学相差显微镜下较为可靠的前庭两型毛细胞辨别标准.方法:对于豚鼠椭圆囊和球囊分离所得的前庭毛细胞,分别对细胞胞体的长、宽以及表皮板纤毛的长度进行测量,并对长宽比和纤毛胞长比进行分析.结果:豚鼠椭圆囊和球囊的两型前庭毛细胞相比,细胞的宽度和纤毛的长度比较差异无统计学意义,Ⅰ型毛细胞的长度明显较长,长宽比较大,纤毛胞长比较小.结论:在既往以是否有明显的颈部来区分两型前庭毛细胞的基础上,结合细胞的长宽比和纤毛胞长比进一步进行辨别,有助于更准确地区分两型毛细胞.     

Cholinesterase activity in vestibular organs of young and old mice.

The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were studied by histochemical methods in the semicircular canal end organs, the utricle and the saccule of young and old mice. AChE was located on the plasma membrane of efferent nerve terminals beneath vestibular hair cells, and along the basement membrane. In the ampulla, stained efferent terminals were more prevalent on the slopes of the crista than in the central region. In all organs examined, there were no discernible differences in AChE activity between young and old mice. BChE activity was observed in the epithelial light cells and supporting cells of the saccule, utricle, and ampulla. Its distribution was similar in both young and old mice in the ampulla, but decreased significantly with age in the utricle. Preliminary data suggest that BChE activity is also weak in old saccular supporting cells. Unlike the utricle, old saccular light cells retained intense BChE activity.     

Compound gravity receptor polarization vectors evidenced by linear vestibular evoked potentials.

The utricle and saccule are gravity receptor organs of the vestibular system. These receptors rely on a high-density otoconial membrane to detect linear acceleration and the position of the cranium relative to Earth's gravitational vector. The linear vestibular evoked potential (VsEP) has been shown to be an effective non-invasive functional test specifically for otoconial gravity receptors (Jones et al., 1999). Moreover, there is some evidence that the VsEP can be used to independently test utricular and saccular function (Taylor et al., 1997; Jones et al., 1998). Here we characterize compound macular polarization vectors for the utricle and saccule in hatchling chickens. Pulsed linear acceleration stimuli were presented in two axes, the dorsoventral (DV, +/- Z axis) to isolate the saccule, and the interaural (IA, +/- Y axis) to isolate the utricle. Traditional signal averaging was used to resolve responses recorded from the surface of the skull. Latency and amplitude of eighth nerve components of the linear VsEP were measured. Gravity receptor responses exhibited clear preferences for one stimulus direction in each axis. With respect to each utricular macula, lateral translation in the IA axis produced maximum ipsilateral response amplitudes with substantially greater amplitude intensity (AI) slopes than medially directed movement. Downward caudal motions in the DV axis produced substantially larger response amplitudes and AI slopes. The results show that the macula lagena does not contribute to the VsEP compound polarization vectors of the sacculus and utricle. The findings suggest further that preferred compound vectors for the utricle depend on the pars externa (i.e. lateral hair cell field) whereas for the saccule they depend on pars interna (i.e. superior hair cell fields). These data provide evidence that maculae saccule and utricle can be selectively evaluated using the linear VsEP.     

Afferent synapses are present in utricular hair cells from otoconia-deficient mice

The head tilt mouse (het/het, abbr. het) is a naturally occurring mutant whose salient phenotypic traits include the complete absence of otoconia in both the utricle and saccule. Cursory histologic evaluation has indicated that the neuroepithelia exhibit a normal appearance. Though evidence exists indicating that utricular function is severely if not completely compromised in these animals, it is not yet known whether afferent synapses exist within utricular hair cells of otoconia-deficient mutants. The absence of synapses would be suggestive of a trophic relationship between stimulus-evoked hair cell activation and the afferent synapse. To address this question, we have conducted an ultrastructural survey of utricular sensory epithelia from confirmed het mice. The specific objective was to determine whether utricular hair cells made synaptic contact with afferent neurons. We found that both type I and II hair cells from utricles of het mice exhibited afferent synapses that were found at numerous sites distributed throughout the utricle. These results indicate that afferent synapses within vestibular hair cells do not critically depend upon stimulus-evoked activity.     

小鼠前庭器官的形态发生

目的了解小鼠前庭器官发育的形态学特征及演变规律.方法利用光镜、扫描电镜、透射电镜观察C57BL/6小鼠从胚胎第8天到刚出生期间前庭器官发育过程中的形态变化.结果 C57BL/6小鼠的前庭器官在胚胎第10.5天开始发育,胚胎第11.5天形成上、后半规管始基,到胚胎第12天水平半规管始基才逐渐形成,椭圆囊已出现许多原始的感觉纤毛,而到胚胎第13天,球囊才开始发育;到胚胎第15天,椭圆囊、球囊、壶腹嵴初具成熟形态,毛细胞和支持细胞也初具雏形,到出生时,前庭器官已基本发育成熟.结论 C57BL/6小鼠前庭感觉细胞在胚胎第12天开始分化,感觉上皮的形态从胚胎第12天至第18天变化最明显.椭圆囊、壶腹嵴的发生较早,随后才形成球囊,到出生时前庭感觉器官已基本发育成熟.     

Morphological evidence of vestibular pathology in long-term experimental diabetes mellitus. II. Connective tissue and neuroepithelial pathology

The influence of long-term experimental diabetes on the saccule and utricle was investigated with light and electron microscopy. Pathological changes involving the connective tissue cells of the subneuroepithelial connective tissue suggest that these cells may play a potentially important role in diabetic pathology of the inner ear. In diabetic animals there was an increased incidence of secondary lysosomes within the connective tissue cells as well as an accumulation of intracellular lipid droplets that increased with the level of hyperglycemia. Five animals with relatively more severe diabetes also had an extensive accumulation of extracellular matrix. Two of these rats with longer diabetes duration had a small number of degenerating type I hair cells scattered through the saccular neuroepithelium, possibly due to chronic stresses from impaired diffusion of oxygen, nutrients and waste material through the dense extracellular matrix. Utricles of these same animals did not have any degenerating hair cells.     

前庭器官超微结构研究及结构图构建

目的通过扫描电镜观察小鼠椭圆囊、球囊、壶腹超微结构,研究分析并据此构建前庭器官新的结构示意图。方法选取3个年龄段小白鼠各10只,分别是年轻组≤2个月、中年组2~12个月、老龄组>12个月。分离出椭圆囊、球囊、壶腹,采用扫描电镜技术样品制备方法制备样品,应用扫描电镜进行样品观察。结果扫描电镜下可以得到:①椭圆囊斑及球囊斑不同层面图片:表面为堆积并相互黏附的"表面耳石",表面耳石下是无结构胶状质;底层表面耳石深入到无结构胶质层里;无结构胶质层下面是毛细胞纤毛及"纤毛间耳石"层,不同纤毛束之间均有纤毛间耳石存在,立于支持细胞表面,表面平坦;蜂窝状胶质物质联接无结构胶质层、纤毛间耳石及毛细胞纤毛。②壶腹超微结构的图片:嵴帽是无结构的胶状质与壶腹外侧壁紧贴,但较易分离,嵴帽和壶腹外侧壁之间有纤细的晶状体物质,在壶腹嵴两侧壁上也有纤细晶体物质(壶腹嵴表面耳石);不同的毛细胞纤毛之间有耳石结构的存在(壶腹嵴纤毛间耳石)。结论通过对前庭器官扫描电镜的观察,发现了椭圆、球囊斑及壶腹的新结构成分,由此构建出新的前庭器官超微结构示意图。     

Vestibular morphology in the mutant mix-mouse

Mix-mice, a new strain of mice with inner ear dysfunction, and their littermates were used in the present study in order to investigate vestibular morphology, visualised by light microscopy (LM) and transmission electron microscopy (TEM). Contrary to the mix-mice, their littermates showed less stained nerve chalices and it was possible to detect that hair cells showed surface herniations and so-called 'blebs' in the apical portion of the epithelium. Sensory hairs showed a disarrayed pattern. The mix-mice had severe microscopical abnormalities: collapse of the membranous cell layer, cavities inside the neuroepithelium, severe loss of hair cells, herniations of the few remaining hair cells and increased supporting cells were evident. In the utricle and saccule, hair cells were missing and the epithelial surface was covered by a single layer of smooth, flattened epithelial cells of hitherto unknown origin.     

Development and evolution of the vestibular sensory apparatus of the mammalian ear

Herein, we will review molecular aspects of vestibular ear development and present them in the context of evolutionary changes and hair cell regeneration. Several genes guide the development of anterior and posterior canals. Although some of these genes are also important for horizontal canal development, this canal strongly depends on a single gene, Otx1. Otx1 also governs the segregation of saccule and utricle. Several genes are essential for otoconia and cupula formation, but protein interactions necessary to form and maintain otoconia or a cupula are not yet understood. Nerve fiber guidance to specific vestibular end-organs is predominantly mediated by diffusible neurotrophic factors that work even in the absence of differentiated hair cells. Neurotrophins, in particular Bdnf, are the most crucial attractive factor released by hair cells. If Bdnf is misexpressed, fibers can be redirected away from hair cells. Hair cell differentiation is mediated by Atoh1. However, Atoh1 may not initiate hair cell precursor formation. Resolving the role of Atoh1 in postmitotic hair cell precursors is crucial for future attempts in hair cell regeneration. Additional analyses are needed before gene therapy can help regenerate hair cells, restore otoconia, and reconnect sensory epithelia to the brain.     

Morphological evidence of vestibular pathology in long-term experimental diabetes mellitus. I. Microvascular changes

The influence of long-term experimental diabetes on the microvasculature of the saccule and utricle was investigated using quantitative light and electron microscopic techniques. Basal lamina thickening or reduplication, typically seen in diabetic microangiopathy, were not observed. However, morphometric analysis did reveal a statistically significant increase in capillary diameters along with an increased vascularization of both the saccule and utricle. Both of these microvascular abnormalities may have been caused by the hemodynamic alterations known to occur in diabetes. These alterations include decreased deformability of red blood cells and increased blood viscosity. Either of these factors can lead to a greater stress on the capillary wall and possibly to a reduced oxygen delivery to the tissues.     

WDR1 expression in the normal and noise-damaged chick vestibule

Unlike mammals, avian cochlear hair cells can regenerate after acoustic overstimulation. The WDR1 gene is one of the genes suspected to play an important role in this difference. In an earlier study, we found that the WDR1 gene is over-expressed in the chick cochlea after acoustic overstimulation. The aim of this study was to compare the expression of WDR1 before and after acoustic overstimulation in the chick vestibule. Seven-day-old chicks were divided into three groups: normal group, damage group, and regeneration group. The damage and regeneration group was exposed to 120 dB SPL white noise for 5-6 hours. The damage group was euthanized shortly after the impulse, but the regeneration group was allowed to recover for 2 days. The utricle, saccule, and the three ampullae of each semicircular canal were dissected and immunohistochemically stained with anti-WD40 repeat protein 1 antibody. For quantitative analysis, immunoreactive densities were measured and quantitative real-time RT PCR was performed. WD40 repeat protein 1 expression was elevated in all the semicircular canals and utricle, two days after an acoustic overstimulation (P=0.001). WDR1 mRNA expression was 1.34 times higher in the regeneration group compared to the normal group, but it was not statistically significant. Exceptionally, WD40 repeat protein 1 expression did not increase in the saccule of the regeneration group. Elevated WDR1 expression in the avian vestibule may have a role in the hair cell regenerating ability as in the avian cochlea. A similar mechanism of hair cell regeneration may exist in the avian cochlea and vestibule.     

Structure and function in the saccule of the goldfish (Carassius auratus): a model of diversity in the non-amniote ear

The vertebrate inner ear is comprised of a remarkable diversity of cell types, including several types of sensory hair cells. In amniotes (reptiles, birds, and mammals), the morphological and physiological characteristics that distinguish these cell types have been well documented, while cellular variation in the ears of non-amniotes (all other vertebrate groups) has remained underrecognized. Since non-amniotes have become increasingly popular models for developmental and genetic research, a more comprehensive understanding of structure and function in the inner ears of these species is warranted. This paper first reviews the large body of data describing the morphology and physiology of hair cells and afferent neurons in the inner ear of the goldfish (Carassius auratus). In particular, we examine the structure of the goldfish saccule, an endorgan that has been the subject of numerous investigations on audition. New data on the structural variation of synaptic bodies in saccular hair cells are also presented, and the functional implications of these data are discussed. Finally, we conclude that hair cell structure varies along the length of the goldfish saccule in a manner consistent with known physiological characteristics of the endorgan. The saccule provides an excellent model for investigating structure-function relationships in the vertebrate inner ear, as well as the development of auditory and vestibular sensory epithelia.     

Vestibular destruction by slow infusion of gentamicin into semicircular canals

Intratympanic or round window application of gentamicin is often used to alleviate disabling vertigo arising from unilateral Meniere's disease; however, treatment is often accompanied by hearing loss because the drug initially enters the cochlea before diffusing to the vestibular system. In order to enhance vestibular damage and reduce the risk of hearing loss, gentamicin was infused directly into the vestibular system. An osmotic pump containing 50, 100, 200 or 400 microg/ml of gentamicin was infused into the superior semicircular canal of the chinchilla for 7 days. Afterwards, vestibular damage was evaluated by measuring the decline in hair cell density in the utricle, saccule and superior semicircular canals. Auditory damage was assessed with distortion product otoacoustic emissions (DPOAE) and outer hair cell (OHC) and inner hair cell (IHC) loss. Infusion with the two lowest gentamicin concentrations resulted in significant hair cell loss and reduced duration of the nystagmus response, but had little or no effect on OHC or DPOAE. Higher doses of gentamicin damaged cochlear hair cells and reduced the DPOAE. In conclusion, slow infusion of a low dose of gentamicin into the semicircular canals mainly damages the vestibular hair cells and inactivates the nystagmus response without damaging cochlear hair cells or DPOAE.     

硫酸链霉素对体外培养大鼠前庭毛细胞的损害作用

目的 探讨硫酸链霉素对离体培养条件下大鼠前庭毛细胞的损害特征.方法 取出生后3～4 d的大鼠球囊斑和椭圆囊斑进行离体前庭器官培养,经培养过夜后再用不同浓度的硫酸链霉素培养液继续培养24 h.应用荧光标记的鬼笔环肽对前庭毛细胞的静纤毛和表皮板进行染色,同时应用Topro-3 DNA探针显示细胞核的形态,在激光共聚焦显微镜下计数和摄像.结果 在离体培养条件下,对照组前庭毛细胞生长良好,随着培养液中硫酸链霉素浓度的增加,毛细胞的密度逐渐降低.培养24 h后,0.25 mmol/L硫酸链霉素可造成大约10%的前庭毛细胞缺损,1 mmol/L硫酸链霉素损害的毛细胞数量在50%左右,3 mmol/L硫酸链霉素破坏的毛细胞数量超过75%.细胞核染色显示硫酸链霉索引起的前庭毛细胞损害伴随着细胞核浓缩或破碎现象.存活毛细胞的数量随着硫酸链霉素浓度的增加而减少,但前庭的支持细胞未发生明显病变.结论 在体外培养条件下,硫酸链霉素可引起剂量依赖性的大鼠前庭毛细胞缺失,其损伤机制可能与凋亡有关.     

Morphometric analysis of the vestibular sensory epithelia of young adult rat

The appearance of vestibular sensory cells and their progressive development has been the subject of many ontogenetic studies. Because deteriorating hair cells are supposed to play a role in balance disorders of the elderly, the final stage of development (i.e. senescence) has been investigated as well. It is generally assumed that the number of hair cells in crista ampullaris, saccule and utricle slowly but steadily decreases with age. However, actual data covering the period between maturation and senescence are scarce. In the present study, rat vestibular epithelia were labeled for actin and tubulin. Morphology was inspected from immediately after weaning until the age of 12 months. Although, postnatal development was no part of this study some data on one day old epithelia are presented for comparison. At postnatal day 1, hair bundles are still shorter than in mature sensory organs, the width of the zonula adherens is less, and the apical cross-sectional area of the epithelial cells is smaller. After one month, maturation is complete. Total cell density is 400-500 per 0.01 mm2, both in the otolith maculae and in the cristae ampullares. During the first year after maturation, no changes in epithelial morphology were observed and cell density remains constant.     

Gentamicin ototoxicity in the saccule of the lizard Podarcis Sicula induces hair cell recovery and regeneration

There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles.     

Three-dimensional morphology of the vestibular cleft and its potential application.

HYPOTHESIS: To delineate quantitatively the spatial relationships of the utricle, saccule, and stapedial footplate, to locate the hole on the footplate, and to analyze the insertion depth into the vestibule and the direction of the piston during stapedotomy. BACKGROUND: The quantitative three-dimensional (3D) configuration of the utricle, saccule, and stapedial footplate is undetermined, and the stapedotomy procedures should be improved. METHODS: Four temporal bones were extracted from the fresh cadavers and were undecalcified polymer-embedded. The specimens were sectioned into serial 50-mum-thickness slices. After image processing and 3D reconstruction, a cartesian coordinate system was established to display the spatial relationships of the utricle, saccule, and stapedial footplate in the 3D Studio Max scene. The configuration of the utricle, the saccule, and the "vestibular cleft" was delineated quantitatively with the contour map method. With this contour map, any distance between one point at the surface of the footplate and another point at the surface of the utricle or saccule and its orientation can be measured. RESULTS: There was a V-shaped cleft between the utricle and the saccule named vestibular cleft. The angle of the cleft was 50.30 degrees +/- 19.90 degrees . The apex of the cleft always directed anterosuperiorly, whereas beneath the posteroinferior part of the footplate was an open and deep "seabed." The vertical distances between points on the tympanic surface of the footplate and points on the surface of the utricle or saccule were measured. The vertical distance from the center point of footplate to the vestibular end organs was 2.20 +/- 0.548 mm, the maximum distance being 3.0 mm, whereas the minimum distance was 1.6 mm. CONCLUSION: The posteroinferior area near the central point of the footplate is the optimal position for the fenestra through which the piston can be inserted relatively safely into a depth of 0.8 to 1.0 mm in the vestibule. If the deep end of the piston is inclined inferiorly and posteriorly by 8 to 10 degrees, respectively, the piston will be farther from the vestibular end organs. These manipulations may enhance surgical safety and efficiency in stapedotomy.     

梅尼埃病前庭功能分级的探讨

目的 建立一种新的梅尼埃病患者前庭功能分级方法,初步探索其临床意义及与梅尼埃病听力分期的相关性。 方法 收集112例单侧梅尼埃病患者资料,所有患者均完成包括纯音测听、cVEMP、oVEMP、vHIT以及冷热试验检查。冷热试验和vHIT的结果中任一项异常均视为半规管功能异常。cVEMP结果异常视为球囊功能异常,oVEMP结果异常视为椭圆囊功能异常。球囊、椭圆囊、半规管功能均正常的患者为前庭功能I级,球囊、椭圆囊、半规管功能其中1项异常者为前庭功能Ⅱ级,2项异常者为前庭功能Ⅲ级,3项均异常者为前庭功能Ⅳ级。 结果 根据听力分期,符合入组标准的95例梅尼埃病患者中Ⅰ期13例、Ⅱ期13例、Ⅲ期52例、Ⅳ期17例。患耳平均听阈为(51.86±21.70)dB HL。前庭功能检测结果cVEMP异常率63.2%,oVEMP24异常率74.7%,vHIT异常率33.7%,冷热试验异常率52.6%。前庭功能分级Ⅰ级9例、Ⅱ级28例、Ⅲ级39例、Ⅳ级19例。前庭功能分级与患者的听力差异有统计学意义(P<0.01, r=0.35)。前庭功能分级与患者梅尼埃病分期差异有统计学意义(P<0.01, r=0.35)。前庭功能分级与患者的病程差异有统计学意义(P=0.02, r=0.24)。前庭功能分级与患者的年龄差异无统计学意义(P=0.084)。 结论 梅尼埃病患者随着病程的进展,耳石器功能和半规管功能检测异常率会逐渐增加。通过对球囊、椭圆囊、半规管低频和高频功能的精细化检测,能够对梅尼埃病患者前庭功能进行精准评估。听力分期与前庭功能分级的同步评估能够反映患者疾病的进展状态,同时能够对患者治疗方案的选择和术后康复的预判起到参考作用。