前庭学

970618哺乳动物前庭迷路的体外发育:周围性眩晕的研究手段/周祥宁…//天津医药一1 996,24(l)一4一7 首次在国内报告小鼠前庭迷路的体外发育。采用周祥宁和Van De Water建立的HEMA水凝胶器官培养法培养第12一13天CBA/C57小鼠胚胎的内耳原基7一8天。内耳原基的上部发育成3个半规管、椭圆囊和球囊。半规管的壶腹晴由一排感觉毛细胞和2一3排支持细胞构成。晴的上方有正在发育的峪顶。椭圆囊斑和球囊斑的表面都有一层耳石膜。这些囊斑内可见一层感觉毛细胞和1一2层支持细胞。超微结构研究发现壶腹峪、椭圆囊斑和球囊斑的毛细胞有排列规则的…     

Smad4 条件基因敲除小鼠前庭终器形态学研究

目的研究Smad4条件基因敲除小鼠前庭终器形忿改变，探讨Smad4基因对于前庭发育的作用。方法利用建立的Smad4条件基测敲除小鼠模型．通过光镜观察Smad4（＋／＋）、Smad4（＋／-）与Smad4（-／-）三种基因型小鼠（0．5、1．5月龄）的球囊及囊斑、椭圆囊及囊斑、半规管及壶腹嵴、前庭神经节、内淋巴管与囊的形态结构、毛细胞的形态及排列缺失情况。通过扫描电镜观察球囊斑、椭圆囊斑和壶腹嵴的形态结构、毛细胞及纤毛的排列缺失情况。结果Smad4（-／-）小鼠个体及内耳明显小于Smad4（＋／＋）和Smad4（＋／-），而后面二者区别不大。所有小鼠的球囊及囊斑、椭圆囊及囊斑、半规管、前庭神经节、内淋巴管与囊的大小和结构正常，淋巴囊腔饱满，囊斑处的感觉上皮、耳石、毛细胞及纤毛排列整齐，没有发现明显的病变．三个基因型间没有差异。Smad4（-／-）小鼠壶腹嵴囊性变多且严重、后半规管壶腹嵴出现明显的副嵴、偶尔可见壶腹嵴感觉上皮空泡样变。壶腹嵴的毛细胞和支持细胞排列整齐，形态无明显改变，未见缺失。扫描电镜示球囊斑、椭圆囊斑、后、外、上半规管壶腹嵴正常，三个基因型之间没有差异。结论Smad4（-／-）小鼠的前庭终器有轻微病理改变，Smad4（＋／＋）与Smad4（＋／-）小鼠的前庭终器形态结构基本正常．表明Smad4对于前庭终器的发育影响可能不明显。     

BMP4/Smad4在C57BL/6小鼠内耳前庭发育不同阶段的表达

目的探讨骨形态形成蛋白4(bone morphogenetic protein 4,BMP4)和Smad4蛋白(drosophila mothers against decapentaplegic protein 4)在C57BL/6小鼠内耳前庭发育不同阶段的表达。方法选择从胚胎第10天(E10)到胚胎第20天(E20)每天的孕鼠各两只(共22只),取E10～E17的胚胎头、E18～E20的胚胎内耳,通过冰冻连续切片、免疫组化或免疫荧光染色,观察小鼠胚胎内耳前庭发育过程中BMP4/Smad4的表达情况。结果 BMP4从E10开始,在间充质浓聚前就有表达;间充质一开始浓聚,BMP4在其中就有较丰富的表达;早期主要表达在前庭囊及前庭囊周围的间充质,在胚胎发育的后期,其在软骨囊及前庭上皮里有广泛阳性表达;Smad4在小鼠内耳前庭的表达情况与BMP4不一致,E10到E12的小鼠内耳Smad4表达阴性,E15时各个壶腹嵴、囊斑里才有较明显的Smad4表达,而内耳周围软骨囊到E16时才有明显的Smad4表达。结论 BMP4与小鼠内耳前庭形态形成及毛细胞分化发育、功能形成有关;Smad4与BMP4的表达时空特征不同,Smad4可能主要参与了小鼠内耳前庭发育后期的形态塑形和功能发育。     

胚胎小鼠耳蜗感觉上皮的形态发生

目的了解胚胎小鼠耳蜗感觉上皮发育的形态学特征及规律。方法利用光镜、扫描电镜、透射电镜观察从胚胎第8天到刚出生期间C57BL/6小鼠耳蜗感觉上皮发育过程中的形态变化。结果C57BL/6小鼠胚胎第10.5天耳蜗开始发育,感觉上皮由高柱状上皮细胞组成,到胚胎第14天才开始分化为原始的毛细胞;胚胎第16天耳蜗底转Corti器原基形成,毛细胞和支持细胞初具雏形;到出生时,耳蜗形态基本成熟,但底转Corti器仅趋向成熟,蜗尖才形成Corti器原基。结论小鼠耳蜗感觉上皮发育的形态变化在胚胎第13天到出生时最明显,感觉细胞都来源于一种高柱状上皮细胞,到胚胎第14天开始分化为毛细胞;而Corti器的分化成熟是从底转向顶转进行的,出生时尚未完全发育成熟。     

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。     

人前庭感觉上皮扫描电镜观察

为了解人前庭器官的超微结构，借助扫描电镜技术观察了５人（１０耳）的前庭感觉上皮。结果显示：①球囊斑呈“Ｌ”形，椭圆囊斑形如打开的贝壳；②Ⅰ型感觉细胞呈烧瓶样，Ⅱ型感觉细胞呈试管状；③感觉细胞纤毛排列呈阶梯状，动纤毛最长，且位于最长的静纤毛一侧，椭圆囊斑最长的静纤毛和动纤毛靠近微纹，其极性也向微纹，球囊斑则背离微纹，水平半规管壶腹嵴的动纤毛朝向椭圆囊侧，上、后半规管壶腹嵴则朝向半规管侧；④耳石形状大小不一致，呈现两端为锥形的圆柱状晶体。     

Smad5 基因在小鼠耳蜗胚胎发育中的作用

目的检测Smad5基因在小鼠耳蜗胚胎发育中的作用。方法选用耳廓反应灵敏、健康的C57BL／6小鼠作为种鼠交配．用观察阴栓方法获得胚胎9天到20天的胎鼠，≤17天取胚胎头，≥18天在显微镜下取耳蜗，胚胎头水平冰冻切片，耳蜗平行于蜗轴冰冻切片，HE染色方法观察小鼠内耳发育形态演变过程，免疫组织化学方法检测Smad5蛋白在小鼠胚胎10～20天的表达情况。结果胚胎10天，听泡发育，胚胎12天听泡下部有蜗管始基形成并开始发育。胚胎18天，蜗管发育了2圈，形成了可以辨认的内、外毛细胞，血管纹开始分化。Smad5在小鼠内耳胚胎发育全程均有阳性表达，且表达比较广泛，尤其早期在整个听囊均有表达。在胚胎15～17天．主要集中在即将发育成基底膜听觉感受器的部分。在胚胎发育中后期在内外毛细胞、螺旋神经节细胞、支持细胞、血管纹、基底膜、前庭膜等也有表达。结论Smad5参与小鼠耳蜗胚胎发育全过程，它可能为听觉的发生所必需的基因。     

人前庭感觉上皮扫描电镜观察

为了解人前庭器官的超微结构，借助扫描电镜技术观察了５人（１０耳）的前庭感觉上皮。结果显示：①球囊斑呈“Ｌ”形，椭圆囊斑形如打开的贝壳；②Ｉ型感觉细胞呈烧瓶样，Ⅱ型感觉细胞呈试管状；③感觉细胞纤毛排列呈阶梯状，动纤毛最长，且位于最长的静纤毛一侧，椭圆囊斑最长的静纤毛和动纤毛造近微纹，其极性也向微纹，球囊斑则背离微纹，水平半规管壶腹嵴的动纤毛朝向椭圆囊侧，上、后半规管过来腹嵴则朝向半规管侧；④耳右形状     

庆大霉素鼓室内注射后在内耳细胞中的分布

目的 观察鼓室内注射庆大霉素后，不同时间庆大霉素在前庭和耳蜗中的分布。方法 将庆大霉素同德州红连接形成庆大霉素-德州红耦联物后，行豚鼠鼓室内注射，注射后12h，1、2、3、4、7、14、28d处死动物，Phalloidin染色后运用激光共聚焦扫描显微镜观察基底膜、椭圆囊、球囊、外半规管壶腹嵴庆大霉素分布情况，并进行荧光分布半定量分析。结果 庆大霉素自注射后12h起在内耳所有细胞均见分布，在基底膜的外毛细胞、椭圆囊、球囊、外半规管壶腹嵴的感觉细胞聚集明显，主要聚集在毛细胞顶端纤毛下方的细胞质中，支持细胞分布较少。注射后第3天庆大霉素在内耳聚集达到最高峰，并在毛细胞内聚集较长时间。结论 庆大霉素-德州红耦联物是一个研究庆大霉素在内耳分布的良好的荧光探针，可用来检测庆大霉素的药代动力学和聚集机制．     

小鼠耳蜗胚胎发育过程中Smad4基因的表达

目的探讨小鼠内耳胚胎发育演变过程，检测Smad4基凶在小鼠耳蜗中的表达情况。方法选用耳廓反应灵敏、健康的C57BL／6小鼠作为种鼠交配，用观察阴栓方法获得适龄胎鼠，≤17天取胚胎头，≥18天在显微镜下取耳蜗，胚胎头水平冰冻切片，耳蜗平行于蜗轴冰冻切片，HE染色方法观察小鼠耳蜗发育形态演变过程，免疫组织化学方法检测Smad4蛋白在小鼠胚胎10～20天耳蜗巾的表达情况。结果胚胎10天，听泡发育．胚胎12天听泡下部有蜗管始基形成并开始发育．胚胎18天，蜗管发育2圈，形成了可以辨认的内、外毛细胞．血管纹开始分化。Smad4从胚胎15天才开始表达，最初表达部位为耳蜗底转将发育成蜗轴以及柱状上皮分化为感觉细胞及支持细胞的区域，但在胚胎早期表达较弱，后期在耳蜗内广泛表达，在螺旋器、血管纹、前庭膜均有表达，且表达逐渐增强，而在蜗轴部位的表达逐渐减弱。结论Smad4在小鼠内耳分化期有阳性表达，且表达具有明显的阶段性和区域性，说明其参与内耳听觉器官的发育过程并且可能存耳蜗的形成过程中起着重要的作用。     

细胞增殖及凋亡与耳蜗感觉上皮的分化

目的观察耳蜗发育中细胞增殖、凋亡的变化规律，探讨它们与耳蜗感觉上皮分化的关系。方法利用透射电镜观察从胚胎第8天到刚出生的C57BL/6小鼠耳蜗感觉上皮的增殖、凋亡及分化的变化。结果胚胎第10天，耳蜗才开始发育；到胚胎第11天耳蜗感觉上皮出现核分裂像并逐渐增多，胚胎第14天时已开始减少；细胞凋亡现象出现在胚胎第13天，从胚胎第14天到胚胎第15天细胞凋亡最明显，后逐渐减少；同时在胚胎第14天毛细胞开始分化形成，到胚胎第16天，Corti器原基形成时，毛细胞和支持细胞形态趋向成熟，但到出生时，Corti器尚未发育成熟。结论细胞增殖和凋亡是耳蜗发育中的必然现象，细胞增殖、凋亡及毛细胞的分化成熟相继发生但又互相重叠；细胞增殖与凋亡的动态平衡在耳蜗感觉上皮分化成熟中起着重要作用。     

Distribution of tyrosine hydroxylase-like immunofluorescence in guinea pig vestibular ganglia and sensory areas.

The distribution of tyrosine hydroxylase (TH)-like immunofluorescence was analyzed in the guinea pig vestibular ganglia and end organs using a monoclonal antibody to TH. TH was chosen as a marker for the sympathetic fibers because TH regulates the first step of catecholamine synthesis by converting tyrosine to dopa. In the vestibular ganglia, there were TH-positive nerve fibers having distinct varicosities surrounding the vestibular ganglion cells. In the sensory areas, there was a sympathetic plexus in the subepithelial tissue of the saccule, the utricle, and the crista ampullaris. We speculated that the sympathetic innervation has a direct influence on the vestibular ganglion cells and diffuse influence on the capillary permeability.     

Morphometric analysis and fine structure of the vestibular epithelium of aged C57BL/6NNia mice

The vestibular organs of young and very old C57BL/6NNia (B6) mice were compared by light and electron microscopy. Hair cell density decreased an average of 14% in the utricle, 19% in the saccule and posterior crista, 23% in the horizontal crista, and 24% in the anterior crista. Hair cell size remained the same throughout the mouse's life span as did the ratio of Type I to Type II hair cells. The most apparent sign of advanced age was dense inclusions found in sensory and supporting cells. Although small inclusions were present at five weeks, by 29 months, additional, larger forms appeared. An unusual melanin-like form was characteristic of old Type I hair cells. Synaptic morphology and synaptic bodies were well preserved even in very old B6 mice. Elongated bars were common in Type I hair cells and spheroid synaptic bodies were the most common form in Type II hair cells. Large clusters of synaptic bodies occurring in both young and old mice were seen only in Type I hair cells. Although the B6 strain suffers from genetically determined early cochlear degeneration, it does not experience early degeneration of the peripheral vestibular organs.     

Development of blood-labyrinth barrier in the semicircular canal ampulla of the rat

Cationic polyethyleneimine (PEI) administered intravenously was transported to anionic sites on the capillary and subepithelial basal laminae (BL) in the vestibular labyrinth. Therefore, changes in the PEI distribution on the BL reflect changes in the transport system in the vestibular labyrinth. A 0.1% PEI solution was administered intravenously (7.5 ml/kg) to developing (1, 4, 7, 14 days after birth) and adult rats in order to investigate the development of the macromolecular transport in the ampulla of the semicircular canal as a function of age. After 1 h, the bony labyrinth was removed and embedded in Epoxy resin. Ultrathin sections of the ampulla were then examined with a transmission electron microscope. In the subepithelial BL in the dark cell area and capillary BL in the crista ampullaris, the PEI distribution in both 1- and 4-day-old rats was markedly increased compared to that in either 7-, 14-day or adult rats. In the sensory cells in 1-, 4-day or 7-day-old rats, PEI density and area was significantly greater than in the adult rats. These findings suggest that the macromolecular transport system in the developing rat ampulla becomes mature by 14 days after birth and that the maturation of its transport system in the ampulla is strongly associated with that in the stria vascularis.     

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.     

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.