C57BL/10J小鼠内耳形态学观察

目的 探讨不同月龄C57BL／10J小鼠内耳形态学变化。方法 取C57BL/10J小鼠2月龄3只（6耳）、8月龄3只（6耳）、12月龄2只（4耳）、27月龄2只（4耳），采用耳蜗铺片和耳蜗图技术，观察耳蜗和前庭毛细胞的变化。结果 2月龄鼠在耳蜗底回和顶回可见有少量的外毛细胞缺失，内毛细胞正常。8月龄鼠外毛细胞缺失主要由耳蜗底回向顶回发展，少量内毛细胞缺失主要在耳蜗底回，对应于20kHz的基底膜区域显示外毛细胞缺失在20％左右。12月龄鼠外毛细胞缺失明显加重，耳蜗底回部分几乎完全缺失，外毛细胞损失区域发展到基底膜中部，并且蜗尖区域外毛细胞缺失达到40％，耳蜗底回内毛细胞缺失达到60％～80％，蜗尖部分内毛细胞基本正常，对应于20kHz的基底膜区域显示外毛细胞缺失近80％，内毛细胞缺失近30％。27月龄鼠耳蜗外毛细胞从基底膜中部至底回完全缺失，顶回缺失60％～80％，内毛细胞缺失逐渐向蜗尖扩展，对应于20kHz的基底膜区域显示外毛细胞完全缺失，内毛细胞缺失超过50％。前庭选取球囊斑，椭圆囊斑和壶腹嵴三个部位进行观察，不同月龄的C57BL／10J鼠前庭毛细胞均正常。结论 本研究首次对C57BL／10J小鼠的内耳形态学进行观察，发现该鼠同C57BL／6J小鼠比较在外毛细胞缺失规律和时间上有明显的差别，明确了内耳毛细胞的变化规律；同时首次观察了前庭毛细胞，为C57BL／10J小鼠作为遗传性和老年性聋研究的动物模型提供了形态学理论依据。     

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。     

小鼠内耳毛细胞基因及其功能

近年分子遗传学和遗传工程的飞速发展，不仅加速确定了许多内耳感觉毛细胞基因，而且为进一步明确这些基因的功能，阐明它们在毛细胞分化、生长和成熟过程中的作用产生了重要的影响。小鼠转基因和通过胚胎干细胞技术的基因敲除，分别揭示了一些毛细胞基因存在基因编码的特异性启动子和它们的功能。本从毛细胞发育和成熟的不同阶段．对部分内耳毛细胞基因的表达和功能进行了概述。     

豚鼠前庭感觉上皮损伤中细胞凋亡的观察

细胞凋亡对细胞增殖、器官发生和功能维持起着重要作用。一定剂量的庆大霉素连续注射，造成豚鼠前庭器官损伤。采用半薄切片，透射电镜（ＴＥＭ）和ＴＵＮＥＬ（ＴｄＴ－ｍｏｄｉｄｅｄ ｂｉｏｔｉｎ－ｄＵＴＰ Ｎｉｃｋ－ｅｎｄ ｌａｂｅｌｉｎｇ，末端脱氧核苷酸转移酶介导的生物素标记）原位杂交技术，特异标记ＤＮＡ片段３＇－ＯＨ末端，原位显示凋亡细胞。在半薄切片和ＴＥＭ观察中发现两种类型的细胞损伤方式：①毛细胞肿胀     

内耳毛细胞离子通道研究进展

耳蜗毛细胞存在调控细胞功能的重要离子通道。目前这类离子通道的类型及结构功能尚不清楚。本就离子通道的类型、特点以及药物对通道的影响进行综述，期望能通过药物干预通道，为临床治愈内耳疾病提供新的思路。     

豚鼠前庭感觉上皮损伤中细胞凋亡的观察

细胞凋亡对细胞增殖、器官发生和功能维持起着重要作用。一定剂量的庆大霉素连续注射，造成豚鼠前庭器官损伤。采用半薄切片，透射电镜（TEM）和TUNEL（TdT－modidedbiotin-dUTPNick-endlabeling，末端脱氧核苷酸转移酶介导的生物素标记）原位杂交技术，特异标记DNA片段3′－OH末端，原位显示凋亡细胞。在半薄切片和TEM观察中发现两种类型的细胞损伤方式：①毛细胞肿胀，胞浆空泡化，细胞体从顶端表面挤出；②毛细胞在上皮内变性，显示出细胞凋亡的形态特征，包括细胞核凝缩，核膜消失，成碎块状，并由支持细胞吞噬。原位杂交显示：细胞凋亡标记阳性细胞主要分布在上皮表层，较高水平标记主要发生于给药后第3到7天。提示细胞凋亡是内耳前庭感觉细胞损伤的一种重要方式，凋亡的主动发生可能是一种潜在的介入方式来减少氨基甙类抗生素对毛细胞造成的急性损伤，并与感觉上皮损伤后的修复过程有关。     

小鼠耳蜗感觉上皮细胞的自然培养诱导毛细胞的产生

目的 培养小鼠耳蜗上皮细胞,寻找听觉毛细胞的前体细胞,从而研究听觉毛细胞的再生。方法 改良细胞培养基和培养技术,建立小鼠耳蜗听觉上皮细胞的培养;用免疫细胞化学方法和BrdU标记法检测培养细胞的性质和分裂状态。结果 培养的听觉上皮细胞表现为大而扁平的上皮细胞形态,并且表达上皮细胞的标志F-actin和cytokeratin,部分新生的细胞可被早期毛细胞的特异标志calretinin着染,表明有听觉毛细胞样的细胞产生,这种现象经3次传代培养后仍然存在。结论 自然细胞培养方法可能诱导小鼠听觉毛细胞的产生,在小鼠的耳蜗内可能存在听觉毛细胞的前体细胞,而这些前体细胞是否是组织特异性干细胞还需要更进一步的研究。     

幼鼠内耳单个器官培养步骤及分类方法

内耳离体培养在研究内耳细胞功能、内耳组织发育再生、细胞损伤退化等方面具有重要价值。但由于内耳的组织结构微小复杂,其取材和培养具有较大难度,使该技术的普及和应用受到一定限制。本文在简要点评不同内耳离体培养方法优劣的基础上,结合本实验室多年来积累的实践经验,详细介绍了新生大鼠各个内耳终器取材及培养的具体步骤。从解剖迷路到分离终器,从凝胶制备到标本铺放,都有详细的操作指南,本文还针对各环节中的技术动作和关键事项进行了具体探讨并配以指导图例,希望这些内容能够弥补以往文献中抽象简略的条款式步骤所造成的实验困难,为开展内耳器官培养研究的初学者提供一定的帮助。     

完整与分段取材法解剖小鼠耳蜗基底膜的比较

目的对完整与分段取材法解剖小鼠耳蜗基底膜进行比较。方法2019年2—3月,将6只10周龄C57BL/6小鼠以随机数字表法分成2组,分别应用完整取材法及分段取材法进行基底膜取材。应用异硫氰酸荧光素标记的鬼笔环肽荧光染色方法标记耳蜗毛细胞并进行计数,观察基底膜的完整性以及荧光染色效果。SPSS 19.0软件进行统计学分析。结果两种方法均能完整或分段将基底膜完全剥离,完整取材法耗时明显短于分段取材法,差异有统计学意义[(16.33±1.86)min比(23.66±3.88)min,t=-4.173,P=0.002]。鬼笔环肽荧光染色显示两组基底膜毛细胞均保存完整,完整取材法及分段取材法取得的基底膜全长比较差异无统计学意义[(5.92±0.22)mm比(5.72±0.16)mm,t=1.822,P=0.099]。结论应用两种取材方法均可较快分离基底膜并保持其结构的完整性。操作者可根据技术熟练程度及实验目的选择不同的方法。     

小鼠内耳毛细胞基因及其功能

近年分子遗传学和遗传工程的飞速发展,不仅加速确定了许多内耳感觉毛细胞基因,而且为进一步明确这些基因的功能,阐明它们在毛细胞分化、生长和成熟过程中的作用产生了重要的影响.小鼠转基因和通过胚胎干细胞技术的基因敲除,分别揭示了一些毛细胞基因存在基因编码的特异性启动子和它们的功能.本文从毛细胞发育和成熟的不同阶段,对部分内耳毛细胞基因的表达和功能进行了概述.     

鸡内耳感觉上皮细胞的自然增殖状态

为研究出生后鸟纲动物内耳感觉上皮细胞是否存在持续的细胞增殖，采用８天龄正常鸡１０只（２只做阴性对照），按１００ｍｇ／ｋｇ剂量腹腔注射ＤＮＡ合成的前体物质５－溴脱氧尿核苷（５－ｂｒｏｍｏｄ－ｅｘｙｕｒｉｄｉｎｅ，ＢｒｄＵ），６小时后处死动物，取颞骨制备石蜡切片，结合免疫细胞化学技术，对鸡基底乳头和前庭器官中的细胞增殖活动进行观察。结果发现，鸡基底乳头切片中未见阳性标记细胞，而椭圆囊斑、球囊斑及壶腹嵴感觉上皮中存在着数量较少的标记支持细胞和标记毛细胞。结论：出生后的鸟纲动物前庭器官存在着较低水平的持续增殖。提示：出生后鸟纲动物前庭器官可能保持着潜在的对抗病理损伤的修复能力。     

电离辐射对内耳的迟发性损害

为探讨电离辐射所致内耳迟发性损伤的特点和机理，观察了分割剂量６０Ｃｏγ射线照射豚鼠颞骨，当总剂量达６０Ｇｙ后８个月期间内耳功能和形态变化。形态研究应用光镜、扫描电镜与透射电镜，而功能测定采用耳蜗电图（ＥＣｏｃｈＧ）和眼震电图（ＥＮＧ）技术。结果发现：辐射耳照射前ＣＡＰ（复合动作电位）反应阈平均３５．６７±６．７８ｄＢ（ｘ±ｓ），总剂量结束后第一天为４１．１７±７．７６ｄＢ，３个月后４７．００±８．８２ｄＢ，８个月后７１．００±７．６３ｄＢ。放射结束后第一天与照射前比较无统计学差异（Ｐ＞０．０５），３个月后ＣＡＰ反应阈上升，８个月后听觉功能损害更明显（Ｐ＜０．０１）。辐射前眼震持续时间４０．００±５．４４秒，辐射后８个月２２．７１±７．３９秒，差异非常显著（Ｐ＜０．０１）。病理变化特点：耳蜗血管纹萎缩、变性，毛细血管数目减少，血管壁增厚，内皮细胞受损，细胞连接破坏；外毛细胞变性、坏死，底回毛细胞损伤率明显高于二、三回；前庭感觉细胞变性。结果表明：放射治疗剂量可致内耳的迟发性损害，内耳微血管辐射损伤后氧供和新陈代谢障碍可能是主要的致病机理。     

A morphometric study of the pallid mutant mouse inner ear

Mice homozygous for the mutant gene pallid (pa/pa) often lack otoconia in some or all of their maculae and are used to study the influences of gravity receptor hypostimulation on vestibular-related behaviors. Since the value of this animal model is based on the assumption that the vestibular sensorineural elements are normal, a morphometric analysis was done on the inner ear of these otoconia-deficient mice to see whether sensori-neural structures are also affected by the pallid gene. In pallid mice lacking all otoconia, the sensory epithelia of the utricle, saccule, and semicircular canal cristae were the same size as in their heterozygous (pa/+) controls. Although the superior and inferior divisions of the vestibular ganglion of the pallid mice were smaller than normal, the first-order neurons within these divisions were normal in size, number, and density. However, the superior divisions in both groups had larger neurons than did the inferior divisions. Within the pallid cochlea, first-order auditory neurons within the spiral ganglion were smaller than normal, but the scala media was larger. Since the significant vestibular influences of the pallid gene are limited primarily to the otoconia, behavioral abnormalities reported for these otoconia-deficient mice are apparently due only to gravity receptor hypostimulation.     

C57BL/6小鼠内耳形态学和年龄相关性听力损失的研究

目的探讨不同周龄C57BL/6小鼠内耳形态学及其ABR阈值变化。方法取C57BL/6小鼠3周、4周、12周、26周各10只,听性脑干反应(ABR)测试双侧2、4、8、16、20 kHz ABR阈值。采用基底膜铺片MyosinⅥ、Neurofilament免疫组化染色,观察耳蜗毛细胞和神经丝的变化。扫描电镜观察耳蜗毛细胞及其静纤毛随年龄的变化。结果随着年龄增长,C57BL/6小鼠各频率ABR阈值明显提高,顶转和底转内毛细胞缺失逐渐增多,神经丝染色渐淡,毛细胞静纤毛逐渐发生数量减少、增粗融合、倒伏等变化。到26周龄时已达到重度聋,各频率较3周组有显著统计学差异。顶转和底转内毛细胞有连续缺失,外毛细胞完全缺失,内毛细胞只有残存的少量静纤毛,粗细不均,倒伏明显。结论本研究对国产C57BL/6小鼠的内耳形态进行观察,明确了其ABR阈值和内耳毛细胞的变化规律,为用国产C57BL/6小鼠进行老年性聋研究提供了依据。     

Distribution of frequenin in the mouse inner ear during development, comparison with other calcium-binding proteins and synaptophysin

Frequenin is a calcium-binding protein previously implicated in the regulation of neurotransmission. We report its immunocytochemical detection in the mouse inner ear, in the adult, and during embryonic (E) and postnatal (P) development. The distribution of frequenin was compared with those of other calcium-binding proteins (calbindin, calretinin, parvalbumin) and synaptophysin. In the adult mouse inner ear, frequenin immunostaining was observed in the afferent neuronal systems (vestibular and cochlear neurons, their processes and endings) and in the vestibular and cochlear efferent nerve terminals. Frequenin colocalized with synaptophysin in well characterized presynaptic compartments, such as the vestibular and cochlear efferent endings, and in putative presynaptic compartments, such as the apical part of the vestibular calyces. Frequenin was not found in vestibular hair cells and in cochlear inner and outer hair cells. During development, frequenin immunoreactivity was first detected on E11 in the neurons of the statoacoustic ganglion. On E14, frequenin was detected in the afferent neurites innervating the vestibular sensory epithelium, along with synaptophysin. On E16, frequenin was detected in the afferent neurites below the inner hair cells in the organ of Corti. The timing of frequenin detection in vestibular and cochlear afferent neurites was consistent with their sequences of maturation, and was earlier than synaptogenesis. Thus in the inner ear, frequenin is a very early marker of differentiated and growing neurons and is present in presynaptic and postsynaptic compartments.     

E2F2过表达使内耳感觉细胞重新进入细胞周期

目的将调控细胞周期和增殖的E2F2基因导入成年哺乳动物耳蜗中,使其过表达,从而使耳蜗内的终末静止细胞重新进入细胞周期,向可分裂细胞转变,达到实现增加毛细胞数量的目的。方法将人来源的E2F2基因构建到腺病毒载体上,通过圆窗导入耳蜗;全耳蜗铺片、耳蜗切片观察E2F2过表达后耳蜗细胞的形态特点,用BrdU标记及ki67免疫组化方法检测其增殖情况。结果腺病毒携带的E2F2基因通过圆窗导入耳蜗后,能够在耳蜗毛细胞和支持细胞上表达。特别是在螺旋神经节细胞表达更强。转染E2F2基因后,耳蜗内的细胞BrdU标记呈阳性结果,且有ki67表达。结论过表达E2F2有可能使处于终末期的耳蜗细胞重新进入细胞周期,此方法为毛细胞再生研究提供了新的思路。     

自身免疫性内耳病动物模型的建立

目的　建立一种自身免疫性内耳病的动物模型 ,其具有可重复性高 ,适于进行深入免疫学分析的特点。方法　提取豚鼠内耳膜迷路组织为抗原 ,与等量完全弗氏佐剂 ,百日咳杆菌一次免疫C5 7BL/6小鼠。检测反应阈、血清免疫学、内耳形态学及免疫组织化学的改变。结果　免疫后小鼠听性脑干反应阈显著提高 ,内耳中出现显著的炎性细胞浸润、内淋巴积水和螺旋神经节细胞变性、数量减少等形态学改变 ,血清中可检测到抗内耳自身抗体 ,鼓阶内浸润细胞中的淋巴细胞主要为CD4 T细胞。结论　应用这种方法在C5 7BL/6小鼠可成功建立自身免疫性内耳病的动物模型     

Two-tone suppression in inner hair cell responses

In an attempt to characterize certain aspects of two-tone suppression (2TS), ac receptor potentials were recorded from mammalian inner hair cells (IHC) in the third turn of the guinea pig cochlea. By comparing magnitude and phase changes occurring during suppression with predictions made on the basis of level-dependent responses to single-tone inputs, it is possible to determine whether 2TS is mimicked by simply attenuating stimulus intensity.

Results indicate that the effects of suppression are not simulated by simple input attenuation for low probe levels which produce responses below saturation. In these situations, the suppressor causes a decrease in the magnitude of the ac receptor potential with the largest deviations measured at the characteristic frequency (CF) of the cell. Thus, frequency response functions become broader. Response phase goes through a lag/lead transition at CF, also opposite to the results expected by simply decreasing input to the cell.

At higher probe levels, within the saturation region, the magnitude reductions produced during 2TS are largest for stimulus frequencies well below and well above CF. This effect partially reverses the broadening of frequency response functions seen at moderate intensities with possible benefits for the processing of complex stimuli at conversational levels. Although the magnitude data obtained at high probe levels are consistent with the attenuation hypothesis, the companion phase measures did not show the expected lead/lag transition through CF since phase changes were generally lags. Consequently, the high-level suppression data suggest that 2TS may reduce input to the IHC but in a way which is not equivalent to the attenuation of a single-input stimulus.     

Lesions to cochlear inner hair cells induced by noise

Summary A total of 28 un-anesthetized rabbits of the small chinchilla strain were unilaterally exposed to noise (2–7 kHz, 135 dB SPL in the ear canal). After a follow-up time ranging from 15 minutes to 10 months, the ears were perfused with glutaraldehyde and prepared for analysis by secondary emission electron microscopy and or transmission electron microscopy. The typical finding was a fusion and clumping of inner hair cell (IHC) sensory hairs. In two of the animals, no loss of outer hair cells (OHC) was observed; in several of the others, only a small local loss of OHC was observed in the 2 and 4 kHz regions in spite of extensive IHC abnormality. A frequency map of the rabbit cochlea was obtained by pure tone lesions to OHC. The extent of IHC abnormalities corresponds to the 1–16 kHz region. The findings may provide a basis for the study of the functional relationship between the IHC and OHC.Supported by grants from the Swedish Medical Research Council (14X-04958-05B, 12X-03156-09C), Stifteisen Tysta Skolan, K A Wallenberg Foundation, The Swedish Work Environment Fund (79/80) and The Karolinska Institute fundsPresented at the 17th Workshop on Inner Ear Biology in Stockholm, June 23–25, 1980