出生后大鼠听毛细胞纤毛发育过程

目的观察新生大鼠（P1、P7、P14、P21）耳蜗Corti器的形态结构及排列方式,以便了解听毛细胞纤毛发育的过程。方法取新乍SD大鼠四个阶段的耳蜗,采刖扫描电镜对出生后大鼠耳蜗Corti器形态结构进行系统观察。结果发现这四个阶段在体犬鼠耳蜗毛细胞从出生至毛细胞完全发育成熟,绝大部分都是以一排内毛细胞和三排外毛细胞的方式排列的,只有少部分在局部发现多出几个外毛细胞形成第四排。毛细胞的形态结构及排列方式随着时间的改变逐渐发育成熟。P14这个阶段是个重要的时期,Corti器表面柱细胞头板增宽,表面的微绒毛减少,Deiter细胞表面微绒毛也减少,动纤毛从底圄至顶圈逐渐退化已经基本结束,P14这个阶段Corti器各个部分发育已经完全成熟：结论出生后14天之内。大鼠听毛细胞静纤毛和动纤毛与听器其他细胞形态结构仍不同程度地处于由不成熟到成熟的逐渐发育过程,为研究大鼠出生后Corti器的发育、听觉功能的建立和完善提供了比较可靠的形态学证据。     

硫酸新霉素对新生大鼠离体培养耳蜗毛细胞的毒性作用研究

目的观察体外培养的新生大鼠耳蜗Corti器经不同浓度硫酸新霉素处理后,各回毛细胞损伤情况。方法取新生大鼠完整耳蜗基底膜,体外培养12小时,存活贴壁后加药处理。对照组10个样本;3个实验组各10个样本,分别用终浓度为1mmol/L、2mmol/L、4mmol/L硫酸新霉素处理24小时。进行MyosinⅦa免疫荧光组织化学染色,在共聚焦显微镜下观察各回毛细胞缺失情况。分别选取10个图片进行毛细胞计数（每张图片截取100μm计数毛细胞个数）,并利用CHISS软件进行统计分析。对照组除不加入硫酸新霉素外,其他培养条件均与实验组相同。结果硫酸新霉素对离体培养的新生大鼠耳蜗毛细胞具有损伤效应,且外毛细胞对硫酸新霉素的毒性作用比内毛细胞敏感。加入硫酸新霉素后,耳蜗毛细胞损失从底回外毛细胞开始,随着药物浓度增加,逐渐累及到中回,最后顶回受累,且损失程度随着浓度的递增而加重,以外毛细胞为甚。单纯体外培养的耳蜗毛细胞没有缺失。经完全随机设计方差分析可得：顶回正常组内毛细胞数量与新霉素1mmol/L组、2mmol/L组无统计学差异,与新霉素4mmol/L组有统计学差异;中、底回正常组内毛细胞数量与新霉素1mmol/L组、新霉素2mmol/L组、新霉素4mmol/L组均有统计学差异;顶回正常组外毛细胞数量与新霉素2mmol/L与4mmol/L组有统计学差异;中、底回正常组外毛细胞数量与新霉素1mmol/L组、新霉素2mmol/L组、新霉素4mmol/L组均有统计学差异。结论新生大鼠体外培养的耳蜗毛细胞随着硫酸新霉素药物浓度的增加损失越严重,且从底回向顶回发展,此与活体动物实验的损害规律基本相同,因此可作为耳毒性药物损伤后毛细胞再生研究的体外模型。     

SD大鼠损伤Corti器中毛细胞样细胞的演变

目的：研究氨基糖类抗生素引起哺乳类动物Corti器损伤及损伤后的修复情况。方法：用新霉素皮下注射致新生SD大鼠Corti器损伤，然后用扫描电镜观察停药后不同时期的Corti器毛细胞的损伤及修复表现。结果：在外毛细胞损伤区有细胞表面长有微绒毛簇的毛细胞样细胞出现，且其在损伤Croti器中有发生-发展-退化的变化过程。结论：毛细胞样细胞的出现是受损伤Corti器的一种修复形式。     

庆大霉素和新霉素经圆窗膜渗入外淋巴

常用滴耳剂中多含有氨基甙类抗生素,对内耳有毒性作用。慢性化脓性中耳炎并发感音性聋时是由细菌毒素引起,还是氨基甙类滴耳剂毒性作用的后果尚不得而知。Brummett(1976)将新霉素直接用于豚鼠耳泡中,每日三次,共30天,发现即令新霉素浓度低至5 mg/ml(相当于常用滴耳剂含量)时,亦可使耳蜗微音电位减低和外毛细胞缺失。另外一些化学品如离子溶液、麻醉剂和肾上腺素等均可从中耳经圆窗膜进入外淋     

速尿与硫酸卡那霉素联合用药对大鼠内耳毒性的实验观察

目的探讨速尿与硫酸卡那霉素联合用药对大鼠内耳的毒性作用,为大鼠药物性耳聋动物模型的制备和相关研究提供参考。方法大鼠静脉注射速尿后,立即肌肉注射硫酸卡那霉素,对照组不做处理。用药7天后行听性脑干反应(auditory brainstem response,ABR)阈值检测、耳蜗铺片免疫荧光染色和颞骨常规切片观察,并对耳蜗进行扫描电镜观察。结果联合注射速尿与硫酸卡那霉素后,大鼠ABR阈值明显提高;铺片和切片观察发现内、外毛细胞完全缺失的大鼠,耳蜗支持细胞大部分完整,前庭毛细胞未见损伤;电镜观察发现耳蜗毛细胞纤毛明显受损。结论速尿和硫酸卡那霉素联合用药可导致大鼠听力和耳蜗毛细胞严重受损,但对耳蜗支持细胞和前庭毛细胞损伤较轻,为耳蜗毛细胞损伤后再生等实验研究提供了一种简单、快速而有效的建立大鼠耳聋动物模型的方法。     

模拟失重条件下飞船内噪声对豚鼠耳蜗形态与功能的影响

目的探讨模拟失重条件下,飞船内稳态噪声对豚鼠耳蜗形态与功能的影响。方法32只豚鼠随机分为单纯失重组16只、失重＋稳态噪声组16只。后肢悬吊法模拟失重,暴露于模拟飞船内在天飞行段的噪声环境,共5天。实验前、实验结束后即刻和实验结束后3天测试脑干诱发电位（ABR）阈值,取耳蜗标本行扫描电镜和透射电镜观察。结果实验组实验结束后即刻ABR阈值较实验前及实验结束后3天均增高（P〈0.01）;实验结束后3天ABR阈值较实验前高（P〈0.05）;实验结束后即刻及实验结束后3天失重＋稳态噪声组ABR阈值均较单纯失重组高（P〈0.01）。扫描电镜观察实验组实验结束后即刻耳蜗内、外毛细胞均受损。实验结束后3天,单纯失重组少数耳蜗各回内、外毛细胞的损伤程度比实验结束即刻加重;失重＋稳态噪声组耳蜗各回内毛细胞损伤较实验结束即刻重,外毛细胞损伤较实验结束即刻轻。实验组各时间段内毛细胞的损伤均重于外毛细胞,自第一回至第四回毛细胞损伤逐渐加重。透射电镜观察实验组耳蜗毛细胞及神经节细胞均可见空泡样改变,线粒体分布减少,细胞核固缩,可见细胞凋亡和细胞坏死两种细胞死亡现象。结论失重及失重＋稳态噪声均可造成豚鼠耳蜗形态和功能损伤,后者造成的损伤更重。失重对耳蜗毛细胞损伤以内毛细胞为重,损伤从底回至顶回逐渐加重。实验结束后3天较实验后即刻的听功能有所恢复但内毛细胞损伤加重。     

高强度低频稳态噪声对大鼠耳蜗形态和功能影响的研究

目的探讨高强度低频稳态噪声对大鼠耳蜗形态和功能的影响。方法 42只SD大鼠随机分为2组,分别暴露于频率100Hz和300Hz,声压级153.7dB和158dB的声场中10分钟。暴露前、暴露后即刻、3天和7天分别测试大鼠听性脑干反应阈值(ABR),并取耳蜗标本行扫描电镜观察。结果 2组大鼠噪声暴露后听性脑干反应阈值均显著提高,脱离声场3天后仅可轻度恢复,7天后无进一步改善。300Hz组听力损伤更重。2组大鼠内外毛细胞均损伤严重,表现为毛细胞静纤毛缺失、融合及团状变,并可见螺旋器表面渗出性改变。由底圈向顶圈毛细胞损伤逐渐减轻,7天损伤程度较即刻有所减轻。300Hz组损伤较100Hz组重。结论高强度低频稳态噪声可造成大鼠耳蜗形态及功能严重损伤。耳蜗形态损伤以底圈最重,外毛细胞损伤较内毛细胞重。     

新霉素损伤后小鼠耳蜗毛细胞的改变[论著]

目的　观察新霉素对小鼠听功能及耳蜗毛细胞形态学变化的影响。方法　选取C57BL/6小鼠16只在出生后第8天开始连续10 d皮下注射硫酸新霉素，对照组（10只）皮下注射等量生理盐水。停药后1、4、8、12 w进行听性脑干反应（ABR）检测。每次ABR检测后，新霉素组随机取3只小鼠，对照组随机取2只小鼠，处死后，取耳蜗进行冰冻切片，用免疫荧光方法观察耳蜗Corti器及毛细形态学变化。结果　新霉素可造成小鼠耳蜗毛细胞严重损伤，且小鼠ARB阈值显著增高，不可恢复；新霉素对耳蜗Corti器毛细胞的损伤顶圈最轻，中圈次之，底圈最重，且随着时间推移Corti器形态结构破坏逐渐加重且不可自我修复。结论　硫酸新霉素造成小鼠Corit器毛细胞的损伤是不可逆的。     

全耳蜗毛细胞定量分析系统

目的 介绍一种计算机辅助下的全耳蜗毛细胞定量分析系统。方法 选取氨基糖甙类药物损伤、噪声性损伤以及老年性聋动物模型的耳蜗制备全耳蜗基底膜铺片，从蜗尖向蜗底逐个视野依次进行内外毛细胞计数，将采集的数据输入到计算机并用毛细胞定量分析系统制备成耳蜗图。结果 氨基糖甙类药物耳蜗损害模型的耳蜗图显示毛细胞缺损自底回向顶回发展，外毛细胞的损伤比内毛细胞严重；强噪声引起的毛细胞破坏局限在与刺激声频率相对应的基底膜区域；6月龄C57BL／6J小鼠的耳蜗图显示老年性聋的早期损害起始于耳蜗底回的起始端，其中外毛细胞的破坏比内毛细胞严重。结论 耳蜗毛细胞定量分析系统可清晰显示全耳蜗毛细胞在基底膜上不同部位的损失程度和破坏范围，并可对应到基底膜上各个频率敏感部位。将传统的耳蜗铺片与计算机技术相结合制备的耳蜗图，具有全面可靠、简便精确、规范等优点。     

蝙蝠耳蜗毛细胞损害后支持细胞转分化为再生毛细胞研究

目的 为探讨哺乳类动物耳蜗毛细胞再生能力的存在和机制,我们选用蝙蝠为动物模型,观察庆大霉素损害耳蜗后毛细胞自发修复再生能力和现象.方法 给蹄蝠连续注射庆大霉素10和14天,造成耳蜗毛细胞损害消失.在注射停后、恢复20和40天分别测定ABR反应阈值,并对耳蜗标本进行扫描和透射电镜观察.结果庆大霉素造成蝙蝠耳蜗毛细胞损害消失,ABR反应阈值升高.恢复20和40天后,蝙蝠耳蜗受损的毛细胞部位支持细胞顶表面长出微绒毛,微绒毛发育成纤毛,替补消失的毛细胞,同时伴随有听功能的恢复.结论 蝙蝠耳蜗毛细胞损害后支持细胞直接转化为新生的毛细胞,证明蝙蝠耳蜗毛细胞具有再生能力,本研究为哺乳类耳蜗毛细胞再生提供了动物模型.     

Neuron-specific enolase immunoreactivity in the developing mouse cochlea

Onset of neuron-specific enolase reactivity was observed on gestation day 17 in spiral ganglion cells of the basal coil, and 2 days later in cochlear inner (IHC) and outer (OHC) hair cells. IHCs and OHCs were similarly reactive up to postnatal day 7, then the reactivity began to decrease in OHCs.     

Dose-dependent changes in the rat cochlea following aminoglycoside intoxication. II. Histological study

The structural damages induced by a range of amikacin doses have been studied with SEM and TEM in the adult rat cochlea. Six groups of rats were given daily subcutaneous amikacin injections of either 200, 400, 600, 700, 800, or 1000 mg/kg per day for five consecutive days. Observations were made one month after the end of treatment, following functional recordings [(1987) Hear. Res. 26, 191-197]. In the group treated with 200 mg/kg per day, neither SEM nor TEM revealed any cochlear abnormalities. In the 400-mg group, the only damages were cytoplasmic abnormalities in the OHCs. Changes in the OHC stereocilia occurred at higher doses and were always associated with severe alterations of the underlying cell body. The first missing hair cells were OHCs in the apical regions of the cochleas of the 600-mg group. In the 700-mg group, the loss of OHCs extended to the middle and basal regions, mainly in the third row. By contrast, in the 800- and 1000-mg groups, the usual pattern of hair-cell degeneration was found. Loss of hair cells was greatest in the basal cochlear regions, where the first row of OHCs was more extensively destroyed. These results indicate that metabolic disorders (only visible with TEM) precede stereociliary alterations. Under SEM, the pattern of hair cell degeneration appeared to depend on the dose of the drug. The study clearly demonstrates that both SEM and TEM are needed to determine the true condition of hair cells after drug intoxication.     

Distribution of F-actin and fodrin in the hair cells of the guinea pig cochlea as revealed by confocal fluorescence microscopy.

We double-stained paraformaldehyde fixed guinea pig cochleas with rhodaminated phalloidin to detect F-actin and with a monoclonal antibody against non-erythroid spectrin (fodrin). The hair cells were studied in surface specimens of the organ of Corti with confocal fluorescence microscopy. In serial optical sections, phalloidin stained the stereocilia, cuticular plate, and a circumferential ring beneath it in the inner and outer hair cells (IHCs and OHCs). The cytoplasm of the IHCs and the OHCs was unlabelled, but the infracuticular network of the OHCs in the upper turns showed a strong reaction. The lateral plasma membrane was unreactive with phalloidin in the IHCs and OHCs, except in the basal turn, where a moderate reaction, probably representing actin of Deiter's cups, was seen along the lateral walls of the basal pole of the OHCs. Fodrin was similarly seen in the cuticular plate, in a circumferential ring beneath it, and in the infracuticular network of the apical OHCs. The most interesting finding was the fodrin-specific distinct labelling of the lateral cell surface in the OHCs of the basal cochlear turn. This staining diminished towards the apex and was practically absent in the OHCs located above the level of 15 mm from the round window. The lateral cell surface of IHCs showed moderate fodrin labelling in all cochlear turns. This staining was much weaker than that seen in the basal OHCs. Fodrin labelling revealed deformation from the regular cylindrical shape in midportion of the OHC bodies in the basal turn of the cochlea.     

内毛细胞缺损对噪声引起外毛细胞损害的潜在影响

目的　观察噪声对正常灰鼠和内毛细胞缺损灰鼠的外毛细胞是否具有不同的破坏效应。方法　 6只正常灰鼠和 12只卡铂致聋灰鼠作为受试对象。提前 30天给 12只灰鼠按照 10 0mg/kg的剂量腹腔注射卡铂 ,以达到预先选择性破坏其内毛细胞的目的。将 6只正常灰鼠和 6只经卡铂处理的灰鼠移入噪声室 ,在强度为 12 0dB、中心频率在 4kHz的脉冲噪声环境暴露 6小时 ;另外 6只经卡铂处理的灰鼠不经噪声暴露作为对照组。在噪声暴露后 30天取出耳蜗 ,常规制备耳蜗铺片 ,然后进行全耳蜗毛细胞计数 ,并将各组动物的毛细胞计数结果制备成平均受损情况分布图 ,观察和比较噪声引起的外毛细胞损害水平在具有正常内毛细胞的灰鼠和丧失内毛细胞的灰鼠是否存在差别。结果　单纯注射卡铂的灰鼠内毛细胞平均损失在 90 %左右 ,但外毛细胞无损害。经噪声暴露的正常灰鼠外毛细胞在耳蜗底回中部约相当于 4kHz共振频率区域平均损失 6 0 % ,但是在该区域两侧靠近底回和顶回的外毛细胞损失少于 2 0 %。预先经卡铂破坏 95 %内毛细胞的灰鼠经噪声暴露后 ,外毛细胞在耳蜗底回中部平均损失达到 90 % ,在该区域两侧的外毛细胞损失百分比大于 4 0 %。结论　内毛细胞缺损可以造成更多的外毛细胞在噪声刺激中被破坏 ,提示正常内毛细胞的存在可能有     

Differences in the distribution of F-actin in outer hair cells along the organ of Corti

There is evidence of differences in the structure, innervation and physiological responses between outer hair cells (OHCs) of the basal and apical turns of the mammalian cochlea. In this study we have used rhodamine-labelled phalloidin to investigate the differential distribution of F-actin in OHCs along the organ of Corti of the guinea pig. Isolated OHCs and surface preparations and cryosections of the organ of Corti were studied. F-actin was observed in stereocilia and the cuticular plate of all OHCs. In addition, some OHCs had a network of F-actin extending from the cuticular plate towards the nucleus. This infracuticular network was observed in most OHCs of the apical cochlear turns but was not seen in any OHCs of the basal turn. These microstructural differences between OHCs of the base and apex could be related to differences in OHC function between the apical and basal portions of the cochlea.     

强脉冲噪声导致的豚鼠耳蜗毛细胞凋亡及P53蛋白的表达

目的 探讨强脉冲噪声暴露引起的豚鼠毛细胞损害模式和P53凋亡蛋白在损伤毛细胞中的表达.方法 12只成年豚鼠,声压级168 dB的强脉冲噪声连续暴露80次,每次间隔时间2 s,分别在噪声暴露后3 h、6 h及12 h各处死4只动物,应用异硫氰酸荧光素(flourescein iso-thiocyanate,FITC)标记的鬼笔环肽(phalloidin)和细胞核DNA荧光染料碘化丙啶(propidium iodide,PI)来施行双重染色,在激光共聚焦显微镜下观察耳蜗毛细胞静纤毛和细胞核的形态学变化;将噪声暴露后12 h的耳蜗基底膜样品进行P53免疫荧光染色,观察受损毛细胞中是否存在P53蛋白的表达;耳蜗琥珀酸脱氢酶染色,制备基底膜铺片,400倍显微镜下行毛细胞计数并绘制耳蜗图.结果 强脉冲噪声暴露后3 h,耳蜗底回末端和第二回起始端已经发生明显的外毛细胞结构破坏,同时伴有细胞核的浓缩;暴露后6 h耳蜗毛细胞的破坏范围扩大,在毛细胞损害的中心区域可见外毛细胞的核碎片;毛细胞损害中心区域在噪声暴露后12 h,多数外毛细胞的细胞核消失,而周边区域的毛细胞可见核浓缩或核碎片.耳蜗图显示,噪声暴露后3、6及12 h,耳蜗毛细胞的破坏范围不断扩大.免疫荧光显示耳蜗第二回外毛细胞损伤的中心部位出现明显的P53阳性表达,在耳蜗底回和第三回外毛细胞中亦出现P53的阳性表达.结论 强脉冲噪声暴露引起耳蜗毛细胞死亡的主要方式是凋亡,损伤的中心部位在耳蜗第二回,并逐渐向耳蜗底回和第三回扩散;P53蛋白在此过程中可能扮演了重要角色.

Abstract：

Objective To explore the pattern of hair cell injury and expression of P53 apoptosis protein in intensive impulse noise injured cochlear hair cells in guinea pigs. Methods Twelve adult guinea pigs were exposed to a series of 40 pairs of impulse noise(2 second intervals) at the intensity of 168 dB (SPL). Animals were terminated at 3, 6 and 12 hours after noise exposure, respectively. Cochlear surface preparations were performed with a double staining of FITC-conjugated phalloidin and propidium iodide for the observations of the stereocilia and the nucleus. P53 immunochemical staining was also performed 12hours post-noise exposure to observe if there was expression of p53 protein in injured hair cells. Results Three hours after noise exposure, the outer hair cells at the end of basal turn and beginning of second turn were destroyed first with a character of nuclear condensation. Six hours post-noise exposure, many hair cells in the center of damage region had nuclear fragmentations, and the damaging area expanded towards to basal turn and apical turn. Twelve hours after noise exposure, the nucleus in most outer hair cells and inner hair cells at the region of damage center were missing. The nuclear condensation and fragmentation were appeared in hair cells in both sides of the center region of degeneration. P53 immunoreactive products were also found in damaged hair cells, not only in the central damage area, but also in the basal turn and the third turn.Conclusions Intensive impulse noise resulted in apoptosis of cochlear hair cells that initiated between the end of basal turn and the beginning of second turn. Hair cell degeneration spread to basal and third turn along the basilar membrane. P53 may play an important role in impulse noise induced-hair cell apoptosis.     

Influence of age on hair cell loss in the rabbit cochlea

The surface preparation technique was used for a study of cochlear hair cell loss as a function of age in rabbits 1 week, 6 months, 1 year, and 4 years old. Cytocochleograms from these animals were prepared which indicated damaged or missing hair cells over the entire spiral organ from the apical to the basal coil. The loss in the mean percentage of total outer hair cells (OHC) and inner hair cells (IHC) appeared to increase linearly with increasing age, with a statistically significant correlation. The cell loss was similar in all three rows of OHC in each of the age groups. In animals up to 1 year of age, areas of OHC loss were localized in the apical and basal coils. In 4-year-old rabbits, OHC degeneration had become progressively accentuated and widespread. The pattern of IHC loss was somewhat similar to that of OHC. Age-related hair cell loss in the rabbit is comparable to that in the rat, monkey, and human cochlea.     

Cochlear morphology of the audiogenic-seizure susceptible (AGS) or genetically epilepsy prone rat (GEPR)

The organ of Corti (OC) of the genetically epilepsy prone rat (GEPR), a strain which is highly susceptible to audiogenic seizures (AGS), was examined by means of the scanning electron microscope (SEM). Ten female GEPRs (seizure intensity score of 2 or 3) and 10 female control rats (seizure intensity score of 0) were used in this study. (Seizure intensity was scored on an ascending scale of 0-9; 0 being no seizure (Jobe et al., 1973).) Each rat was perfused with buffered glutaraldehyde and the temporal bones fixed for one week in formalin. After decalcification, staining and microdissection, the entire OC was prepared for scanning electron microscopy (SEM). The GEPR organ of Corti contained several morphological differences when compared with controls. 1) In all 10 GEPRs, the headplates forming the top of the tunnel of Corti exhibited some form of structural abnormality. 2) Five animals had some form of stereocilia aberration of the inner (IHC) and/or outer (OHC) hair cells. 3) In 4 animals, significant numbers (10-15%) of IHC's were missing in large segments of all cochlear turns. 4) In 2 GEPRs, all OHC's were absent from the middle turn to the hook. In these 2 animals, IHC's were present in the upper middle turn but became less numerous and completely absent in the basal turn and hook. 5) One set of cochleas had 1000 more OHC's than had those of control rats. Since GEPRs are genetically susceptible to seizures, the preceding cochlear abnormalities are probably genetically-induced developmental defects. It is likely that the abnormally long stereocilia, mis-shaped stereocilia and deficits in hair cell populations are a consequence of the distorted headplates. The elongated stereocilia could be a compensatory attempt to contact the tectorial membrane during development. The mis-shaped stereocilia and hair cell deficits could represent a failure of compensatory mechanisms. The cochlear abnormalities may play a role in both susceptibility and intensity of audiogenic seizures.