双醋酸荧光素—碘化丙啶双染色法鉴别豚鼠外毛细胞活性

为提供一种准确判断单离细胞活性的方法，采用双醋酸荧光素（ＦＤＡ）－碘化丙啶（ＰＩ）双染色，荧光显微镜观察，鉴别了豚鼠耳蜗单个外毛细胞的活性。在激发光一染色后的活细胞显绿色，失活细胞核呈红色。在室浊下计数 ５只豚鼠（１０侧听泡）在断头１、２、３、５和７ｈ后的单离细胞平均存活百分率分别为８５．０％、７８．０％、７０．０％、２３．５％和０。本方法为一种鉴别细胞活性的客观可靠的方法。     

丫啶橙-碘化丙啶双染色法观察大鼠耳蜗基底膜毛细胞活性

目的探讨大鼠耳蜗基底膜毛细胞活性鉴别的实验观察方法。方法采用丫啶橙-碘化丙啶(acri-dine orange-propidiumiodide,AO/PI)双染色法,荧光显微镜观察,检测经组织培养后新生大鼠耳蜗基底膜毛细胞的活性。结果在激发光下染色后的活细胞核是亮绿色,失活细胞核呈橙红色。当染液pH为6.5,AO浓度为0.1‰时,细胞染色不足,浓度超过1.5‰时,出现“过染”,最适染色浓度为1‰;当1‰AO的pH值低于5.0时毛细胞蒙上红晕,背景模糊,当pH值高于7.4时,不能区分死亡细胞与活细胞,pH值6.5~7.0时,染色效果最好。结论用pH为6.8的1‰AO、0.1‰PI染色,可清晰分辨死/活细胞。     

双醋酸荧光素－碘化丙啶双染色法鉴别豚鼠外毛细胞活性

为提供一种准确判断单离细胞活性的方法，采用双醋酸荧光素（ＦＤＡ）－碘化丙啶（ＰＩ）双染色，荧光显微镜观察，鉴别了豚鼠耳蜗单个外毛细胞的活性。在激发光下染色后的活细胞显绿色，失活细胞核呈红色。在室温下计数５只豚鼠（１０侧听泡）在断头１、２、３、５和７ｈ后的单离细胞平均存活百分率分别为８５．０％、７８．０％、７０．０％、２３．５％和０。本方法为一种鉴别细胞活性的客观可靠的方法。     

锰诱导体外培养耳蜗毛细胞的损伤与凋亡研究

目的探讨锰诱导体外培养耳蜗毛细胞损伤与凋亡的关系。方法将出生3天的新生Sprague Dawley大鼠的耳蜗器官体外培养,将贮存浓度为100m M氯化锰用SFM稀释到终浓度为1m M,3m M,5m M后进行体外染毒处理耳蜗基底膜。应用鬼笔环肽染色特异性显示耳蜗毛细胞的静纤毛,同时用β-Tubulin对神经纤维进行染色,用TUNEL试剂盒;Cleaved caspase-3抗体对耳蜗基底膜进行染色,在激光共聚焦显微镜下分别观察耳蜗基底膜的毛细胞,神经纤维。结果耳蜗基底膜培养24小时,1m M氯化锰对耳蜗毛细胞及神经纤维损伤甚微,差异无统计学意义(P>0.05),内,外毛细胞排列整齐规律,听神经纤维分布均匀,成束排列。最高浓度5m M氯化锰处理24小时后对神经纤维造成明显的损害,与对照组相比,差异有统计学意义(P<0.05)。当浓度逐渐增加,损伤愈加显著并呈浓度依赖性。2m M氯化锰处理,对耳蜗基底膜底转损伤较中转、顶转明显。不同浓度氯化锰处理24小时TUNEL染色阳性细胞随着氯化锰浓度的增加而增多;Cleaved caspase-3染色阳性细胞亦随着氯化锰浓度的增加而增多。结论通过大鼠耳蜗器官体外培养方法,发现氯化锰会造成毛细胞缺失,神经纤维变细变少,而这种损伤的机制与细胞凋亡密切相关。     

氧自由基与耳蜗毛细胞的DNA损伤

在内耳中有关氧自由基的研究已经较多，但关于氧自由基与毛细胞DNA损伤的研究在起步阶段。本文就氧自由基与耳蜗毛细胞的DNA损伤作一综述。     

新霉素损伤后小鼠耳蜗毛细胞的改变

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、12w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...     

年龄相关性听力损失小鼠耳蜗毛细胞的凋亡机制

目的观察年龄相关性听力损失小鼠耳蜗毛细胞的死亡方式,探讨老年性聋的分子机制。方法随机选用5～7只28、30和60天龄的NMF308nmf/nmf小鼠,应用ABR和DPOAE检测听功能,用免疫荧光染色组织化学技术TUNEL、Caspase-3和碘化丙啶（PI）染色标记并观察耳蜗毛细胞。结果NMF308nmf/nmf小鼠从1月龄开始发生听功能减退和毛细胞改变,到2月龄时出现明显的TUNEL阳性标记,是毛细胞凋亡的最早表现;Caspase-3阳性表达的毛细胞凋亡现象稍晚出现;PI标记可见2～3月龄开始出现毛细胞细胞核固缩和碎片;到3月龄时听功能基本丧失,耳蜗毛细胞严重缺失。结论老年性聋的早期首先出现耳蜗毛细胞出现DNA单链断裂,随后Caspase-3信号途径激活,导致耳蜗毛细胞凋亡。     

噪声暴露后耳蜗凋亡与坏死毛细胞琥珀酸脱氢酶活性的变化

目的揭示噪声暴露后耳蜗凋亡、坏死毛细胞线粒体功能的变化。方法将灰鼠暴露于155dBSPL的脉冲噪声75次，噪声暴露后5h解剖取双侧耳蜗。采用琥珀酸脱氢酶（SuccinateDehydrogenase，SDH）染色法进行耳蜗基底膜细胞线粒体染色，细胞核DNA荧光染料碘化丙啶（propidiumiodide，PI）双重染色耳蜗基底膜，以未受噪声暴露动物为对照，显微镜下观察噪声暴露后耳蜗基底膜核固缩和核肿胀毛细胞琥珀酸脱氢酶染色的变化。结果噪声暴露损伤区的耳蜗外毛细胞SDH着色变浅．蓝色颗粒物存在于PI标记固缩的耳蜗毛细胞核周围，而肿胀的毛细胞核周围缺少SDH阳性染色物。结论强脉冲噪声暴露后，凋亡的耳蜗外毛细胞依然存在不同程度的线粒体功能，而坏死的外毛细胞线粒体功能受损严重。     

耳蜗半薄切片细胞凋亡检测

目的尝试在耳蜗半薄切片上用TUNEL方法检测耳蜗组织的细胞凋亡.方法基因敲除Smad5小鼠耳蜗,分别作常规石蜡切片和环氧树脂包埋的半薄切片,用TUNEL方法检测耳蜗组织的细胞凋亡.结果在半薄切片上Smad5基因敲除小鼠,耳蜗内有大量的凋亡细胞产生,主要集中在螺旋神经节细胞、血管纹上的细胞,以及基底膜上的间皮细胞等,而且能够更清楚的显示凋亡的毛细胞.而在石蜡切片上只在螺旋神经节细胞、血管纹上的细胞,以及基底膜上的间皮细胞检出凋亡细胞,但是毛细胞没有凋亡细胞的检出.结论用TUNEL方法检测耳蜗组织的细胞凋亡半薄切片明显好于石蜡切片.     

Contacts between hair cells in the avian cochlea

In the avian papilla basilaris, contacts between hair cells are a common feature. With few exceptions, they only occur between tall hair cells (THC), and they are more frequent in the apical half of the papilla. In this quantitative study, four types of contacts are defined: Protrusion contacts, touch contacts, fusion contacts and multiple contacts. There are strong indications that these contacts are dynamic structures. The fact that avian hair cells probably communicate with each other means that their function can only be fully understood if cell groups are studied.     

Extra inner hair cells in the developing rabbit cochlea

Development of the rabbit inner ear was analysed with respect to the presence of extra inner hair cells (XIHCs) in phalloidin-impregnated cochleas of newborn rabbits 0, 3 and 5 days of age. The number of XIHCs ranged from four to 77. The distribution was asymmetrical with a peak in the apical 3 mm of the cochlea. There was no general disorganisation in the vicinity of the XIHCs, and the numbers were highly symmetrical between the two ears. The number was significantly larger (P<0.001) in newborns than in adults. There was no correlation between number of XIHCs and cochlear length, making it unlikely that the presence of XIHCs is due to lack of space in the ordinary row of IHCs. The possible relation of the XIHCs to growth factors, molecular genetics and regeneration is discussed.     

Salicylate-induced morphological changes of isolated inner hair cells and outer hair cells from guinea-pig cochlea

Objectives

The aim of this study is to investigate the salicylate-induced morphological changes of cochlear inner hair cells (IHCs) and outer hair cells (OHCs).

Methods

IHCs and OHCs were acutely isolated from the guinea-pig cochlea. Cells were observed under the inverted microscope and 10 mmol/L sodium salicylate solutions or 0.01 mmol/L dexamethasone-plus-salicylate solutions were applied. The cell length or the ratio between the length and width was the indices of the morphological changes in cells.

Results

Isolated IHCs did not demonstrate any significant changes in sodium salicylate solutions in 20 min and in 40 min, whereas OHCs were shortened by the 10 mmol/L sodium salicylate to 83% in 20 min and 75% in 40 min. There were no significant differences between in the dexamethasone-plus-salicylate solutions and in the control solutions after 20 min and 40 min both in IHCs and OHCs.

Conclusions

Although salicylate affected the isolated OHCs from guinea-pig cochlea, IHCs were not changed morphologically by sodium salicylate applications. Dexamethasone inhibited the salicylate-induced morphological changes of OHCs.     

Electrically evoked auditory nerve responses in the cochlea with normal outer hair cells

As hybrid cochlear implant devices are increasingly used for restoring hearing in patients with residual hearing, it is important to understand electrically evoked responses in cochleae having functional hair cells. To test the hypothesis that extracochlear electrical stimulation (EES) from sinusoidal current can provoke an auditory nerve response with normal frequency selectivity, the EES-evoked compound action potential(ECAP) was investi-gated in this study. Brief sinusoidal electrical currents, delivered via a round window electrode, were used to e-voke ECAP. The ECAP waveform was observed to be the same as the acoustically evoked CAP(ACAP), except for a shorter latency. The input/output and intensity/latency functions of ACAPs and ECAPs were also similar. The maximum acoustic masking for both ACAP and ECAP occurred near probe frequencies. Since the masked tuning curve of a CAP reflects the frequency selectivity of neural excitation, these data demonstrate a highly specific acti-vation of the auditory nerve, which would result in high degree of frequency selectivity. This frequency selectivity likely results from the cochlear traveling wave caused by electrically stimulated outer hair cells.     

豚鼠耳蜗外毛细胞的钾通道

目的研究外毛细胞钾离子通道的生物物理学和药理学特性.方法用细胞贴附式和内面向外式膜片钳技术,研究豚鼠耳蜗单离外毛细胞底侧膜的钾离子单通道电流.结果记录到两种钾离子单通道电流:(1)大电导型钙激活钾通道,在等渗140mmol/L KC1溶液时电导为161 26pS(内面向外式)或155±27pS(细胞贴附式),反转电位为0mV;浴液为Hanks液而电极内液为140mmo/L KC1液时,电导为133±9pS(细胞贴附式).当膜内面浴于无钙液时通道活动消失.通道呈簇状发放或快速簇状发放方式,其开启和关闭时程直方图均可用三阶指数方程拟合.双氢链霉素能够可逆性地抑制外毛细胞的大电导型钙激活钾通道,表现为电流幅度减少,在链霉素浓度增大以及细胞膜电位偏正时更明显,新霉素的抑制作用更强.(2)～44pS钾通道.结论豚鼠耳蜗外毛细胞底侧膜具有两种钾离子通道,研究了其大电导型钙激活钾通道的生物物理学和药理学特性.     

Nerve maintenance and regeneration in the damaged cochlea

Following the onset of sensorineural hearing loss, degeneration of mechanosensitive hair cells and spiral ganglion cells (SGCs) in humans and animals occurs to variable degrees, with a trend for greater neural degeneration with greater duration of deafness. Emergence of the cochlear implant prosthesis has provided much needed aid to many hearing impaired patients and has become a well-recognized therapy worldwide. However, ongoing peripheral nerve fiber regression and subsequent degeneration of SGC bodies can reduce the neural targets of cochlear implant stimulation and diminish its function. There is increasing interest in bio-engineering approaches that aim to enhance cochlear implant efficacy by preventing SGC body degeneration and/or regenerating peripheral nerve fibers into the deaf sensory epithelium. We review the advancements in maintaining and regenerating nerves in damaged animal cochleae, with an emphasis on the therapeutic capacity of neurotrophic factors delivered to the inner ear after an insult. Additionally, we summarize the histological process of neuronal degeneration in the inner ear and describe different animal models that have been employed to study this mechanism. Research on enhancing the biological infrastructure of the deafened cochlea in order to improve cochlear implant efficacy is of immediate clinical importance.     

Tip-link organization in anomalously-oriented hair cells of the guinea pig cochlea

Stereocilia are described in a group of guinea pigs with abnormal conformation of the outer hair cell stereociliary bundles. Rows of Stereocilia were found which were circular instead of V- or W-shaped, and rotated or even reversed in orientation with respect to normal. The Stereocilia have however a normal gradation in heights of the rows of Stereocilia, and have tip links running in the direction of gradation in height.