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。     

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。     

定量观察卡铂引起的灰鼠耳蜗毛细胞和神经纤维的早期损害过程

目的：定量观察和比较灰鼠耳蜗毛细胞和缰孔内神经纤维数量在卡铂耳中毒早期损害过程中的变化。方法：在耳蜗基底膜铺片的基础上制备耳蜗毛细胞图；从骨性螺旋板切片上对缰孔内的神经纤维计数。结果：缰孔内的神经纤维数量在注射卡铂后24h内明显减少。而内毛细胞的缺失是发生在注射上学铂后72h。结论：在卡铂耳中毒早期，缰孔内神经纤维的破坏早于内毛细胞的缺失。     

谷氨酸调节耳蜗内毛细胞游离钙的实验观察

目的 探讨谷氨酸（glutamate,Glu）对离体耳蜗内毛细胞（inner hair cells,IHC)内钙信号的调控作用及其生理病理意义。方法 在激光共聚焦显微镜下用钙敏荧光探针Fluo－3作为指示剂,观察外源性谷氨酸对分离的10个豚鼠耳蜗IHC胞内游离钙离子浓度（[Ca^2 ]i)的影响。结果 分离好的正常IHC呈烧瓶形态,有明显的颈部,皮板上可观察到静纤毛,大球形的细胞体中间可见圆形的细胞核。形态完好的IHC大约存活2h,Fluo-3 钙敏荧光探 针染色后IHC胞体、胞核及表皮板有明显的染色梯度,表明游离Ca^3 浓度从细胞 核向细胞逐渐减少。终浓度为3.85umol/L的Glu对游离IHC内[Ca^2 ]i有增高趋势,而对游离外毛细胞（outer hair cells,OHC)内[Ca^2 i]浓度无影响。观察10个IHC,发现9个[Ca^2 ]i浓度增加,1个无变化;观察10个OHC,发现7个[Ca^2 ]i无变化,3无略有下降。当Glu浓度增高后,IHC内[Ca^2 ]i先是迅速升高,继而逐渐下降,IHC外形由烧瓶状逐渐变成球形,提示IHC水种变性。结论 Glu可选择性调控IHC内[Ca^2 ]i ,而对OHC内[Ca^2 ]i无影响,而过量的Glu刺激,可造成IHC[Ca62 ]i的堆积,从而IHC水肿变性。     

C57BL/6J小鼠听力及耳蜗毛细胞活性的年龄相关性研究

目的 建立年龄相关性听力损失(age-related hearing loss,AHL)的小鼠动物模型,探讨C57BL/6J小鼠发生AHL与毛细胞活性变化的关系,并初步对C57BL/6J小鼠AHL模型进行AHL的病理分类.方法 按3、8、9、10、17、18月龄段分6组培育C57BL/6J小鼠,各组分别进行听性脑干反应(ABR)测试,对耳蜗毛细胞行琥珀酸脱氢酶染色并作基底膜硬铺片,观察各年龄段小鼠内外毛细胞线粒体琥珀酸脱氢酶的活性.结果 C57BL/6J小鼠随年龄增大,ABR阈值明显增高,在3月龄到9月龄期间ABR平均反应阈值增大比较缓慢,差异无统计学意义;在10月龄时,出现明显的听力下降,平均阈值比3月龄时约高18～23 dB,差异有统计学意义(click:t=5.78,P＜0.01;6 kHz:t =3.93,P＜0.01;8 kHz:t=3.01,P＜0.05).10月龄后小鼠听力继续下降,21月龄时平均阈值比3月龄时增高约60～68 dB,差异有显著统计学意义(click:t=31.23,P＜0.01;6 kHz:t=30.44,P＜0.01;8 kHz:t=33.83,P＜0.01).琥珀酸脱氰酶染色显示,随年龄增大,毛细胞线粒体活性丧失逐渐加重:先是底回外毛细胞活性下降,接着发生活性消失,并逐渐向顶回发展,最后累及内毛细胞.结论 C57BL/6J小鼠具有典型的年龄相关性听力损失特点,其听力下降的原因早期可能主要足外毛细胞及内毛细胞活性的丧失,晚期可能是由于基底膜结构混乱,导致电生理屏障消失,致耳蜗内电位(EP)不能维持而引起.C57BL/6J小鼠可作为感音型老年性听力损失动物模型.     

卡铂引起的灰鼠耳蜗内毛细胞缺损模式

目的:研究卡铂损害灰鼠耳蜗内毛细胞(IHCs)的模式,即IHCs何时出现缺损、给药后不同时间基底膜上IHCs缺损的范围、基底膜上IHCs最大缺损部位以及致绝大部分IHCs缺损所需时间等。方法:对成年灰鼠采用一次性卡铂腹腔注射(100mg/kg),给药后不同时间处死动物,常规制备耳蜗铺片和耳蜗图,以定量观察灰鼠耳蜗毛细胞的缺损情况。结果:注射卡铂后从24h～3个月,灰鼠的外毛细胞基本无缺损。IHCs损伤模式的特征:①注射卡铂后24h耳蜗毛细胞完整无损,IHCs从注药后第3天开始出现缺损;②从注射卡铂后3d到3个月期间,IHCs的最大缺损率均出现在第1回和第2回交界处;③IHCs缺损从第1回和第2回交界处开始,范围逐渐向底回和顶回扩展,与顶回相比,底回的IHCs缺损出现早且更严重;④至注射后3个月基底膜上IHCs的缺损呈平坦型。结论:100mg/kg卡铂腹腔注射对各回IHCs的破坏是不均等的,对卡铂的敏感度不一致可能造成了各回IHCs缺损在时间上的差异。     

谷氨酸调节耳蜗内毛细胞游离钙的实验观察

目的探索谷氨酸(glutamate,Glu)对离体耳蜗内毛细胞(innerhaircells,IHC)内钙信号的调控作用及其生理病理意义。方法在激光共聚焦显微镜下用钙敏荧光探针Fluo-3作为指示剂,观察外源性谷氨酸对分离的10个豚鼠耳蜗IHC胞内游离钙离子浓度(［Ca2+］i)的影响。结果分离好的正常IHC呈烧瓶形状,有明显的颈部,皮板上可观察到静纤毛,大球形的细胞体中间可见圆形的细胞核。形态完好的IHC大约存活2h,Fluo-3钙敏荧光探针染色后IHC胞体、胞核及表皮板有明显的染色梯度,表明游离Ca2+浓度从细胞核向细胞质逐渐减少。终浓度为3.85μmol/L的Glu对游离IHC内［Ca2+］i有增高趋势,而对游离外毛细胞(outerhaircells,OHC)内［Ca2+］i浓度无影响。观察10个IHC,发现9个［Ca2+］i浓度增加,1个无变化;观察10个OHC,发现7个［Ca2+］i无变化,3个略有下降。当Glu浓度增高后,IHC内［Ca2+］i先是迅速升高,继而逐渐下降。IHC外形由烧瓶状逐渐变成球形,提示IHC水肿变性。结论Glu可选择性调控IHC内［Ca2+］i,而对OHC内［Ca2+］i无影响,而过量的Glu刺激,可造成IHC［Ca2+］i的堆积,从而IHC水肿变性。     

年龄相关听力损失BALB／c小鼠耳蜗形态学观察

目的 探讨年龄相关听力损失小鼠耳蜗毛细胞形态学变化,建立年龄相关听力损失动物模型.方法 分别取3、6、12、18月龄BALB/c小鼠测定其听性脑干反应(ABR)阈值,应用耳蜗铺片和扫描电镜技术,观察不同月龄鼠耳蜗毛细胞计数和形态的变化.结果 3、6、12月龄鼠8 kHz ABR反应阈分别为(24.8±5.1)、(51.5±6.7)和(92.5±7.5)dB SPL.18月龄组120 dB SPL刺激声无诱发反应.耳蜗铺片毛细胞计数自6月龄组外毛细胞出现显著缺失,随月龄增加而加重,由底回逐渐向顶回发展,至12月龄时耳蜗底回和中回外毛细胞几乎完全丧失,内毛细胞显著缺失.扫描电镜显示6月龄组小鼠耳蜗毛细胞静纤毛可见不同程度的缺失、转位、散乱、倒伏、融合、变短现象,随月龄增加而逐渐加重.结论 BALB/c小鼠听力损失、耳蜗毛细胞缺失和纤毛损害随年龄增加而逐渐加重,可作为年龄相关听力损失研究的合适动物模型     

二甲基亚砜对在体耳蜗毛细胞的毒性作用

目的研究二甲基亚砜(DMSO)对在体耳蜗毛细胞的毒性作用。方法取清洁级健康、ABR阈值正常SD大鼠32只,雌雄不限,体重100-120g。随机分成4组,人工外淋巴液对照组(即0%组)8只;0.1%DMSO溶液组8只;1%DMSO溶液组8只;5%DMSO溶液组8只。所有动物均取右耳作为实验耳。通过耳蜗鼓阶打孔显微注射向每个实验耳注入不同浓度DMSO溶液4ul。术前1天和术后7天分别进行ABR(click和toneburst)检测。激光共聚焦显微镜观察DMSO溶液导入7天后的毛细胞变化。结果人工外淋巴液对照组click、4kHz、8kHz、16kHz处阈移平均值均<5dBSPL,仅于32kHz处有约13dBSPL的阈移,形态方面未见明显损伤;0.1%浓度组在click、4kHz、8kHz处阈移平均值均<5dBSPL,而32kHz处阈移约25dBSPL,与人工外淋巴液对照组比较提示有统计学意义,激光共聚焦显微镜观察底回时偶见少数内毛细胞胀大;1%浓度即可引起OHC大量丢失,造成相应纤毛表皮板缺如,且以底回最重,各频率ABR阈移均>15dBSPL,32kHz处阈移>30dBSPL;当浓度增加到5%时,不仅损伤耳蜗底回的外毛细胞,也导致内毛细胞的丢失,所造成的听力损失在32kHz处较1%组严重。结论 DMSO对毛细胞的损伤存在剂量依赖性,损伤程度自耳蜗底回向顶回逐渐减轻。     

豚鼠耳蜗各回外毛细胞的分离

目的 :探讨豚鼠耳蜗各回外毛细胞 (OHC)的分离方法。方法 :选豚鼠 8只 ,解剖耳蜗各回组织 ,获得Corti器并采用酶消化后机械分离。结果 :各回均可获得一定数量、活性良好的 OHC,第 1、2、3、4回单离 OHC的长度依次为 2 3.81、34.5 0、6 0 .48和 71.37μm。结论 :熟悉耳蜗各回解剖、组织特性并按操作要点进行是成功分离出各回 OHC的关键。     

Reversible contraction of isolated mammalian cochlear hair cells

Outer hair cells were isolated from the guinea pig cochlea using a micromechanical non-enzymatic procedure. Depolarization of outer hair cells in the presence of 25-125 mM K+ was accompanied by a longitudinal contraction of the isolated cells. A decrease of [K+] to 5.4 mM interrupted contraction and induced a relaxation. Individual hair cells were able to undergo as many as 5 cycles of contraction and relaxation. External Ca2+ was required for relaxation of the contracted hair cells. The contractile event led to the production of a visible cytoplasmic network between the supranuclear area and the cuticular plate.     

Modulation of copper transporters in protection against cisplatin-induced cochlear hair cell damage

Cisplatin belongs to platinum-based drugs and is widely used in cancer chemotherapy.Ototoxicity is one of the major dose limiting side-effects of cisplatin.For toxicity to occur cisplatin must first be...     

“Neural” responses to acoustic stimulation after destruction of cochlear hair cells

Summary Electrophysiological and histological observations in guinea pig's cochleas after amikacin treatment (14 × 450 mg/kg) confirm the results obtained in a former experiment: clear, short-latency, click-evoked responses were recorded in cochleas with only very few hair cells remaining at the extreme apex. Detailed analysis of these responses strongly indicates a neural origin and confirms their low-frequency sensitivity. Careful histological observations confirm the extensive hair cell loss and the preservation of nerve fibers in the remnants of the organ of Corti and of the vestibular sense organs. These results suggest that the acoustical vibrations either stimulate the vestibular receptors or act directly or through some kind of mechano-electrical transduction on the remaining cochlear nerve fibers.This work was supported by INSERM grant 8-ASR-6     

Relationship of gross cochlear potentials to hair cell pathology in the waltzing guinea pig

Electrophysiological recordings were made from the hearing organs of young waltzing guinea pigs of different ages. Age-related morphological changes in the cochleas of the same animals were studied with scanning and transmission electron microscopy. Results were compared with the results of studying the cochleas of normal guinea pigs of the same age. Waltzing guinea pigs are born with a hearing loss, as can be concluded from the magnitude of the whole-nerve action potential and their lower than normal summating potentials. However, their cochlear microphonics are nearly normal until the time in the degeneration process when hair cells start to disappear. This degeneration process begins at the tops of the hair cells and is evident upon electron microscopical examination of these structures.     

老年性聋患者人工耳蜗植入效果分析

目的 探究老年性聋患者人工耳蜗植入术后听觉言语康复效果及生活质量的变化。方法 对31例行人工耳蜗植入的老年性聋患者进行听力学、言语功能及生活质量评估并比较差异。听力学评估采用助听听阈,言语能力评估采用词表识别率,评估时间为术前、开机后6个月及开机后12个月。生活质量评估采用Nijmegen 人工耳蜗植入量表(NCIQ),评估时间为术前及开机后12个月。结果 共纳入31例患者,31例患者术前、开机后6个月及开机后12个月助听听阈分别为(62.55±3.69)、(46.58±5.14)、(38.68±4.26)dBHL,差异具有统计学意义(P＜0.05)。术前、开机后6个月及开机后12个月单音节词识别率分别为(9.55±5.81)%、(54.77±8.90)%、(68.52±7.21)%,差异具有统计学意义(P＜0.05);术前、开机后6个月及开机后12个月双音节词识别率分别为(19.87±9.72)%、(64.00±6.53)%、(74.26±6.79)%,差异具有统计学意义(P＜0.05);术前、开机后6个月及开机后12个月短句识别率分别为(28.00±10.58)%、(68.52±7.78)%、(77.61±8.59)%,差异具有统计学意义(P＜0.05)。术前及开机12个月NCIQ总量表得分分别为(35.90±5.80)、(65.16±8.18)分,差异具有统计学意义(P＜0.05)。结论 人工耳蜗植入可以改善老年性聋患者的听觉言语能力及生活质量,对于重度以上听力损失且助听器效果不佳的老年性聋患者可以选择人工耳蜗植入。     

Two-tone suppression in cochlear hair cells

The phenomenon of two-tone suppression that is known to occur at the level of the auditory nerve is shown to also occur in the receptor potential of single presumed inner hair cells in the first turn of the guinea pig cochlea.     

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     

Protective effect of corticosteroid against the cytotoxicity of aminoglycoside otic drops on isolated cochlear outer hair cells

OBJECTIVES: Otic drops are commonly used not only for otitis externa but also for otorrhea in the presence of tympanic membrane perforation or tympanostomy tube. Many studies demonstrated the ototoxicity of aminoglycoside. In our previous study, we observed that gentamicin (GM), when activated with liver extract, demonstrated significant cytotoxicity. The purpose of this study was to assess the protective effect of corticosteroid against the cytotoxicity of GM and tobramycin drops using isolated cochlear outer hair cells (OHCs) in vitro with liver extract. METHODS: OHCs from adult chinchilla cochleae were exposed to standard bathing solution, liver extract alone, and aminoglycoside otic drops with and without corticosteroid and liver extract. All experiments were performed at an osmolality of 305 +/- 5 mOsm, at room temperature, and for up to 60 minutes. The images of OHCs were recorded using an inverted microscope and analyzed on the Image Pro-Plus 3.0 program. Time to cell death and change of cell length were measured and analyzed. RESULTS: The time to cell death and percent change in cell length observed was significantly longer in the GM + liver extract + dexamethasone group than the GM + liver extract group (P <.05). The Tobradex + liver extract group showed an insignificant increase in percent change of cell length (P >.05) and significantly increased time to cell death than the tobramycin + liver extract group (P <.05). CONCLUSION: This study demonstrated that dexamethasone significantly reduced aminoglycoside cytotoxicity.     

实验动物前庭感觉毛细胞的定量观察

目的 测量常用实验动物内耳前庭感觉区的实际面积和量化分析前庭各个感觉区的毛细胞总数或密度。方法 ①制作CBA/CaJ小鼠、裸鼠、SD大鼠、豚鼠、南美栗鼠、新西兰白兔和非洲黑长尾猴的球囊斑铺片和椭圆囊斑铺片及壶腹嵴铺片,所有铺片样品来自每种受试动物的6个颞骨,在放大100倍的光学显微镜下拍摄2个囊斑铺片的整体照片;②应用Image J软件的图像测量程序,测量了上述7种常用实验动物球囊斑和椭圆囊斑的实际面积;③用网格将球囊斑铺片和椭圆囊斑铺片照片上的前庭感觉区划分为一个个方块区域。在放大400倍的光学显微镜下准确计数每个方格内的毛细胞数量,然后将每个方格的毛细胞计数结果相加以获得每种受试动物球囊斑和椭圆囊斑上的毛细胞总数;④应用前庭小视野定量观察技术计算出前庭各个感觉区小视野范围内的毛细胞密度。结果 ①从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑面积依次为(0.193±0.009)、(0.216±0.008)、(0.323±0.010)、(0.528±0.035)、(0.687±0.065)、(1.237±0.075)、(1.371±0.032)mm²;椭圆囊斑的面积依次为(0.193±0.020)、(0.208±0.013)、(0.321±0.011)、(0.526±0.034)、(0.795±0.017)、(1.224±0.082)、(1.388±0.048)mm²;②从小鼠、裸鼠、大鼠、豚鼠、南美栗鼠、白兔到猴的球囊斑毛细胞的总数依次为(2476.3±64.4)、(2389.8±47.8)、(3135.3±191.6)、(4882.2±208.7)、(6128.5±242.9)、(10572.2±464.4)、(10992.7±397.4)个;椭圆囊斑毛细胞的总数依次为(2491.4±54.8)、(2368.0±46.1)、(3218.8±82.9)、(4925.3±271.1)、(7794.0±386.1)、(11347.4±435.7)、(11114.5±410.6)个;③从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的球囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.007mm²)依次为101.0±5.79(微纹区)/120.8±4.15(周边区),95.5±3.91(微纹区)/109.2±5.26(周边区),78.4±6.54(微纹区)/94.8±4.38(周边区),60.0±4.74(微纹区)/84.6±2.61(周边区),57.2±3.83(微纹区)/80.0±3.54(周边区),53.8±4.21(微纹区)/68.0±4.18(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的椭圆囊斑微纹区和周边区的毛细胞密度(毛细胞数量/0.007mm²)依次为103.8±5.02(微纹区)/119.2±3.70(周边区),91.2±2.49(微纹区)/106.4±4.16(周边区),74.1±3.54(微纹区)/90.8±3.56(周边区),60.4±4.98(微纹区)/81.6±2.07(周边区),57.8±1.92(微纹区)/77.8±3.70(周边区),54.0±2.74(微纹区)/66.4±2.51(周边区)。从小鼠、大鼠、豚鼠、南美栗鼠、白兔和猴的壶腹嵴毛细胞密度(毛细胞数量/0.007mm²)依次为112.4±6.38,105.5±3.51,95.2±3.42,84.0±7.16,78.2±2.86,70.8±2.39。可见由于体型较小动物毛细胞的细胞体比体型较大动物毛细胞的细胞体小,因而体型较小动物的前庭毛细胞密度高于体型较大动物的前庭毛细胞密度。另外,每种实验动物球囊斑和椭圆囊斑微纹区的毛细胞密度相似,周边区的毛细胞密度也大致相同,但是同种实验动物囊斑微纹区的毛细胞密度却低于周边区的毛细胞密度。此外,壶腹嵴毛细胞的密度与球囊斑和椭圆囊斑周边区的毛细胞密度几乎相同。鉴于某些损害因素往往具有选择性破坏囊斑微纹区毛细胞的表现,因此囊斑微纹区的毛细胞密度应该与囊斑周边区的毛细胞密度区分开来进行统计,必要时甚至需要把Ⅰ型毛细胞和Ⅱ型毛细胞也区分开来分别予以病理学改变的定量评估。结论 本研究采用的前庭测量方法和获得的前庭各个感觉区的测量数据和毛细胞总数及毛细胞密度,为前庭病理学研究的定量分析提供了有益的参考经验和必要的参考数据。