Calpain在噪声损害毛细胞中的角色

目的：了解Calpain在噪声损害耳蜗毛细胞中所扮演的角色。方法观察Calpain免疫细胞化学反应产物在噪声损害中的变化。结果正常对照耳蜗样品中未见Calpain活性显示,但噪声损害耳蜗样品中可见外毛细胞底部及其神经末梢区域出现大量的Calpain。结论噪声引起的耳蜗毛细胞破坏可能与Calpain的激活及其降解功能有关。     

Loss of hair cells and threshold sensitivity during prolonged noise exposure in normotensive albino rats

The relation between hearing loss and loss of hair cells after prolonged exposure to a simulated industrial noise environment was determined in normotensive albino rats. Hearing loss was assessed behaviorally by a conditioned suppression technique and electrophysiologically by auditory brainstem response to pulses of 1/3 octave filtered full cycle sine waves. The ears were analyzed in surface preparations and the hair cells counted. Sixty-two ears from 46 animals were analyzed after 1, 3, 7, and 15 months of exposure. A hearing loss of up to approximately 30 dB was consistently found with minimal loss of hair cells. Between 30 and 60 dB hearing loss there was a fair correspondence between loss of function and loss of hair cells. After 15 months exposure a relatively larger loss of hair cells was observed than expected from decrease of threshold sensitivity. The results indicate the existence of a systematic although not directly proportional relation between hair cell loss and loss of sensitivity during prolonged noise exposure. It is suggested that the correlation between hearing loss and hair cell loss is influenced e.g., by duration and level of exposure, degree of hearing loss, and location along the basilar membrane.     

强噪声暴露后耳蜗毛细胞的病理形态变化

目的观察强噪声暴露后,耳蜗毛细胞的纤毛、胞体、及胞核的损伤情况。方法听力正常豚鼠,体重在250～300g之间,经强度为170dB SPL的脉冲噪声暴震200次后,取材作全耳蜗铺片行免疫组化检测（prestin和phalloidin）;扫描电镜及透射电镜,观察受损毛细胞的病理变化。同时做毛细胞的纤毛、胞体及胞核的标记染色。荧光显傲镜和电镜下观测结果：结果强噪声暴露后,毛细胞纤毛大部分消失,只有小部分表皮板存在,同时细胞核蹿裂消失,而此时胞体大部分存在,但有肿胀及变形等变化。结论噪声损伤后,毛细胞的纤毛及胞核首先发生变化,随后胞体肿胀溶解,因此毛细胞的胞核及纤毛变化最先反映毛细胞受损情况,但纤毛的缺失并不代表毛细胞的缺失和死亡。如果单从一种形态观测指标评估毛细胞的损伤病理状况是不全面的。     

睫状神经营养因子防治强脉冲噪声对豚鼠内耳损伤的实验研究

目的探索应用睫状神经营养因子（ｃｉｌｉａｒｙｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ,ＣＮＴＦ）防治强脉冲噪声对豚鼠内耳损伤的可能性,为强脉冲噪声致聋的防治提供新的方法。方法制作强脉冲噪声致聋豚鼠模型３６只,１８只应用ＣＮＴＦ肌内注射３周,１８只等量的生理盐水作对照,正常对照豚鼠１８只,进行耳蜗铺片计算机图像分析毛细胞计数,螺旋神经节细胞计数、耳蜗乙酰胆碱酯酶染色铺片观察和ＡＢＲ反应阈测定。结果正常对照组、噪声暴露后２１ｄ的生理盐水对照组和ＣＮＴＦ组耳蜗毛细胞平均总数（ｘ±ｓ,下同）分别为９０９４±１０３．６、７３５１±１９６．５、８５２２±２０３．１;而螺旋神经节细胞计数在耳蜗中轴切片第二回下段３组间差异有显著性,分别为５１±４．７２、２７±６．９４、３７±１０．４。乙酰胆碱酯酶染色铺片结果示ＣＮＴＦ组较生理盐水对照组耳蜗乙酰胆碱酯酶活性损失轻、范围小。结论ＣＮＴＦ在一定程度上能防止受损的螺旋神经节细胞及外毛细胞发生变性坏死,并可能促进其损伤的修复。     

Apoptosis of guinea pig cochlear hair cells following chronic aminoglycoside treatment

Although aminoglycosides have been investigated for their cochleotoxicity, it has still not been determined whether apoptosis or necrosis results in cochlear hair cell death following aminoglycoside treatment. To study possible mechanisms of cell death, we used in situ DNA break-labeling to examine guinea pig cochleae affected by kanamycin ototoxicity. Chronic kanamycin treatment induced DNA fragmentation that was detectable in both outer and inner hair cells, suggesting the occurrence of apoptosis. These findings suggest that apoptosis achieves deletion of affected hair cells without disrupting tissue architecture in the organ of Corti. Received: 22 April 1997 / Accepted: 31 July 1997     

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。     

Cytodifferentiation of cochlear hair cells

Cytodifferentiation of a limited number of cochlear hair cells in the mouse starts on the 14th gestational day. One day later, the number of identified hair cells increases considerably. Cytodifferentiation apparently occurs in a gradient from the hair cell surface to the base. First, the irregular microvilli covering the future hair cell surfaces begin to show a regular pattern but are of the same thickness and length as microvilli on supporting cells. Second, a polarization of sensory hairs occurs with a stepwise increase in stereocilia length in the different rows toward the kinocilium. Finally, the cuticle is formed, giving an anchorage for sensory hair rootlets in the hair cell. At birth some hair cells can be found with immature surface morphologic features, e.g., stereocilia of the same length on the entire hair cell surface and lack of a cuticular plate. The onset of hair cell differentiation takes place without morphologic contact with ingrowing nerve fibers.     

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.     

白噪声暴露对豚鼠耳蜗毛细胞感受器电位非线性特性的影响

为了探讨正常及在噪声暴露过程中耳蜗毛细胞感受器电位非线性特性的变化规律，采用玻璃微电极在体毛细胞胞内记录的实验方法，记录了７只豚鼠（７耳）正常状态和白噪声暴露后耳蜗外毛细胞（ＯＨＣ）胞内交流感受器电位输入－输出曲线（Ｉ－Ｏ曲线）。正常豚鼠耳蜗ＯＨＣ交流感受器电位幅度在刺激声强度较低时呈线性增长，声强达到５０～７０ｄＢＳＰＬ时幅度增长变慢，在８０～１００ｄＢＳＰＬ时，幅度不再随刺激强度的增加而继续增加，出现饱和现象。测试耳在用１００ｄＢＳＰＬ时白噪声暴露１０～２０分钟后，感受器电位幅度普遍下降，Ｉ－Ｏ曲线的线性段延长，非线性段缩短，但高强度感受器电位幅度增大，出现“幅度重振现象”，与临床上观察的响度重振现象具有相似的特点。从而推测ＯＨＣ感受器电位非线性特性减弱，发生幅度重振，可能是临床上主观响度重振现象的客观来源之一。     

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。     

豚鼠冲击波负压暴露后耳蜗毛细胞损害定量观察

目的 探讨冲击波负压（blast underpressure,BUP）暴露后豚鼠耳蜗毛细胞损害特点.方法将豚鼠暴露实验性BUP 14天后处死,硝酸银染色硬铺片法计数观察耳蜗基底膜毛细胞损伤情况.结果压力峰值介于-22.4kPa和-63.3kPa之间的实验性BUP暴露后,豚鼠耳蜗外毛细胞出现了明显的病理性改变,损伤的程度以第二转最重,第二排和第三排的病变比第一排更为严重.BUP强度越高,毛细胞损害越重.各实验组动物的外毛细胞总缺失率明显高于正常对照组（P＜0.01）;重复暴露3次的动物外毛细胞缺失率明显高于暴露1次的动物（P＜0.01）.结论BUP暴露可引起明显的豚鼠耳蜗外毛细胞缺失等损害,其损害程度与负压峰值及暴露次数密切相关;毛细胞损害越重,ABR阈移也就越明显.     

荧光下豚鼠听觉离体毛细胞的凋亡改变

目的：探讨离体耳蜗毛细胞的凋亡改变。方法：采用胶原酶制备离体耳蜗毛细胞、0．01％吖啶橙进行荧光染色,荧光显微镜观察凋亡改变。结果：凋亡毛细胞的主要形态特征包括：胞核皱缩;荧光显微镜下凋亡毛细胞呈红色或红黄色荧光,毛细胞形态完整,无胞膜破裂或胞质溢出。结论：离体耳蜗毛细胞的凋亡改变使细胞呈红色或红黄色荧光,胞膜完整。     

耳蜗毛细胞死亡引发耳蜗内延迟性继发病变的研究

本文讨论了由耳蜗毛细胞死亡引发的耳蜗内一系列延迟性继发病变。在外毛细胞遭到破坏的早期阶段,外指细胞即刻膨胀开来并堵塞了螺旋器表面的穿孔。外指细胞的膨胀有效阻止了含高钾浓度的内淋巴液进入到螺旋器的内部,从而使剩余的毛细胞和支持细胞避免了钾中毒损害。膨胀的外指细胞随后分化成高大柱形细胞并继续支撑起耳蜗螺旋器的整个外形结构。在发生散在性外毛细胞缺失的耳蜗损害模型,由外指细胞转化的高大柱形细胞在外毛细胞缺失的位置永久支撑起耳蜗螺旋器的结构,使周围剩余的存活外毛细胞继续发挥其放大和转换声学振动信号的功能以维持残余听力。在发生大面积外毛细胞死亡的耳蜗损害模型,转化成高大柱形细胞的前外指细胞在外毛细胞缺失后30d左右死亡,随后导致整个耳蜗螺旋器的结构坍塌和内毛细胞及其他支持细胞的继发性死亡,最后在耳蜗基底膜上仅存一层扁平上皮。无论是在内外毛细胞被同时破坏的实验模型还是在外毛细胞大面积死亡后继发支持结构坍塌和内毛细胞死亡的实验模型,耳蜗传出神经和传入神经都会在丧失内外毛细胞后数周内发生继发性破坏。位于蜗轴螺旋管内的螺旋神经节随后也因丧失神经刺激信号和缺乏神经营养因子而引发延迟性螺旋神经节死亡,螺旋神经节的死亡使与耳蜗核相连的听觉神经中枢端轴突也发生不可逆的破坏,最终导致耳蜗周围系统与中枢听觉系统的神经连接永久中断。     

噪声刺激致豚鼠外毛细胞凋亡时半胱氨酸天冬氨酸蛋白酶3激活和凋亡诱导因子转移

目的探讨噪声损伤后豚鼠外毛细胞发生凋亡的通路及其机制。方法分离解剖对照组（12只）和噪声暴露组（12只）豚鼠耳蜗，利用免疫荧光抗体和免疫荧光染料，分别染色细胞核、线粒体、凋亡诱导因子（apoptosis inducing factor，AIF）和半胱氨酸天冬氨酸蛋白酶3（Caspase 3），按表面制备法行耳蜗铺片，观察凋亡和坏死毛细胞内的荧光信号。结果对照组正常耳蜗毛细胞内没有激活的Caspase 3，AIF分布在毛细胞的线粒体内。实验组动物暴露于声压级120dB的白噪声环境中每天4h，连续2d后，耳蜗外毛细胞出现死亡，表现为坏死和凋亡两种方式，但以凋亡为主。在凋亡的外毛细胞中，Caspase 3被激活，AIF从线粒体转移到细胞核中；而在坏死的外毛细胞中，没有Caspase 3的激活，只有AIF自线粒体向细胞核的转移。结论噪声损伤后耳蜗外毛细胞的凋亡以Caspase依赖型的通路为主；线粒体释放的AIF在外毛细胞死亡通路中起重要作用。     

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.     

Comparison of low- and high-side two-tone suppression in inner hair cell and organ of corti responses

Two-tone interactions were measured from inner hair cells and from the organ of Corti fluid space in the third turn of the guinea pig cochlea. At relatively low stimulus levels, a low-side suppressor caused frequency response functions to become broader. Phase changes exhibited a lag/lead transition around the characteristic frequency in harmony with the change in magnitude. These patterns are similar to those previously documented for a high-side suppressor (Cheatham and Dallos 1989) and suggest that suppression is not simply an attenuation phenomenon since level reductions for single-tone inputs produce response patterns which are mirror images of those obtained for the two-tone conditions. In contrast to the low-level results, data measured at moderately high stimulus levels indicate that the magnitude changes produced by both low- and high-side suppressors are qualitatively similar to changes generated by reducing the input sound level. In other words, ac frequency response functions become narrower, partially reversing the broadening of these functions which occurs as sound level increases. Companion phase measures, however, demonstrate that lowand high-side suppressors, in spite of producing similar changes in filter shape, do not produce similar changes in response phase. In fact, neither of the two-tone conditions produce response patterns similar to the one associated with reducing the input sound level.     

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。     

Comparative ultrastructure of subsurface cisternae in inner and outer hair cells of the guinea pig cochlea

Summary The subsurface cisternal systems of outer hair cells (OHCs) from different cochlear regions have been compared with the subsurface cisternal system of inner hair cells. Three main observations have been made: (1) the number of cisternal layers, when there is more than one present, is reduced along the length of an individual outer hair cell; (2) basal outer hair cells may have only one fenestrated cisternal layer; and (3) the inner hair cells possess a lateral cistern and associated pillar and filament complexes which are very similar to those of some basal OHCs. These observations are discussed in relation to hypotheses regarding the role of these structures in hair cell motility.Presented at the 25th Workshop on Inner Ear Biology in London, England, 4–7 September 1988     

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。     

Structure,pharmacology and function of GABAA receptors in cochlear outer hair cells

Summary There is evidence that the inhibitory neurotransmitter -aminobutyric acid (GABA) is released from some efferent olivocochlear nerve endings terminating at outer hair cells (OHCs). Using monoclonal antibodies against postsynaptic GABA_A receptor from bovine cerebral cortex we confirm the presence of GABA and benzodiazepine bindings sites of - and -subunits of GABA_A receptors at the basal pole of isolated OHCs. Whole-cell recording with viable OHCs revealed that the application of 10^–3–10^–8 M GABA to the cell surface was followed by a concentration-dependent hyperpolarization of the outer cell membrane. Hyperpolarization was increased in the presence of 2.5 × 10^–5 M chlorazepate, a benzodiazepine derivative. Electrophysiological effects caused by GABA alone or in combination with chlorazepate were specifically inhibited by 10^–6 M of the GABA-receptor antagonist picrotoxin. Moreover, 10^–5–10^–7 M GABA caused reversible slow elongation of the cylindrical hair cell body in OHCs examined. These neurotransmitter-induced motile responses were specifically blocked by 10^–4 M picrotoxin. The results suggest that a subpopulation of OHCs express - and -subunits of GABA_A receptors which both form a GABA/benzodiazepine-receptor complex at the basal pole of isolated OHCs. These receptors are thought to allow GABA which is released from efferent auditory nerve terminals to bind to the cell surface of OHCs, resulting in GABA_Areceptor activation. This probably gates a GABA_A-receptor-associated chloride channel in the postsynaptic OHC membrane, allowing hyperpolarization and elongation of the cell. Correspondence to: P.K. Plinkert