庆大霉素对内侧橄榄耳蜗传出神经毒性作用的形态学观察

目的 观察庆大霉素慢性耳中毒前后蒙古沙鼠人侧橄榄耳蜗传出神经的形态改变及其与细胞损害的关系,以探讨ＭＯＣ传出神经在氨基糖甙类抗生素慢性耳中毒中的重要性。方法 采用改良的乙酰胆碱酯酶组化染色和甲苯胺蓝＝苏木素染色法,全耳蜗铺片观察健康对照组和庆大霉素组耳蜗ＭＯＣ传出神经和外毛细胞的分布特征,并测量耳蜗ＭＯＣ传了神经纤维、末梢及ＯＨＣ的数量。结果 停经后３周出现耳蜗ＭＯＣ纤维和末梢损害,损害程度随时间     

庆大霉素慢性耳中毒对听觉和传出神经功能的影响

目的：探讨庆大霉素慢性耳中毒对听觉和传出神经功能的影响。方法：在庆大霉素应用前后通过观察对侧噪声（ＣＬＮ）对听神经复合动作电位（ＣＡＰ）的影响确定内侧橄榄耳蜗（ＭＯＣ）系统功能,通过测试在４,６,８,１０和１２ｋＨｚ的ＣＡＰ反应阈确定听功能。结果：注射庆大霉素后３周和１１周。ＣＬＮ对ＣＡＰ的抑制效应呈进行性、不可逆性消除,且以１１周最明显,ＣＡＰ反应阈分别上升１０和２５ｄＢ,与耳蜗传出神经和毛细胞     

庆大霉素对豚鼠耳蜗橄榄束传出神经影响的研究

目的探讨庆大霉素对豚鼠耳蜗橄榄束传出神经的损害。方法采用AchE组织化学染色,全耳蜗铺片观察对照组和庆大霉素组停药后不同时间耳蜗橄榄束传出神经的分布特征,并测量耳蜗橄榄束传出神经纤维的数量。实验前后行畸变产物耳声发射对侧声抑制测试。结果肌注庆大霉素2周后耳蜗AchE终末反应物减少,神经纤维连续性中断,耳声发射对侧声抑制幅度下降,损害随观察时间延长而加重,8周后损害最为严重,耳声发射未引出。结论庆大霉素可对豚鼠内侧橄榄耳蜗束传出神经系统产生不可逆性的损害。     

自由基清除剂二甲基亚砜对庆大霉素耳蜗毒性的影响

观察豚鼠同时注射二甲基亚砜（ＤＭＳＯ）与庆大霉素（ＧＭ）及单独注射ＧＭ等几组动物后，耳蜗听功能、扫描电镜所见及耳蜗和血清中脂质过氧化物（ＬＰＯ）丙二醛（ＭＤＡ）含量的变化。结果表明ＧＭ＋ＤＭＳＯ组动物ＡＰ阈值较ＧＭ组明显降低，ＡＰ（Ｎ＿１）潜伏期ＧＭ组较ＧＭ＋ＤＭＳＯ组显著延长，耳蜗扫描电镜显示ＧＭ＋ＤＭＳＯ组毛细胞受损程度较ＧＭ组明显为轻。耳蜗中ＭＤＡ检测表明，ＧＭ组耳蜗中ＭＤＡ含量较ＧＭ＋ＤＭＳＯ组明显增高（Ｐ＜０．０１）。提示自由基引起耳蜗ＬＰＯ可能是ＧＭ耳毒性机制之一；ＤＭＳＯ可减轻ＧＭ的耳毒性。     

川芎嗪对庆大霉素耳中毒耳蜗外毛细胞和血管纹的保护作用

目的：探讨在庆大霉素（gentamycin，GM）耳中毒情况下，川芎嗪（tetramethylpyrazine，TMP）对豚鼠耳蜗外毛细胞和血管纹边缘细胞的保护作用。方法：12只豚鼠随机分为GM组、联合用药组、TMP组及对照组，用药十天后处死，采用透射电镜观察耳蜗外毛细胞及血管纹边缘细胞超微结构，扫描电镜观察血管纹边缘细胞表面形态。结果：透射和扫描电镜显示，联合用药组外毛细胞及血管纹边缘细胞超微及表面结构破坏不均明显轻于庆大霉素组，特别是其中的线粒体结构破坏与数目减少更显著轻于庆大霉素组。结论：川芎嗪具有保护庆大霉素耳中毒耳蜗外毛细胞和血管纹结构的作用，从而拮抗庆大霉素耳毒性。     

水杨酸钠豚鼠耳蜗单离外毛细胸游离钙浓度影？…

目的 观察水杨酸钠对豚鼠耳蜗外毛细胞（ｏｕｔｅｒ ｈａｉｒ ｃｅｌｌｓ，ＯＨＣ）内游离钙浓度「Ｃａ＾２＋」ｉ的影响，并对钙通道调控药物的拮抗作用进行观察，以深入探讨水杨酸钠耳毒性的分子生物学机制。方法 豚鼠全身麻醉后断头活杀，酶－机械法分离获得耳蜗单离ＯＨＣ。１０ｕｍｏｌ／Ｌ的Ｅｌｕｏ－３／ＡＭ负载３０ｍｉｎ后用ＡＣＡＳＵｌｔｉｍａ型激光扫描共聚焦显微镜观察不同药物灌流下ＯＨＣ「Ｃａ＾２＋」ｉ的变     

在体耳蜗毛细胞电反应记录

为了解耳蜗内、外毛细胞基本电生理特性，建立了经耳蜗外侧壁入路在体记录耳蜗毛细胞电反应的方法，１２０只豚鼠（１２０耳）中成功记录到１８个细胞的胞内反应，对耳蜗第三回内毛细胞（ＩＨＣ）、外毛细胞（ＯＨＣ）的电生理特性进行了观察。ＩＨＣ、ＯＨＣ的静息膜电位均值分别为－３５±８．８３ｍＶ（ｎ＝３）和－７１±７．４４ｍＶ（ｎ＝７）；支持细胞负值更大。毛细胞感受器电位有交流和直流两种成分，交流反应峰－峰均值ＩＨＣ和ＯＨＣ分别为１２．４ｍＶ和５．６ｍＶ，直流成分ＩＨＣ平均为２．１６ｍＶ，ＯＨＣ平均为１．９９ｍＶ。胞内记录失败的常见原因为微电极质量较差和耳蜗外侧壁手术等造成耳蜗功能损伤。     

庆大霉素致豚鼠耳蜗毛细胞凋亡时Caspase-3的表达

目的探讨庆大霉素所致耳蜗外毛细胞的损伤特点及耳毒性机制。方法豚鼠随机分3个组,分别腹腔注射庆大霉素、庆大霉素加速尿、生理盐水各连续6天,比较用药前后各组豚鼠畸变产物耳声发射（distortion product otoacoustic emissions,DPOAE）变化、耳蜗铺片、毛细胞凋亡情况及Caspase-3在毛细胞的表达。结果注射庆大霉素和庆大霉素加速尿2个组,用药后DPOAE明显下降,耳蜗铺片见毛细胞损伤明显,细胞凋亡发生及Caspase-3免疫复合物阳性表达。结论庆大霉素可致豚鼠耳蜗毛细胞损伤并加速凋亡,Caspase-3在毛细胞凋亡过程中起重要作用。     

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。     

水杨酸钠对豚鼠单离外毛细胞外向钾电流和静息电位及膜电容影响

目的　探讨水杨酸钠对豚鼠单离外毛细胞（ｏｕｔｅｒｈａｉｒｃｅｌｌ,ＯＨＣ） 钾电流（ｐｏｔａｓｓｉｕｍ ｃｕｒｒｅｎｔ,ＩＫ）、静息电位（ｒｅｓｔｉｎｇ ｐｏｔｅｎｔｉａｌ,ＲＰ） 和膜电容（ｍｅｍｂｒａｎｅ ｅｌｅｃｔｒｉｃ ｃａｐａｃｉｔａｎｃｅ,ＭＥＣ） 的影响,分析水杨酸钠影响毛细胞功能的细胞电生理机制。方法　应用膜片钳全细胞记录技术,测试０．１ 和１ ．０ ｍｍｏｌ／Ｌ水杨酸钠用药前后ＯＨＣ的ＩＫ、ＲＰ和ＭＥＣ 变化。结果　水杨酸钠对ＩＫ 的影响具有时效和量效关系,即用药后有ＩＫ 先增加后降低的双相作用,而高浓度水杨酸钠无论是对ＩＫ 的增加作用还是降低作用都比低浓度时明显。ＲＰ平均约为－６０ ｍＶ,ＭＥＣ 平均约为３９ ｐＦ。水杨酸钠有降低ＲＰ和ＭＥＣ的作用,且均存在着量效关系,即高浓度时的作用大于低浓度时。水杨酸钠对ＲＰ和ＭＥＣ影响的时效关系不明显。结论　水杨酸钠具有影响ＯＨＣ两侧膜的Ｋ＋ 电导和细胞内外Ｋ＋ 分布的作用,并通过对ＩＫ、ＲＰ和ＭＥＣ的影响改变ＯＨＣ的兴奋性和机械活动特性,可能是水杨酸影响毛细胞功能的耳蜗机制之一。     

Modulation of hair cell efferents

Outer hair cells (OHCs) amplify the sound-evoked motion of the basilar membrane to enhance acoustic sensitivity and frequency selectivity. Medial olivocochlear (MOC) efferents inhibit OHCs to reduce the sound-evoked response of cochlear afferent neurons. OHC inhibition occurs through the activation of postsynaptic α9α10 nicotinic receptors tightly coupled to calcium-dependent SK2 channels that hyperpolarize the hair cell. MOC neurons are cholinergic but a number of other neurotransmitters and neuromodulators have been proposed to participate in efferent transmission, with emerging evidence for both pre- and postsynaptic effects. Cochlear inhibition in vivo is maximized by repetitive activation of the efferents, reflecting facilitation and summation of transmitter release onto outer hair cells. This review summarizes recent studies on cellular and molecular mechanisms of cholinergic inhibition and the regulation of those molecular components, in particular the involvement of intracellular calcium. Facilitation at the efferent synapse is compared in a variety of animals, as well as other possible mechanisms of modulation of ACh release. These results suggest that short-term plasticity contributes to effective cholinergic inhibition of hair cells.     

Olivocochlear efferents: anatomy, physiology, function, and the measurement of efferent effects in humans

This review covers the basic anatomy and physiology of the olivocochlear reflexes and the use of otoacoustic emissions (OAEs) in humans to monitor the effects of one group, the medial olivocochlear (MOC) efferents. MOC fibers synapse on outer hair cells (OHCs), and activation of these fibers inhibits basilar membrane responses to low-level sounds. This MOC-induced decrease in the gain of the cochlear amplifier is reflected in changes in OAEs. Any OAE can be used to monitor MOC effects on the cochlear amplifier. Each OAE type has its own advantages and disadvantages. The most straightforward technique for monitoring MOC effects is to elicit MOC activity with an elicitor sound contralateral to the OAE test ear. MOC effects can also be monitored using an ipsilateral elicitor of MOC activity, but the ipsilateral elicitor brings additional problems caused by suppression and cochlear slow intrinsic effects. To measure MOC effects accurately, one must ensure that there are no middle-ear-muscle contractions. Although standard clinical middle-ear-muscle tests are not adequate for this, adequate tests can usually be done with OAE-measuring instruments. An additional complication is that most probe sounds also elicit MOC activity, although this does not prevent the probe from showing MOC effects elicited by contralateral sound. A variety of data indicate that MOC efferents help to reduce acoustic trauma and lessen the masking of transients by background noise; for instance, they aid in speech comprehension in noise. However, much remains to be learned about the role of efferents in auditory function. Monitoring MOC effects in humans using OAEs should continue to provide valuable insights into the role of MOC efferents and may also provide clinical benefits.     

Course and distribution of efferent fibers in the cochlea of the mouse.

The course, distribution and termination of single efferent fibers to the cochlea has been described in only a few animals and relatively few fibers have been studied with knowledge of their ipsilateral or contralateral origin. In order to examine the efferent fibers in the mouse, the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophoretically injected into one side of the brain stem near the location of known efferent nuclei. Examination of surface preparations of the cochlea revealed detailed information for both the lateral olivocochlear (LOC) and medial olivocochlear (MOC) systems. Many, but not all, fibers entered the cochlea within the intraganglionic spiral bundle (IGSB). The LOC fibers were restricted to the ipsilateral cochlea and rarely branched within the IGSB and osseous spiral lamina (OSL). In the organ of Corti, they traveled either basally or apically in the region of the inner hair cells (IHCs), spanning lengths up to 130 microns (basally) and 890 microns (apically). Terminal swellings of these fibers were ca 3.0 microns in diameter. Numerous en passant swellings were present where the fibers formed a plexus in the area of the IHCs. The MOC fibers followed a similar course in the IGSB and OSL, and within the OSL the fibers had few branches. Within the organ of Corti they traveled apically (up to 70 microns) in the nerve bundles located in the IHC area before they crossed the tunnel of Corti. In the region of the OHCs, 9% of the traceable fibers branched to innervate two to three OHCs while 91% appeared to innervate only one OHC. There was no discernible difference in the distribution of contralateral and ipsilateral MOC projections in terms of cochlear region or outer hair cell rows.     

Reconstructions of efferent fibers in the postnatal hamster cochlea

It is well known that adult-like physiological functioning of the mammalian postnatal cochlea occurs coincidentally with the presence of efferent synapses on outer hair cells (OHCs). This study described the cochlear innervation patterns of thick efferent fibers traveling in the vestibular nerve in postnatal hamsters ranging in age from day zero to day 10. At least three kinds of efferent fibers were labeled via an in vitro horseradish peroxidase (HRP) technique: varicose, thin efferents; nonvaricose, thin efferents; and nonvaricose, thick efferents. Nonvaricose thick efferents were reconstructed from the basal third of the cochlea. Reconstructed efferent fibers traversed in the intraganglionic spiral bundle (IGSB) on the peripheral edge of the spiral ganglion and branched profusely in the osseous spiral lamina (OSL). From day zero to day five, large (greater than 1.0 microns) diameter nonvaricose efferent fibers gave rise to branches that either terminated underneath inner hair cells or appeared to end blindly in the OSL. Efferent fibers also had branches that traveled in the inner spiral bundle (ISB) and tunnel spiral bundle (TSB). In cochleae from hamsters six to eight days old, some thin and thick diameter efferent fibers contacted both inner and outer hair cells. By the tenth day, large diameter fibers traveled radially across the tunnel of Corti to terminate on one to five OHCs. As early as day seven, large diameter fibers also appear to terminate preferentially on OHCs in row one. These observations are consistent with the notion that the end of the first postnatal week represents a critical period in the formation of adult-like synapses on the OHCs. The data also suggest a developmental transition period when efferent fibers contact both hair cell types before contacting OHCs separately.     

Effects of gentamicin and pH on [Ca2+]i in apical and basal outer hair cells from guinea pigs.

Aminoglycosides are widely used antibiotics and frequently produce acute ototoxicity. In this study we attempted to comparatively investigate the effects of gentamicin on Ca2+ influx of apical and basal outer hair cells (OHCs) isolated from guinea-pig cochlea. Since the solution of gentamicin sulfate salt is acidic (pH 3.1-3.3), we also explored the effect of external acidification on Ca2+ influx. By means of fura-2 microspectrofluorimetry, we measured the intracellular calcium concentration ([Ca2+]i) of OHCs bathed in Hanks' balanced salt solution (pH 7.40) during either a resting state or high K+-induced depolarization. Our results show that at the resting state, the baseline [Ca2+]i in apical OHCs (94+/-2.0 nM) was slightly lower than that in basal OHCs (101.1+/-2.4 nM). By contrast, the increase in [Ca2+]i evoked by high K+ depolarization in apical OHCs was about two-fold greater than that in basal OHCs. Nifedipine (30 microM) abolished the increased [Ca2+]i in both types of OHCs, suggesting that Ca2+ influx was mainly through L-type Ca2+ channels of OHCs. While gentamicin and extracellular acidification (pH 7.14) can separately attenuate this increase in [Ca2+]i in both types of OHCs, their suppressive effects are additive in basal OHCs, but not in apical OHCs. The implications of these findings are that: (1) apical and basal OHCs behave differently in response to depolarization-increased [Ca2+]i, and (2) basal OHCs are more vulnerable to the impairment of Ca2+ entry during depolarization by a combination of gentamicin and extracellular acidification, which is correlated with the clinical observation that ototoxicity of aminoglycosides at the basal coil of OHCs is more severe than that at the apical coils. Moreover, the possibility that extracellular acidification may enhance the acute ototoxic effects of aminoglycosides should be considered especially in topical applications.     

Hidden cochlear impairments

Pure tone audiometry is a routine clinical examination used to identify hearing loss. A normal pure tone audiogram is usually taken as evidence of normal hearing. Auditory deficits detected in subjects with normal audiograms, such as poor sound discrimination and auditory perceptual disorders, are generally attributed to central problems. Does the pure tone audiogram truly reflect cochlear status? Recent evidence suggests that individuals with normal audiogram may still have reduced peripheral auditory responses but normal central responses, indicating that the pure tone audiometry may not detect some types of cochlear injuries. In the cochlea, the outer hair cells (OHCs), inner hair cells (IHCs), and the spiral ganglion neurons that synapse with IHCs are the 3 key cochlear components in transducing acoustical vibrations into the neural signals. This report reviews three types of cochlear damage identified in laboratory animals that may not lead to overt hearing loss. The first type of cochlear impairment, such as missing a certain proportion of IHCs without damage to OHCs, may reduce the cochlear output and elevate response threshold; however, the reduced peripheral auditory sensitivity may be restored along the auditory pathway via central gain enhancement. The second type of cochlear impairment, such as selective damage to the synapses of the high-threshold thin auditory nerve fibers (ANFs), reduces cochlear output at high stimulation levels with no effect on response threshold. In this case the reduced cochlear output may be compensated along the auditory pathway as well. The third type of cochlear impairment, such as missing a certain number of OHCs without damage to others, may not even affect cochlear function at all. These “hidden” cochlear impairments do not result in overt hearing loss, but they may increase the vulnerability of the cochlea to traumatic exposure and lead to disrupted central auditory processing.     

Anatomical correlates of functional recovery in the avian inner ear following aminoglycoside ototoxicity

Tucci and Rubel have demonstrated functional recovery of the chick cochlea following aminoglycoside ototoxicity. The cochleae of these same animals were examined by scanning electron microscopy (SEM) in order to further understand this recovery process. Hatchling chicks were given daily doses of gentamicin for 10 days. Auditory-evoked potential measurements and examination of the cochlea by scanning electron microscopy were performed after survival periods of 5 days to 20 weeks. After 5 days of gentamicin exposure, there was near complete basal hair cell loss associated with a high-frequency hearing loss. Apical progression of damage with a broad-band hearing loss occurred over 4 weeks. At 20-weeks, hair cell counts were normal with a small high-frequency hearing loss. Hair cell regeneration played a major role in the functional recovery of the cochlea.     

Noise exposure induced cochlear hair cell death pathways in guinea pig

Objective To understand the mechanism of noise exposure induced outer hair cells(OHCs) death pathways. Methods Thirty two guinea pigs were used in this study. The animals were either exposed for 4 h/day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused with MNNG. After auditory test,the cochleae of animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. F-actin staining was used to determine missing OHCs. Caspase-3 was detected in living organ of Corti whole mounts using the fluorescent probe. The single strand DNA (ssDNA) in apoptotie OHCs in guinea pigs and apoptosis inducing factor (AIF) in hair cells in guinea pigs were examined by immunohistology method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. Results (1) Both apoptotic and necrotic hair cells appeared following noise exposure. (2) Noise exposure induced single strand DNA in apoptotic OHCs but not in the normal OHCs. (3)Either after noise exposure or after MNNG perfusion, apoptotic OHCs were featured by nuclear condensation or fragmentation with caspase-3 activation, whereas necrotic OHCs were characterized by nuclear swelling without caspase-3 activation. (4) In normal organ of Corti, AIF was located in the mitochondria areas. After noise exposure,AIF was translocated from mitochondria in apoptotic and necrotic OHCs. Conclusion These findings indicate that noise exposure damages DNA in the OHC, which triggers action of Caspase-3. Subsequently, AIF is translocated to the nucleus, leading to DNA damage and OHCs death.