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.     

Evaluation of inner hair cell and nerve fiber loss as sufficient pathologies underlying auditory neuropathy

Auditory neuropathy is a hearing disorder characterized by normal function of outer hair cells, evidenced by intact cochlear microphonic (CM) potentials and otoacoustic emissions (OAEs), with absent or severely dys-synchronized auditory brainstem responses (ABRs). To determine if selective lesions of inner hair cells (IHCs) and auditory nerve fibers (ANFs) can account for these primary clinical features of auditory neuropathy, we measured physiological responses from chinchillas with large lesions of ANFs (about 85%) and IHCs (45% loss in the apical half of the cochlea; 73% in the basal half). Distortion product OAEs and CM potentials were significantly enhanced, whereas summating potentials and compound action potentials (CAPs) were significantly reduced. CAP threshold was elevated by 7.5 dB, but response synchrony was well preserved down to threshold levels of stimulation. Similarly, ABR threshold was elevated by 5.6 dB, but all waves were present and well synchronized down to threshold levels in all animals. Thus, large lesions of IHCs and ANFs reduced response amplitudes but did not abolish or severely dys-synchronize CAPs or ABRs. Pathologies other than or in addition to ANF and IHC loss are likely to account for the evoked potential dys-synchrony that is a clinical hallmark of auditory neuropathy in humans.     

Hearing with selective inner hair cell loss

In a comparative model study, the effect of selective inner hair cell loss on hearing threshold was investigated. Functional testing was related to a detailed morphological evaluation. Inner ears of homozygous Bronx waltzer mice (bv/bv) were investigated using light microscopy with Nomarski interference contrast optics and transmission electron microscopy. Hearing thresholds were determined using click evoked auditory brainstem responses at different ages. The histological evaluation was performed immediately after determination of the hearing threshold. The functional and histological findings were compared to the normal controls of the same age.

Scattered loss of up to 70% of inner hair cells was found to cause only moderate elevation of hearing threshold.     

Noise-induced hearing loss in normotensive and spontaneously hypertensive rats

Hearing loss was investigated in normotensive (N) and spontaneously hypertensive (SH) rats after prolonged exposure to a simulated industrial noise environment. Exposure was initiated in a group of young rats (3 months old) and continued up to 18 months of age, and in a group of old rats (15 months old) and followed up to 18 months of age. Hearing thresholds were determined behaviorally with a conditioned suppression technique before and after 1, 2, 3 and, for some groups, also after 15 months of exposure. A frequency-modulated noise band sweeping from 3 to 30 kHz at a frequency of 0.5 Hz was presented 10 hours daily at a level of 100 dB L_eq (lin). The results showed that young N and SH rats not exposed to noise had identical hearing thresholds. Noise exposure induced a significantly greater hearing loss in SH rats than in N rats; SH animals were more susceptible to noise than were young ones; no difference was seen between males and females. The histology of the inner ears of the rats was examined by light microscopy after the end of the experiment. The young noise-exposed N rats showed no abnormal loss of hair cells in spite of the fact that they sustained hearing losses of 30–40 dB. The SH rats showed a significantly greater loss of hair cells than did the N rats. The stereocilia were found to be fused on a large number of inner hair cells in the basal turns of both N and SH animals. It was concluded that SH and N rats constitute an interesting model for the investigation of biological mechanisms behind individual differences in susceptibility to noise-induced hearing loss.     

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。     

Auditory brainstem response thresholds in a mouse mutant with selective outer hair cell loss

Summary Mutant animals with a particular type of cochlear pathology are an excellent model for studying the functional role of various cells of the cochlea. In homozygous W^V/W^V mutant mice we found a selective loss of outer hair cells as a constant defect with no progressive degeneration of the organ of Corti. The mice were followed throughout their lives and exhibited auditory brainstem responses that were elevated to about 50 dB SPL as compared to normal control animals. Sequential temporal bone studies showed that there was a selective loss of outer hair cells throughout the entire cochlea as seen in surface preparations. The inner hair cells were present in normal numbers and appeared to be essentially normal.Presented at the 25th Workshop on Inner Ear Biology in London, England, 4–7 September 1988     

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。     

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。     

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.     

Age-related functional and histopathological changes of the ear in the MPS I mouse

OBJECTIVE: Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by a mutation in the gene encoding the enzyme alpha-L-iduronidase. This enzyme is responsible for degradation of dermatan and heparan sulfates. Enzyme deficiency results in their accumulation in lysosomes of virtually all organs, resulting in severe somatic and neurological changes. Clinical findings of otitis media with mixed hearing loss are common. Cellular and molecular mechanisms of ear pathology and hearing loss are not understood. The purpose of this study is to describe the age-related audiologic and histopathologic changes of the ear in the mouse model of MPS I. METHODS: Auditory brainstem responses (ABR) were obtained to clicks and tone bursts at 1-32kHz, and pathological changes to middle and inner ears were studied with light and electron microscopy in 53 mice that included: (1) wild type (+/+)-five at 2 months, five at 4-6 months, and five at 13-19 months; (2) heterozygotes (+/-)-four at 2 months, five at 4-6 months, and eight at 13-19 months; and (3) homozygotes (-/-)-five at 2 months, six at 4-6 months, and five at 13-19 months. Histopathology was also done on five newborn -/- mice. RESULTS: In newborns, no lysosomal storage was observed and the ear appeared age appropriately normal. In all other -/- mice, cells with lysosomal storage vacuoles were observed in spiral ligament, spiral prominence, spiral limbus, basilar membrane, epithelial and mesothelial cells of Reissner's membrane, endothelial cells of vessels, and some ganglion cells; their number increased with aging. Hair cell loss was not observed at 2 or 6 months, but there was total loss of the organ of Corti in year-old mice. Hearing of -/- mice was significantly decreased at all ages compared to +/+ and +/-. Hearing loss progressed from mild to moderate loss at 2 months to profound at 6 months and total deafness by 1 year of age. CONCLUSIONS: Progressive age-related changes suggest early therapeutic intervention to prevent sensory cell damage and hearing loss.     

补肾药对老年大鼠听力影响的实验研究

目的观察右归丸对老年大鼠听力自然衰退的影响.方法30只18月龄Wistar大鼠随机分为两组,对照组按10ml·kg-1·d-1的剂量予生理盐水灌胃;右归丸组按3.3g·kg-1·d-1的剂量予右归丸灌胃,连续6个月后,观察听觉脑干反应及内耳毛细胞损失情况.结果右归丸组ABR反应阈较对照组低(P＜0.05);右归丸组耳蜗底回近钩端部位外毛细胞损失较对照组少(P＜0.05).结论右归丸对老年性聋的发生具有延缓作用.     

卡那霉素和速尿联合用药后的毛细胞死亡*

目的观察卡那霉素和速尿联合用药后豚鼠耳蜗毛细胞的死亡时程和方式.方法选用健康成年白色红目豚鼠,雌雄不限,随机分成健康对照组和药物致聋后6 h、9 h组(实验组),每组5只.实验组在选取的时间点完成听性脑干反应(ABR)检测后行耳蜗基底膜铺片、PI荧光染色,共聚焦显微镜下观察毛细胞.对照组不做处理.结果实验组半数豚鼠致聋(ABR阈值>95 dB SPL),致聋豚鼠耳蜗可见外毛细胞核固缩和核肿胀,与给药6 h组相比,给药9 h组外毛细胞核肿胀数目增多.检测给药6 h组和9 h组豚鼠末端脱氧核苷酸转移酶介导的dUTP缺口末端标记物(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL),没有观察到阳性着色细胞.正常对照组所有豚鼠ABR正常,其耳蜗各回毛细胞没有出现毛细胞核固缩或者核肿胀.结论卡那霉素和速尿联合用药后数小时即可导致豚鼠耳蜗毛细胞死亡,毛细胞损害为两种类型,即坏死和凋亡.     

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。     

Relation between outer hair cell loss and hearing loss in rats exposed to styrene

The relationship between outer hair cell (OHC) loss and cochlear sensitivity is still unclear, because in many animal models there exist surviving but dysfunctional OHCs and also injured/dead inner hair cells (IHC). Styrene is an ototoxic agent, which targets and destroys OHCs starting from the third row to the second and first rows depending on the exposure level. The remaining cells may be less affected. In this experiment, rats were exposed to styrene by gavage at different doses (200-800mg/kg/day) for varying periods (5 days/week for 3-12 weeks). An interesting finding was that the cochlear sensitivity was not affected in a few rats with all OHCs in the third row being destroyed by styrene. A further loss of OHCs was usually accompanied with a linear input/output (I/O) function of cochlear compound action potentials (CAP), indicating the loss of cochlear amplification. However, normal CAP amplitudes at the highest stimulation level of 90dB SPL were often observed when all OHCs were destroyed, indicating normal function of the remaining IHCs. The OHC-loss/hearing-loss relation appeared to be a sigmoid-type function. Initially, styrene-induced OHC losses (<33%) did not result in a significant threshold shift. Then CAP threshold shift increased dramatically with OHC loss from 33% to 66%. Then, CAP threshold changed less with OHC loss. The data suggest a tri-modal relationship between OHC loss and cochlear amplification. That is, under the condition that all surviving OHCs are ideally functioning, the cochlear amplifier is not affected until 33% of OHCs are absent, then the gain of the amplifier decreases proportionally with the OHC loss, and at last the amplifier may fail completely when more than 67% of OHCs are lost.     

低频强声对巴马香猪听功能及耳蜗毛细胞表面结构的影响

目的 了解高强度低频率噪声(低频强声)对巴马香猪听功能及耳蜗毛细胞表面结构的影响.方法 将8只巴马香猪随机分为对照组(2只)及实验组(6只),均于实验前行听性脑干反应(auditory brainstem response,ABR)检测后,将实验组再分为噪声暴露后即刻、36小时及84小时组,每组2只;实验各组动物暴露于50Hz、142 dB SPL的低频噪声中5 min,再于暴露后即刻、36小时、84小时分别行ABR检测,然后分别在扫描电镜下观察各组动物耳蜗毛细胞表面结构的形态变化.对照组不给予噪声暴露,其他步骤同实验组.结果 8只(16耳)巴马香猪低频强声暴露前ABR反应阈值为91.25±10.72 dB SPL;实验组低频强声暴露后即刻、36 h及84 h所有动物双耳ABR均未能引出.实验组噪声暴露后即刻扫描电镜下可见内外毛细胞气球样变,外毛细胞静纤毛融合及散在性缺失;噪声暴露后36 h可见内外毛细胞轻度气球样变、静纤毛散乱;噪声暴露后84 h可见耳蜗底回内外毛细胞缺失,中回及顶回内外毛细胞静纤毛缺失,且以第三排外毛细胞静纤毛缺失最重;噪声暴露后听毛细胞表面结构损伤以基底回和中回为主,顶回损伤较轻.结论 50 Hz、142 dB SPL噪声暴露后巴马香猪双耳ABR反应阈值较暴露前明显升高;扫描电镜下可见内、外毛细胞静纤毛融合、散乱及缺失等改变.     

C57BL/6小鼠内耳形态学和年龄相关性听力损失的研究

目的探讨不同周龄C57BL/6小鼠内耳形态学及其ABR阈值变化。方法取C57BL/6小鼠3周、4周、12周、26周各10只,听性脑干反应(ABR)测试双侧2、4、8、16、20 kHz ABR阈值。采用基底膜铺片MyosinⅥ、Neurofilament免疫组化染色,观察耳蜗毛细胞和神经丝的变化。扫描电镜观察耳蜗毛细胞及其静纤毛随年龄的变化。结果随着年龄增长,C57BL/6小鼠各频率ABR阈值明显提高,顶转和底转内毛细胞缺失逐渐增多,神经丝染色渐淡,毛细胞静纤毛逐渐发生数量减少、增粗融合、倒伏等变化。到26周龄时已达到重度聋,各频率较3周组有显著统计学差异。顶转和底转内毛细胞有连续缺失,外毛细胞完全缺失,内毛细胞只有残存的少量静纤毛,粗细不均,倒伏明显。结论本研究对国产C57BL/6小鼠的内耳形态进行观察,明确了其ABR阈值和内耳毛细胞的变化规律,为用国产C57BL/6小鼠进行老年性聋研究提供了依据。     

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。     

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.     

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