Immediate and Delayed Cochlear Neuropathy after Noise Exposure in Pubescent Mice

Moderate acoustic overexposure in adult rodents is known to cause acute loss of synapses on sensory inner hair cells (IHCs) and delayed degeneration of the auditory nerve, despite the completely reversible temporary threshold shift (TTS) and morphologically intact hair cells. Our objective was to determine whether a cochlear synaptopathy followed by neuropathy occurs after noise exposure in pubescence, and to define neuropathic versus non-neuropathic noise levels for pubescent mice. While exposing 6 week old CBA/CaJ mice to 8-16 kHz bandpass noise for 2 hrs, we defined 97 dB sound pressure level (SPL) as the threshold for this particular type of neuropathic exposure associated with TTS, and 94 dB SPL as the highest non-neuropathic noise level associated with TTS. Exposure to 100 dB SPL caused permanent threshold shift although exposure of 16 week old mice to the same noise is reported to cause only TTS. Amplitude of wave I of the auditory brainstem response, which reflects the summed activity of the cochlear nerve, was complemented by synaptic ribbon counts in IHCs using confocal microscopy, and by stereological counts of peripheral axons and cell bodies of the cochlear nerve from 24 hours to 16 months post exposure. Mice exposed to neuropathic noise demonstrated immediate cochlear synaptopathy by 24 hours post exposure, and delayed neurodegeneration characterized by axonal retraction at 8 months, and spiral ganglion cell loss at 8-16 months post exposure. Although the damage was initially limited to the cochlear base, it progressed to also involve the cochlear apex by 8 months post exposure. Our data demonstrate a fine line between neuropathic and non-neuropathic noise levels associated with TTS in the pubescent cochlea.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

噪声短时暴露所致的耳蜗电位改变

本实验观察115dB(SPL)白噪声暴露20min对豚鼠耳蜗直流电位(EP),复合听神经动作电位(CAP),微音器电位(CM)的影响。发现此种噪声暴露确可提高源于血管纹的正EP(P-EP),说明有血管纹功能的代偿性增强;而负EP(N-EP)变化不大。AP及CM输入-输出函数的变化说明噪声首先影响外毛细胞的主动运动功能。EP与耳蜗电图的对照分析表明,血管纹功能的改变确能影响噪声性听损伤的发展。     

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.     

稳态白噪声对豚鼠耳蜗电位影响的研究

本工作利用面神经管慢性埋植电极在清醒动物上对噪声暴露前后听神经动作电位(AP)和微音器电位(CM) 进行了研究,结果表明:当改变测试声强和频率时CM应起始点会发生移位(CM-shift); 123dBSPL稳态白噪声暴露1小时后.AP各参数(幅值、潜伏期和阈值)均发生明显变化.而CM各参数(幅值、CM-shift和检测阈)变化则基本未达显著性.说明AP对产损伤的敏感性要高于CM另外发现噪声暴露后AP的N_2峰的恢复比N_1峰快、CM在恢复过程中会出现幅值加大现象.     

Hidden hearing loss is associated with loss of ribbon synapses of cochlea inner hair cells

The present study aimed to observe the changes in the cochlea ribbon synapses after repeated exposure to moderate-to-high intensity noise. Guinea pigs received 95 dB SPL white noise exposure 4 h a day for consecutive 7 days (we regarded it a medium-term and moderate-intensity noise, or MTMI noise). Animals were divided into four groups: Control, 1DPN (1-day post noise), 1WPN (1-week post noise), and 1MPN (1-month post noise). Auditory function analysis by auditory brainstem response (ABR) and compound action potential (CAP) recordings, as well as ribbon synapse morphological analyses by immunohistochemistry (Ctbp2 and PSD95 staining) were performed 1 day, 1 week, and 1 month after noise exposure. After MTMI noise exposure, the amplitudes of ABR I and III waves were suppressed. The CAP threshold was elevated, and CAP amplitude was reduced in the 1DPN group. No apparent changes in hair cell shape, arrangement, or number were observed, but the number of ribbon synapse was reduced. The 1WPN and 1MPN groups showed that part of ABR and CAP changes recovered, as well as the synapse number. The defects in cochlea auditory function and synapse changes were observed mainly in the high-frequency region. Together, repeated exposure in MTMI noise can cause hidden hearing loss (HHL), which is partially reversible after leaving the noise environment; and MTMI noise-induced HHL is associated with inner hair cell ribbon synapses.     

Structure of the stereocilia side links and morphology of auditory hair bundle in relation to noise exposure in the chinchilla

Stereocilia side links are directly involved in the maintenance of stereociliary bundle integrity in hair cells. The structure of the stereocilia side links and morphology of the auditory hair bundle in relation to noise exposure in the chinchilla was investigated by transmission electron microscopy. The outer hair cell (OHC) stereocilia side link was suggested to consist of extracellular, juxta-membrane and thin filamentous regions. Two beaded filaments were folded at their distal ends and fastened in one globule in the center between stereocilia. An intracellular, submembraneous layer appeared to form a bridge between the actin core and the extracellular, juxta-membrane region of the side link. In normal physiological conditions, most OHC stereocilia had a regular distribution of side links, forming a ‘zipper-like’ lattice between stereocilium shafts. Side links of the inner hair cell (IHC) stereocilia had a similar filamentous appearance, but were observed less commonly and had decreased structural organization compared to those of the OHC stereocilia. Ultrastructural analysis of OHC and IHC stereocilia showed that a large number of the side links could survive acoustic stimulation of 114 dB SPL for 2 hrs or 123 dB SPL for 15 min, that resulted in temporarily elevated hearing thresholds in all animals. Disarray, separation, close attachment and fusion of stereocilia were more frequently observed for IHC stereocilia and OHC stereocilia that were poorly connected or that lacked side links. Most disarrayed OHC and IHC stereocilia recovered to a normal erect state with restored orientation of the side links after 14–28 days, which correlated with near-complete recovery of auditory sensitivity. However, direct attachment of plasma membranes, ruptured links, fusion and blebs were seen on some stereocilia even after 28 days and appear to be permanent.     

TEMPORARY THRESHOLD SHIFTS AFTER NOISE EXPOSURE IN THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) MEASURED USING EVOKED AUDITORY POTENTIALS

The time course of recovery from temporary threshold shift (TTS) was measured in a bottlenose dolphin, Tursiops truncatus , using an evoked-potential procedure. The envelope-following response (EFR), which is a rhythmic train of auditory brainstem responses (ABR) to sinusoidally amplitude-modulated tones, was used as an indicator of the sound reception by the animal. Variation of the intensity of the stimulus allowed us to measure the animal's hearing via EFR thresholds. During each session, following an initial measure of threshold, the trained animal voluntary positioned itself within a hoop 1 m underwater while a 160 dB re 1 μPa noise of a 4–11 kHz bandwidth was presented for 30 min. After the noise exposure, thresholds were measured again at delays of 5, 10, 15, 25, 45, and 105 min. Measurements were made at test frequencies of 8, 11.2, 16, 22.5, and 32 kHz. The maximum TTS occurred 5 min after exposure and rapidly recovered with a rate of around 1.5 dB per doubling of time. TTS occurred at test frequencies from 8 to 16 kHz, with the maximum at 16 kHz. TTS was negligible at 22.5 kHz and absent at 32 kHz.     

Model of cochlear microphonic explores the tuning and magnitude of hair cell transduction current

The mammalian cochlea relies on the active forcing of sensory outer hair cells (OHCs) to amplify traveling wave responses along the basilar membrane. These forces are the result of electromotility, wherein current through the OHCs leads to conformational changes in the cells that provide stresses on surrounding structures. OHC transducer current can be detected via the voltage in the scala tympani (the cochlear microphonic, CM), and the CM can be used as an indicator of healthy cochlear operation. The CM represents a summation of OHC currents (the inner hair cell contribution is known to be small) and to use CM to probe the properties of OHC transduction requires a model that simulates that summation. We developed a finite element model for that purpose. The pattern of current generators (the model input) was initially based on basilar membrane displacement, with the current size based on in vitro data. The model was able to reproduce the amplitude of experimental CM results reasonably well when the input tuning was enhanced slightly (peak increased by ∼6 dB), which can be regarded as additional hair bundle tuning, and with a current/input value of 200–260 pA/nm, which is ∼4 times greater than the largest in vitro measures.     

Reduction in Noise-Induced Functional Loss of the Cochleae in Mice with Pre-Existing Cochlear Dysfunction Due to Genetic Interference of Prestin

Various cochlear pathologies, such as acoustic trauma, ototoxicity and age-related degeneration, cause hearing loss. These pre-existing hearing losses can alter cochlear responses to subsequent acoustic overstimulation. So far, the knowledge on the impacts of pre-existing hearing loss caused by genetic alteration of cochlear genes is limited. Prestin is the motor protein expressed exclusively in outer hair cells in the mammalian cochlea. This motor protein contributes to outer hair cell motility. At present, it is not clear how the interference of prestin function affects cochlear responses to acoustic overstimulation. To address this question, a genetic model of prestin dysfunction in mice was created by inserting an internal ribosome entry site (IRES)-CreER^T2-FRT-Neo-FRT cassette into the prestin locus after the stop codon. Homozygous mice exhibit a threshold elevation of auditory brainstem responses with large individual variation. These mice also display a threshold elevation and a shift of the input/output function of the distortion product otoacoustic emission, suggesting a reduction in outer hair cell function. The disruption of prestin function reduces the threshold shifts caused by exposure to a loud noise at 120 dB (sound pressure level) for 1 h. This reduction is positively correlated with the level of pre-noise cochlear dysfunction and is accompanied by a reduced change in Cdh1 expression, suggesting a reduction in molecular responses to the acoustic overstimulation. Together, these results suggest that prestin interference reduces cochlear stress responses to acoustic overstimulation.     

Noise‐induced cochlear F‐actin depolymerization is mediated via ROCK2/p‐ERM signaling

Our previous work has suggested that traumatic noise activates Rho‐GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise‐induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho‐associated kinases (ROCKs), and the targets of ROCKs, the ezrin‐radixin‐moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise‐induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G‐actin ratio. The levels of cochlear ROCK2 and p‐ERM decreased 1 h after either TTS‐ or PTS‐noise exposure. In contrast, ROCK2 and p‐ERM in OHCs decreased only after PTS‐, not after TTS‐noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G‐actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down‐regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p‐ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2‐mediated ERM‐phosphorylation signaling cascade modulates noise‐induced hair cell loss and NIHL by targeting the cytoskeleton.

     

白噪声对听觉脑干电反应(ABR)和耳蜗电图(ECochG)的影响

本文通过20例听力正常人和10例听力正常豚鼠研究了白噪声对耳蜗电图(ECochG)和听觉脑干电反应(ABR)的干涉作用。实验结果表明,白噪声比短声(信号)的声强级低30dB(SL)以上时,ECochG和ABR的振幅仅轻微减小。白噪声与短声的声强级相等时,ECochG与ABR的振幅和出现率会明显受到干涉而减小,甚至完全消失。但是,此时的耳蜗微音器电位(CM)并未观察到有明显的变化。这意味着白噪声对ECochG和ABR的干涉作用主要与围绕毛细胞基底部的突触产生的抑制密切相关。由于白噪声对ABR各波的干涉有些差异,所以认为这种抑制,可能既包括脑中抑制也包括侧方抑制。     

变压器噪声暴露对SD大鼠听力及应激状态的影响研究

目的:探究短时间内低声级强度低频的变压器噪声暴露对SD大鼠听力及应激状态方面的影响。方法:选取90只SPF级健康无听力障碍的(雌雄各半)SD大鼠作为实验对象,随机分为实验A、B组和对照C组,A、B组分别给予声级上限为65 dB SPL、60 dB SPL(频谱范围:100~800 Hz)的变压器噪声,噪声暴露时程为8周,每日噪声给予时间为22点至次日8点,C组在相同条件下饲养,不给予噪声暴露。噪声暴露结束后,通过DPOAE(畸变耳声发射)、ABR(听性脑干反应)检测、耳蜗铺片及毛细胞计数对SD大鼠听力学状况进行评估;通过血清中促肾上腺皮质激素(ACTH)、血清皮质醇(CORT)对SD大鼠的应激状态进行评估。结果:在变压器噪声暴露的8周内,各组大鼠生长状况良好,体重均呈正常生理性增长,组间无明显差异(P0.05);在变压器噪声暴露8周后,对A、B、C三组大鼠的听力学指标进行两两比较,组间均无明显差异(P0.05),对大鼠血清中促肾上腺皮质激素(ACTH)、血清皮质醇(CORT)的含量进行三组间比较,组间差异均无统计学意义(P0.05)。结论:连续暴露于声压级上限65/60 dB SPL,频谱范围为100~800 Hz的变压器噪声下8周(10小时/天)对SD大鼠听力未产生明显影响,未引发SD大鼠应激状态。     

催产素对内耳外毛细胞功能的调控作用

采用耳蜗外淋巴液灌流催产素(OXT),记录由鼓阶电极引导的听神经复合动作电位(CAP)及耳蜗微音电位(CM)的输入—输出(I/O)函数。发现OXT可在90dB(SPL)以下各声强提高短纯音诱发的CM振幅以及短声诱发的CAP振幅,而当声强高于90dB时CM变化不明显。但在用含氯化筒箭毒(dTC)的外淋巴液灌流以阻断橄榄耳蜗束胆碱能传出控制后,OXT不再引起CM改变,而对CAP的作用在低声强段(<60dB)依然存在。这些结果提示OXT可能调节传出神经对内耳的控制,并可能对外毛细胞(OHC)的运动能力有直接影响。     

Reg3b在大鼠耳蜗的分布及噪声刺激后的表达变化

目的：探讨Reg3b在大鼠耳蜗中的分布情况及在噪声刺激前后的表达变化,为治疗噪声性聋提供新思路。方法：30只健康成年SD大鼠,分为噪声暴露组和正常对照组,利用110dBSPL宽频稳态白噪声对噪声组进行噪声暴露,通过免疫组织荧光技术,观察Reg3b在正常及噪声刺激后成年sD大鼠耳蜗内的分布情况。采用实时定量PCR技术（Realtime-PCR）方法检测大鼠接受噪声刺激前后Reg3b在耳蜗内的表达变化。结果：免疫组织荧光技术提示,Reg3b在噪声暴露后主要表达于大鼠耳蜗的内毛细胞、外毛细胞,以及螺旋神经节处,而正常大鼠耳蜗中Reg3b表达不明显或呈阴性表达。与噪声刺激前相比,噪声刺激后,Reg3b在mRNA水平表达较噪声前明显提高。结论：Reg3b在耳蜗内的分布及在噪声刺激后的表达显著升高提示其在噪声诱导的细胞死亡及对抗噪声损伤方面具有一定作用,可能成为治疗感音神经性聋的新靶点。     

Shaping the mammalian auditory sensory organ by the planar cell polarity pathway

The human ear is capable of processing sound with a remarkable resolution over a wide range of intensity and frequency. This ability depends largely on the extraordinary feats of the hearing organ, the organ of Corti and its sensory hair cells. The organ of Corti consists of precisely patterned rows of sensory hair cells and supporting cells along the length of the snail-shaped cochlear duct. On the apical surface of each hair cell, several rows of actin-containing protrusions, known as stereocilia, form a "V"-shaped staircase. The vertices of all the "V"-shaped stereocilia point away from the center of the cochlea. The uniform orientation of stereocilia in the organ of Corti manifests a distinctive form of polarity known as planar cell polarity (PCP). Functionally, the direction of stereociliary bundle deflection controls the mechanical channels located in the stereocilia for auditory transduction. In addition, hair cells are tonotopically organized along the length of the cochlea. Thus, the uniform orientation of stereociliary bundles along the length of the cochlea is critical for effective mechanotransduction and for frequency selection. Here we summarize the morphological and molecular events that bestow the structural characteristics of the mammalian hearing organ, the growth of the snail-shaped cochlear duct and the establishment of PCP in the organ of Corti. The PCP of the sensory organs in the vestibule of the inner ear will also be described briefly.     

Conservation of endocochlear potential in mice with profound hearing loss induced by co-administration of kanamycin and furosemide

Research in mammalian hair cell regeneration is hampered by a lack of in vivo model of adult mouse inner ear injury. In the present study we investigated the effects of a combination of a single dose of aminoglycoside followed by a loop diuretic in adult mice. The auditory brainstem response threshold shift, extent and defining characteristics of the cochlear lesion were assessed and verified at different time points post-treatment. Our data indicated that this drug combination caused the rapid and extensive death of outer hair cells (OHCs). OHC death presented throughout the cochlea that commenced in the basal turn by 24 h and progressed apically. In contrast, inner hair cell (IHC) loss was delayed and mild. Terminal deoxynucleotidyl transferase dUTP nick end labelling-positive nuclei demonstrated that the majority of OHCs died via an apoptotic pathway. Auditory threshold shifts of up to 90 dB SPL indicated a profound hearing loss. In addition, the endocochlear potential (EP) in the drug-treated animals displayed a significant decline at 12 h post-treatment followed by recovery by 48 h post-treatment. Despite this recovery, there was a significant and progressive decrease in strial vascularis thickness, which was predominantly due to atrophy of marginal cells. The present study reproduced an adult mouse model of aminoglycoside-induced hearing loss. The mechanism underlying the recovered EP in the model with extensive hair cell death is discussed.     

强噪声暴露后听觉脑干电反应及皮层电反应的变化

为了观察噪声作用豚鼠后,在暂时性或移和永久性阈移期间,听觉末梢和听觉各级中枢电反庆的变化规律,我们进行了６２ｄ的测定。听神经电反应振幅减小２９％（Ｐ＜０．０５）,耳蜗核电反应振幅减小２８％（Ｐ＜０．０５）,上橄榄核电反应反而增加２１％（Ｐ＜０．０５）,中脑下丘的电反应振幅进而增大３７％（Ｐ＜０．０５）,听觉皮层电反应振幅却意外地增加１３１％（Ｐ＜０．００１）。这说明强噪声（１２５ｄＢ １５０ｍｉｎ     

Adenosine amine congener mitigates noise-induced cochlear injury

Hearing loss from noise exposure is a leading occupational disease, with up to 5% of the population at risk world-wide. Here, we present a novel purine-based pharmacological intervention that can ameliorate noise-induced cochlear injury. Wistar rats were exposed to narrow-band noise (8–12 kHz, 110 dB SPL, 2–24 h) to induce cochlear damage and permanent hearing loss. The selective adenosine A₁ receptor agonist, adenosine amine congener (ADAC), was administered intraperitoneally (100 μg/kg/day) at time intervals after noise exposure. Hearing thresholds were assessed using auditory brainstem responses and the hair cell loss was evaluated by quantitative histology. Free radical damage in the organ of Corti was assessed using nitrotyrosine immunohistochemistry. The treatment with ADAC after noise exposure led to a significantly greater recovery of hearing thresholds compared with controls. These results were upheld by increased survival of sensory hair cells and reduced nitrotyrosine immunoreactivity in ADAC-treated cochlea. We propose that ADAC could be a valuable treatment for noise-induced cochlear injury in instances of both acute and extended noise exposures.     

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.