Basic and Clinical Studies of Noise-Induced and Age-Related Hearing Loss

描述（由申请人提供）：声学过度暴露是一个日益严重的问题，了解其长期后果对公众健康至关重要。我们最近在噪音和衰老的小鼠模型中进行的研究表明，适度的噪音暴露，最初似乎是可逆的，不会导致急性或慢性毛细胞损失，但在暴露数月后，会引起螺旋神经节细胞（SGCs）的缓慢损失。共聚焦免疫组织化学表明，许多SGC外周末梢及其内毛细胞上的突触在最初几天或几小时内消失，与声过度暴露的急性兴奋毒性作用一致。我们假设这种急性树突回缩破坏了耳蜗上皮中毛细胞、支持细胞和神经元之间正常的神经营养因子信号传导，并且这种中断启动了SGCs中缓慢的细胞死亡级联。提出的目的通过描述神经元变性和相关病理生理的性质和时间过程（目的1），通过操纵急性兴奋毒性（目的2）或神经营养因子表达（目的3）和评估对耳蜗神经变性的影响来验证这一假设。神经病变的量化（Aim 1a）将跟踪暴露后时间内从突触到外周轴突到细胞体的变性。在群体反应和单纤维水平上与病理生理学的相关性（Aim 1b）将验证我们是否已经确定了功能上重要的结构变化，并将测试噪音导致低自发率神经元优先丧失的假设。为了测试急性兴奋性毒性是否是神经病变的关键上游诱发因子，我们利用小鼠耳蜗灌注技术，1)用谷氨酸拮抗剂DNQX （Aim 2a）阻断噪声诱导的兴奋性毒性，2)用谷氨酸激动剂AMPA （Aim 2b）模拟它，或3)用靶向缺失谷氨酸转运体GLAST （Aim 2c）的小鼠增强它。为了测试神经营养因子在细胞死亡的缓慢级联过程中的作用，我们将检测（通过qRT-PCR、免疫组织化学和nt3报告小鼠）神经营养因子信号通路中关键分子的基因表达水平作为暴露后时间的函数（Aim 3a），并尝试在毛细胞或支持细胞中诱导神经营养因子过表达的小鼠系（Aim 3b）进行拯救实验（减少/防止神经元损失；促进完整ihc的再神经化）。了解噪声暴露小鼠慢发性神经变性的性质、病因和可能的预防对人类听力有重要影响。这表明原发性神经元损失是获得性感音神经性听力损失的一个更常见和重要的方面。这也引起了人们对明显良性声学过度暴露可能产生的长期后果的重要关注：缓慢发作的噪声引起的神经退行性变现象可能在很大程度上导致老年人的主要听力相关疾病，即在嘈杂环境中理解语言的问题。我们最近在老鼠身上的研究表明，噪音暴露，即使是那些看起来会导致完全可逆的阈值变化的噪音，实际上也会启动一个缓慢的细胞死亡级联，导致耳蜗大区域中大约一半的神经元件最终丢失。如果普遍适用于哺乳动物的耳朵（有充分的理由相信这将是事实），缓慢发作的、噪声引起的原发性耳蜗神经丧失现象可能是一个非常普遍的问题，具有重大的公共卫生影响。我们提出的实验提供了一个强大的平台来研究这一现象并探索其机制，使用与人类状况高度相关的耳蜗损伤（即噪音）。

DESCRIPTION (provided by applicant): Acoustic overexposure is a growing problem, and understanding the long-term consequences is critical to public health. Our recent work in a mouse model of noise and aging shows that moderate exposures, which initially appear reversible and cause no acute or chronic hair cell loss, elicit a slow-onset loss of spiral ganglion cells (SGCs) when followed for months post-exposure. Confocal immunohistochemistry suggests that many SGC peripheral terminals, and their synapses on inner hair cells, disappear within the first days or hours, consistent with acute excitotoxic effects of acoustic overexposure. We hypothesize that this acute dendritic retraction disrupts normal neurotrophin signaling among hair cells, supporting cells and neurons in the cochlear epithelium, and that this interruption initiates the slow cell-death cascade in SGCs. The proposed Aims test this hypothesis by characterizing the nature and time course of neuronal degeneration and associated pathophysiology (Aim 1), and by manipulating acute excitotoxicity (Aim 2) or neurotrophin expression (Aim 3) and assessing the effects on cochlear neurodegeneration. Quantification of neuropathy (Aim 1a), will track degeneration over post-exposure time as it progresses from synapse, to peripheral axon, to cell body. Correlations with pathophysiology at population-response and single-fiber levels (Aim 1b) will verify if we have identified the functionally important structural changes, and will test the hypothesis that noise causes a preferential loss of neurons with low spontaneous rates. To test if acute excitotoxicity is the key upstream elicitor of the neuropathy, we exploit our techniques for cochlear perfusion in mouse to either 1) block noise-induced excitotoxicity with the glutamate antagonist DNQX (Aim 2a), 2) mimic it with the glutamate agonist AMPA (Aim 2b) or 3) enhance it using mice with targeted deletion of the glutamate transporter GLAST (Aim 2c). To test the role of neurotrophins in the slow cascade of cell death, we will assay (via qRT-PCR, immunohistochemistry and a NT3-reporter mouse) gene expression levels of key molecules in the neurotrophin signaling pathway as a function of post-exposure time (Aim 3a), and attempt a rescue experiment (reduce/prevent loss of neurons; promote re-innervation of intact IHCs) using mouse lines with inducible neurotrophin overexpression in either hair cells or supporting cells (Aim 3b). Understanding the nature, etiology and possible prevention of slow-onset neurodegeneration in our noise- exposed mice has important ramifications for human hearing. It suggests that primary neuronal loss is a more common and important aspect of acquired sensorineural hearing loss than previously thought. It also raises important concerns re possible long-term consequences of apparently benign acoustic overexposures: the phenomenon of slow-onset noise-induced neurodegeneration may contribute in a major way to the main hearing-related complaint in aging humans, i.e. problems understanding speech in a noisy environment. PUBLIC HEALTH RELEVANCE Our recent work in mouse shows that noise-exposures, even those that appear to result in fully reversible threshold shifts, actually set in motion a slow cell death cascade leading to the ultimate loss of roughly half of the neural elements throughout large regions of the cochlea. If generally applicable to the mammalian ear, as there is every reason to believe it will be, the phenomenon of slow-onset, noise-induced, primary, cochlear-nerve loss is potentially a very common problem with significant public health implications. Our proposed experiments provide a powerful platform to study the phenomenon and to probe its mechanisms, using a cochlear insult (i.e. noise) that is highly relevant to the human condition.