Trophic Interactions in the Developing and Adult Inner Ear

描述(申请人提供)：最近对噪音导致和年龄相关的听力损失的研究表明，耳蜗神经末梢和内毛细胞(IHC)之间的突触丢失，而不是毛细胞损伤，通常是第一个退行性事件。噪声暴露只会引起暂时性的阈值移动，毛细胞不会丢失，但会导致IHC突触的快速和永久性丧失，随后是螺旋神经节神经元(SGN)的缓慢死亡。对于老化的耳朵，突触的重要性也出现了类似的观点。即使没有刻意的噪音暴露，小鼠体内IHC突触的丢失在整个生命过程中也会稳步进行，早在毛细胞和SGN丢失之前，对人类耳朵的尸检研究也出现了类似的发现。因此，了解IHC-SGN突触形成、维持和再生的机制，对于了解获得性听力损失的细胞和分子基础，从而开发合理的治疗方法是关键。在几个新的转基因小鼠品系中，我们利用细胞特异的、可诱导的基因重组，证明了来自耳蜗支持细胞的神经营养素3(NT-3)是新生儿IHC突触形成和维持的关键调节因子，而新生儿NT-3在支持细胞中的过度表达促进了成年鼠在声过度暴露后突触和功能的恢复。在这里，我们将检验如下假设：1)NT-3对调控至关重要 2)年龄相关或噪声诱导的耳蜗神经病变可以通过上调或下调NT-3来调节。目的1研究NT-3基因在成年耳蜗内诱导支持细胞NT-3过度表达或缺失，以及在短(Wks)和长(月)存活时间内对耳蜗结构和功能的影响，以确定NT-3在成年和衰老耳部IHC突触调节中的作用。目的2利用遗传学和药理学方法验证耳蜗神经NT-3水平的增加或减少可以影响噪声所致耳蜗神经病的严重程度或恢复的假说。我们将在神经性噪声暴露前1周或暴露后1天通过遗传手段在支持细胞中过度或低表达NT-3，并通过ABR和DPOAE以不同的间隔监测耳蜗功能，最长可达6个月。对于药理学方法，NT-3将通过在缓释凝胶中的圆形窗口应用来传递。对于这两个目标，我们将通过ABR和DPOAEs监测耳蜗功能的变化，并收集耳蜗组织用于NT-3表达的RT-PCR分析和/或毛细胞和SGN计数的组织学分析以及IHC突触数量和形态。

DESCRIPTION (provided by applicant): Recent studies of noise-induced and age-related hearing loss show that loss of synapses between cochlear nerve terminals and inner hair cells (IHCs), rather than hair cell damage, is often the first degenerative event. Noise exposures causing only temporary threshold shifts, and no loss of hair cells, nevertheless cause rapid and permanent loss of IHC synapses, followed slowly by death of spiral ganglion neurons (SGNs). A similar view of the importance of synaptopathy has emerged with respect to the aging ear. Even without purposeful noise exposure, loss of IHC synapses in mice progresses steadily throughout life, long before the loss of hair cells and SGNs, and similar findings are emerging from post-mortem studies of human ears. Thus, understanding the mechanisms that underlie the formation, maintenance, and regeneration of the IHC-SGN synapse are key to understanding the cellular and molecular basis of acquired hearing loss and thus in the development of rational therapies. Using cell-specific, inducible gene recombination in several novel mouse transgenic lines, we showed that neurotrophin 3 (NT-3) derived from cochlear supporting cells is a key regulator of IHC synapse formation and maintenance in the neonate, and that neonatal NT-3 overexpression in supporting cells enhances synaptic and functional recovery after acoustic overexposure in the adult. Here we will test the hypotheses that 1) NT-3 is critical for regulation of IHC synapses in the adult, and 2) that age- related or noise-induced cochlear neuropathy can be modulated by up- or down-regulating NT-3. Aim 1 will determine the roles of NT-3 in regulating IHC synapses in the adult and aging ear by genetically inducing supporting-cell NT-3 overexpression or deletion in the adult cochlea and assessing the effects on cochlear structure and function over short (wks) and long (months) survival. Aim 2 will test the hypothesis that increases or decreases in cochlear NT-3 can influence the severity of, or recovery from, noise-induced cochlear neuropathy using genetic and pharmacological approaches. We will over- or under-express NT-3 in supporting cells by genetic means either 1 wk before or 1 day after a neuropathic noise exposure and monitor cochlear function via ABRs and DPOAEs at different intervals, out to 6 months. For the pharmacological approach, NT-3 will be delivered via round window application in a slow-release gel. For both aims, we will monitor changes in cochlear function via ABRs and DPOAEs, and cochleas will be collected either for RT-PCR analysis of NT-3 expression and/or for histological analysis of hair cell and SGN counts as well as IHC synapse number and morphology.