Pathways towards regenerating the mammalian cochlea

描述（申请人提供）：耳蜗变性是感音神经性听力损失（SNHL）的主要原因，缺乏自发再生导致SNHL的不可逆性。先前的研究已经检查了毛细胞在退化耳蜗中的强制分化的效用，但尚不清楚通过细胞增殖恢复细胞数量是否也有助于耳蜗再生。最近的研究表明，通过遗传或药理学操作激活典型Wnt信号可诱导新生儿耳蜗的增殖。此外，在耳蜗支持细胞和基底膜下的鼓室边界细胞中也观察到响应Wnt信号的增殖能力。本研究的目的是研究补充Wnt信号是否能刺激新生儿和成熟耳蜗毛细胞变性后的增殖和/或再生。我们将在体外和体内应用氨基糖苷或通过转基因策略启动变性，同时在体外和体内绘制支持细胞和鼓室边界细胞的命运图。在多个转基因小鼠品系的平行实验中，我们将研究针对感觉毛细胞或支持细胞亚型的消融。通过在转基因小鼠中使用Cre-Lox系统或局部应用Wnt激动剂实现Wnt激活，并检查它们是否会通过支持细胞和鼓室边界细胞启动增殖和再生。对全动物耳蜗的损伤程度和可能的恢复情况进行组织学检查，并与治疗前后的听觉生理相关。为了获得对受损耳蜗遗传特征的公正见解，通过流式细胞术从未受损和受损耳蜗中分离支持细胞和鼓室边缘细胞并进行基因阵列分析。所有转基因小鼠品系、药物制剂、在体外和体内操作和检查耳蜗的专业知识，以及分离和富集耳蜗细胞的技术都近在咫尺。总之，我们的研究将确定1)补充Wnt是否有助于启动耳蜗再生，2)在新生儿和成熟耳蜗中增强这种再生过程的其他靶点。

DESCRIPTION (provided by applicant): Cochlear degeneration is a major cause of sensorineural hearing loss (SNHL), and the lack of spontaneous regeneration contributes to the irreversible nature of SNHL. Prior studies have examined the utility of forced differentiation of hair cells in the degenerating cochlea, yet it remains unclear whether a restoration in cell number via cell proliferation can also aid cochlear regeneration. Recent work demonstrates that activation of canonical Wnt signaling via genetic or pharmacologic manipulation induces proliferation in the neonatal cochlea. Also, the competence to proliferate in response to Wnt signals is observed in both cochlear supporting cells and tympanic border cells below the basilar membrane. The goal of this proposal is to investigate whether supplementation of Wnt signals can stimulate proliferation and/or regeneration after hair cell degeneration in both the neonatal and mature cochleae. We will initiate degeneration with aminoglycoside application or via a transgenic strategy and concurrently fate-map supporting cells and tympanic border cells in vitro and in vivo. In parallel experiments using multiple transgenic mouse strains, we will study ablation targeting sensory hair cells or supporting cell subtypes. Wnt activation is achieved by using the Cre-Lox system in transgenic mice or local application of Wnt agonists and whether they will initiate proliferation of and regeneration by supporting cells and tympanic border cells are examined. The degree of damage and possible recovery in the cochlea are examined histologically and correlated with pre- and post-treatment auditory physiology in the whole animal. To gain an unbiased insight into the genetic signature of the damaged cochlea, supporting cells and tympanic border cells from undamaged and damaged cochlea are isolated via flow cytometry and subjected to gene array analyses. All transgenic mouse strains, pharmacologic agents, expertise to manipulate and examine the cochlea in vitro and in vivo, and techniques to isolate and enrich cochlear cells are at hand. Together, our research will determine 1) whether Wnt supplementation can help initiate cochlear regeneration and 2) additional targets to enhance this regenerative process in both the neonatal and mature cochleae.