Pathways towards regenerating the mammalian cochlea

描述(申请人提供)：耳蜗性变性是感觉神经性听力损失(SNHL)的主要原因，缺乏自发再生是SNHL不可逆转的本质。以前的研究已经检验了毛细胞强制分化在退化耳蜗中的作用，但尚不清楚通过细胞增殖恢复细胞数量是否也能帮助耳蜗再生。最近的工作表明，通过遗传或药物操作激活规范的Wnt信号可以诱导新生儿耳蜗组织的增殖。此外，在耳蜗基底膜下的支持细胞和鼓膜边缘细胞中也观察到了对Wnt信号反应的增殖能力。这项建议的目的是研究补充Wnt信号是否可以刺激新生和成熟耳蜗毛细胞退化后的增殖和/或再生。我们将通过应用氨基糖苷类药物或通过转基因策略启动退变，并在体外和体内同时进行支持细胞和鼓膜边缘细胞的命运图。在使用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激动剂来实现WNT的激活，并检测它们是否会通过支持细胞和鼓膜边缘细胞启动增殖和再生。组织学检查耳蜗的损伤程度和可能的恢复，并与整个动物治疗前和治疗后的听觉生理学相关联。为了无偏见地了解受损耳蜗的遗传特征，通过流式细胞仪分离了正常和受损耳蜗组织的支持细胞和鼓膜边缘细胞，并进行了基因芯片分析。所有转基因小鼠品系、药理学试剂、在体外和体内操纵和检查耳蜗组织的专业技术，以及分离和浓缩耳蜗细胞的技术都近在咫尺。总而言之，我们的研究将确定1)补充WNT是否有助于启动耳蜗再生，以及2)在新生儿和成熟耳蜗中增强这一再生过程的额外目标。

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.