Regeneration of stereocilia of cochlear hair cells

描述（由申请人提供）：听觉和平衡感依赖于毛细胞，内耳的感觉受体。毛细胞将机械刺激转化为电活动。由于衰老或暴露于噪音和耳毒性药物中，毛细胞的损失或对毛细胞表面的传导装置，即立体纤毛束的损伤是导致非先天性听力和平衡缺陷的主要原因。过去二十年的研究表明，在鸟类和低等脊椎动物中，失去的毛细胞可以被产生新的毛细胞所取代。哺乳动物的毛细胞再生是有限的，但有证据表明，受损的毛细胞可以存活并进行细胞内修复。在这个提议中，我们将确定哺乳动物耳蜗毛细胞是否可以在失去立体纤毛后存活。如果毛细胞能够存活一段时间，这个机会之窗对于基因或化学治疗干预至关重要，以修复毛束。因此，我们将探讨利用基因治疗方法再生立体纤毛的可行性。我们的探索性/发展性研究有两个具体目标。在Aim 1中，我们将检查哺乳动物毛细胞在有意去除其立体纤毛后的命运，以确定毛细胞是否可以存活和发育。在Aim 2中，我们将确定myosin-15a和whirlin这两个参与立体纤毛形态发生的基因的过表达是否可以促进毛束的修复/再生，以及新再生的立体纤毛是否具有功能。实验将在培养的新生沙鼠Corti器官中进行。在Corti器官的指定区域外毛细胞的毛束将被一个小的吸液器移除。毛细胞存活/发育和立体纤毛形态/功能将使用免疫组织化学（共聚焦显微镜）、延时成像、扫描电子显微镜和电生理学进行检查。研究损伤毛细胞的维持、抢救和修复策略，对毛细胞相关性听力损失和耳聋的生物修复和预防具有重要意义。公共卫生相关性：因为大多数听力损失的美国人，总共约有3000万，都有某种类型的毛细胞损伤，了解损伤修复的细胞过程，探索挽救和修复受伤毛细胞的可能性，对于毛细胞相关听力损失和耳聋的生物修复和预防至关重要。

DESCRIPTION (provided by applicant): The senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Hair cells transduce mechanical stimuli into electrical activity. Loss of hair cells or damage to the transduction apparatus, the stereocilium bundle on the surface of the hair cell, as a result of aging or exposure to noise and ototoxic drugs is the major cause of noncongenital hearing and balance deficits. Studies in the last two decades have demonstrated that lost hair cells can be replaced by production of new hair cells in birds and lower vertebrates. Hair cell regeneration is limited in mammals, but there is evidence to suggest that damaged hair cells may survive and undergo intracellular repair. In this proposal, we will determine whether mammalian cochlear hair cells could survive after loss of stereocilia. If the hair cells can survive for some time, this window of opportunity can be crucial for genetic or chemical therapeutic interventions in order to repair hair bundles. Thus, we will explore the feasibility of regenerating stereocilia using a gene therapy approach. We have two specific aims for this Exploratory/Developmental research. In Aim 1, we will examine the fate of mammalian hair cells after their stereocilia have been intentionally removed to determine whether hair cells can survive and develop. In Aim 2, we will determine whether over-expression of myosin-15a and whirlin, two genes that are involved in stereocilium morphogenesis, can promote hair-bundle repair/regeneration and whether the newly regenerated stereocilia are functional. The experiments will be carried out in the cultured organ of Corti from neonatal gerbils. The hair bundles of outer hair cells in a designated area of the organ of Corti will be removed by a small suction pipette. Hair-cell survival/development and stereocilium morphology/function will be examined using immunohistochemistry (with confocal microscopy), time-lapse imaging, scanning electron microscopy, and electrophysiology. This project is highly significant, since exploring the strategies of maintaining, rescuing, and repairing injured hair cells are essential to the biological remediation and prevention of hair cell-related hearing loss and deafness. PUBLIC HEALTH RELEVANCE: Because the majority of Americans with hearing loss, some 30 million in all, have some kind of hair cell damage, understanding the cellular process of damage repair and exploring the possibilities of rescuing and repairing injured hair cells are essential to the biological remediation and prevention of hair cell-related hearing loss and deafness.