Pilot Studies of the Effect of Aging on Mucociliary Clearance

描述（由申请人提供）：呼吸道感染是老年人发病和死亡的主要原因。在这一人群中，肺炎是传染病导致死亡的主要原因，社区获得性肺炎的发病率每十年增加一次。抗呼吸道感染最重要的先天防御机制之一是粘膜纤毛清除（MCC）系统。具有MCC遗传缺陷的个体遭受慢性肺部感染，表明MCC对宿主防御的重要性。重要的是，有充分的证据表明，MCC随着年龄的增长而下降。因此，老年人MCC发病率的降低可能会增加他们对呼吸道感染的易感性。然而，迄今为止报道的研究都没有确定老年受试者中MCC发病率降低的机制，也没有探索改善MCC治疗的有效性。令人惊讶的是，在小鼠模型中还没有研究衰老对MCC的影响。缺乏成熟的小鼠模型是一个重大障碍，无论是研究老年人MCC发病率降低的机制，还是开发和测试旨在改善该人群MCC的治疗方法。因此，本课题的目标是：1)在已建立的衰老小鼠模型中准确评估衰老对MCC的影响；2)检查衰老对MCC关键成分的影响，从而开始确定老年受试者MCC发病率下降的机制。具体目标1。为了验证衰老小鼠MCC减少的假设。通过跟踪内源性粘液颗粒的运动或注入荧光珠，测量3、6、12和24月龄C57BL/6小鼠气管和鼻咽部粘膜纤毛清除率。具体目标2。研究年龄对MCC各成分的影响。2a)验证老年小鼠MCC纤毛成分存在缺陷的假设。通过视频显微镜测量3、6、12和24月龄小鼠气管和鼻腔的睫状体搏动频率（CBF）。纤毛细胞的数量将由组织学决定，轴突结构缺陷的发生率将通过电子显微镜量化。2b)验证老年小鼠MCC粘液成分存在缺陷的假设。鼻腔和气管粘膜细胞的数量将由不同年龄小鼠样本的组织学决定。用琼脂糖凝胶电泳和免疫印迹法测定肺内气道粘蛋白的分泌量和总量。2c)验证老年小鼠在离子转运调节方面存在缺陷的假设。上呼吸道和下呼吸道的离子传输特性将被确定，因为这些反映了ASL的水化状态。3、6、12和24个月大的小鼠的组织样本将在Ussing实验室内进行研究。

DESCRIPTION (provided by applicant): Respiratory tract infections are a major cause of morbidity and mortality in the elderly population. In this population, pneumonia is the leading cause of death due to infectious disease, and the incidence of community-acquired pneumonia increases with every decade of life. One of the most important innate defense mechanisms against respiratory infections is the mucociliary clearance (MCC) system. Individuals with genetic defects in MCC suffer from chronic pulmonary infections, demonstrating the importance of MCC to host defense. Importantly, there is good evidence to suggest that MCC declines with age. Therefore it is likely that a reduced rate of MCC in the elderly, increases their susceptibiliy to respiratory infections. However, none of the studies reported to date has identified the mechanism(s) responsible for the reduced rate of MCC in older subjects, nor have they explored the effectiveness of treatments to improve MCC. Surprisingly, there have been no studies of the effect of aging on MCC in a mouse model. The lack of an established mouse model is a significant barrier, both to studies of the mechanisms responsible for the reduced rate of MCC in the elderly and to the development and testing of therapeutics designed to improve MCC in this population. Therefore the goals of this proposal are to 1) accurately assess the effect of aging on MCC in an established mouse model of aging, and 2) to examine the effect of aging on the key components of MCC to begin to identify the mechanisms responsible for the decreased rate of MCC in older subjects. Specific aim 1. To test the hypothesis that MCC is reduced in aged mice. Mucociliary clearance will be measured in the trachea and nasopharynx of 3, 6, 12, and 24 month old C57BL/6 mice by tracking the movement of endogenous mucus particles or instilled fluorescent beads. Specific aim 2. To examine the effect of age on individual components of the MCC. 2a) To test the hypothesis that aged mice exhibit defects in the ciliary component of MCC. Ciliary beat frequency (CBF) will be measured by video microscopy in the trachea and nasal cavity of 3, 6, 12, and 24 month old mice. The number of ciliated cells will be determined by histology, and the incidence of axonemal structural defects will be quantified by electron microscopy. 2b) To test the hypothesis that aged mice exhibit defects in the mucus component of MCC. The number of mucous cells in the nasal cavity and trachea will be determined by histology in samples from mice of different ages. The amount of secreted and total airway mucin in the lung will be quantified by agarose gel electrophoresis and Western blotting. 2c) To test the hypothesis that aged mice exhibit defects in the regulation of ion transport. The ion transport properties of the upper and lower airways wil be determined, as these are reflective of the hydration status of the ASL. Tissue samples from mice 3, 6, 12, and 24 months old will be studied in Ussing chambers.