Pilot Studies of the Effect of Aging on Mucociliary Clearance

描述（由申请方提供）：呼吸道感染是老年人群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随着年龄的增长而增加。对抗呼吸道感染的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遗传缺陷的个体患有慢性肺部感染，表明MCC对宿主防御的重要性。重要的是，有很好的证据表明MCC随着年龄的增长而下降。因此，老年人MCC发生率的降低可能会增加他们对呼吸道感染的易感性。然而，迄今为止报告的研究均未确定老年受试者中MCC发生率降低的机制，也未探索治疗改善MCC的有效性。令人惊讶的是，在小鼠模型中没有研究衰老对MCC的影响。缺乏已建立的小鼠模型是一个重大障碍，无论是对老年人MCC发病率降低机制的研究，还是对旨在改善该人群MCC的治疗方法的开发和测试。因此，本提案的目标是：1）在已建立的衰老小鼠模型中准确评估衰老对MCC的影响，以及2）检查衰老对MCC关键组分的影响，以开始确定老年受试者中MCC发生率降低的机制。具体目标1.验证老年小鼠MCC减少的假设。通过跟踪内源性粘液颗粒或滴注荧光珠的运动，测量3、6、12和24月龄C57 BL/6小鼠气管和鼻咽中的粘液纤毛清除率。具体目标2。检查年龄对MCC单个组分的影响。2a）检验老年小鼠表现出MCC纤毛组分缺陷的假设。将通过视频显微镜测量3、6、12和24月龄小鼠气管和鼻腔中的纤毛搏动频率（CBF）。纤毛细胞的数量将通过组织学确定，轴丝结构缺陷的发生率将通过电子显微镜定量。2b）检验老年小鼠表现出MCC粘液组分缺陷的假设。将通过组织学方法测定不同年龄小鼠样品中鼻腔和气管中粘液细胞的数量。通过琼脂糖凝胶电泳和蛋白质印迹定量肺中分泌的和总气道粘蛋白的量。2c）验证老龄小鼠在离子转运调节方面表现出缺陷的假设。将确定上呼吸道和下呼吸道的离子转运特性，因为这些特性反映了ASL的水合状态。将在Ussing室中研究3、6、12和24月龄小鼠的组织样本。

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.