M.Z.和 K.S.F. 和这项工作得到了匹兹堡大学临床和转化科学研究所在皮特创新挑战 （PInCh） 下授予的赠款的支持，NHLBI R01 授予 HL153407，授予 M.Z.

匹兹堡大学机构动物护理和使用委员会在进行这些动物实验之前，批准了本出版物中指定的所有动物协议。
注:此协议详细说明了如何使用双模SPECT/CT扫描仪进行放射性核素成像的体内粘膜清除研究。所展示的技术包括运行系统校准、麻醉小鼠、小鼠气管插管、向肺部灌输同位素、双模成像、这些图像的共同注册以及分析。
1. 光谱/CT 系统设置
<ol><li>设计一个合适的工作流程，并在使用活体动物进行实验之前设置。<ol>
<li>使用由 60 个投影组成的 SPECT 采集，在旋转半径为 40 厘米的投影之间步幅为 6o。CT 采集由 220 个投影组成，投影之间角度为 1.6o。</li>
	</ol>
</li>
	<li>确保系统为小鼠提供正确的 MWB 校准器和 SPECT 成像。如果安装了不适当的校准器，请使用对准器向导安装正确的对准器。</li>
	<li>运行必要的系统校准，为系统准备使用。 注:扫描仪的SPECT和CT组件需要校准。使用源调理和暗/光 （D/L） 校准来校准 CT 组件，每 2 周进行一次中心偏移 （COS） 校准，并每月评估 X 射线硬件。SPECT 组件需要每年校准一次。<ol>
<li>要评估 X 射线硬件，请检查 CT 校准期间 的评估 X 射线硬件 框（补充 CT 校准菜单）。</li>
		<li>要执行源调理，请在 CT 校准期间检查 执行源调理 框（补充 CT 校准菜单）。</li>
		<li>要执行 D/L 校准，请检查 CT 校准期间实验中使用的 CT 采集协议旁边的 D/L 框。取消检查所有其他协议（补充CT校准菜单）。</li>
		<li>要执行 COS 校准，请用校准环工具替换床，调整床型设置以匹配运动控制设置，并在 CT 校准期间的实验中使用的 CT 采集协议旁边检查 COS 框。取消检查所有其他协议（补充CT校准菜单，补充校准环）。</li>
	</ol>
</li>
</ol>2. 鼠标灌输和灌输
<ol><li>称重要扫描的鼠标。如果扫描多个鼠标，请注意使用耳击或尾部标记等方法为识别目的标记小鼠。</li>
	<li>使用 1.5% 异氟化物麻醉鼠标，气体流量为 2 L/min O2，在气室中持续约 5 分钟，以产生足够深度的麻醉，直到呼吸速度减慢到每分钟16 次 55-65次呼吸（图 1A）。</li>
	<li>将鼠标从腔室中取出，在 45o斜坡的灌管支架上由前切口悬挂。用鼻锥装备插管支架，以确保小鼠在插管过程中麻醉（图1B）。</li>
	<li>将 50μm 光纤线的一端连接到光源，并用电线将 20 光纤管穿线（图 1C）。</li>
	<li>打开鼠标的嘴，用钝钳把舌头向前拉。照亮导线，并用它来可视化声带（图1D）。</li>
	<li>通过声带的导线，使电线刚刚超越声带，并在上气管休息。沿着钢丝向前滑动1英寸的管子，给老鼠做管子，通过足够深的管子，使其中心与动物的切口（图1E）相抗使。取出留下管子的电线。</li>
	<li>通过用手指短暂地插入手杖并检查呼吸变化来测试插管。停止呼吸或呼吸紧张，同时堵塞和释放后加速呼吸是适当的气管插管的迹象。如果堵塞手杖后呼吸模式没有变化，后者很可能在食道中。</li>
	<li>在10μL的体积中准备 9900万个技术硫胶体（99mTc-Sc）的0.2 mCi，并将移液器放入管子中。让老鼠在1-2分钟（图1F）内自发地吸入肺部。在将鼠标转移到扫描仪托盘之前取出手杖。 注:放射性核素是由红衣主教健康公司制备和过滤的。</li>
</ol>3. 光谱/CT 成像
<ol><li>将鼠标转移到带鼻锥的25毫米托盘中，用胶带固定，注意不要将胸部和腹部粘得太紧，以免损害呼吸。请小心删除连接到鼠标的任何金属耳标。</li>
	<li>在 200 μL 中制备由 0.05 mCi 组成的放射性幻影，并将此量放置在 0.2 mL PCR 管中。通过将管子贴在小鼠下腹部下方的托盘上，避免与肺部重叠，从而放置管子。 注:幻影用于共同注册CT和SPECT图像，以及清除的负控制。</li>
	<li>将鼠标插入SPECT/CT系统，选择成像工作流程并运行 "设置"。</li>
	<li>设置鼠标上探测器的定位，并运行成像工作流程。</li>
	<li>为手术后接受放射性的老鼠准备一个笼子，不受限制地获得食物和水，并使用辐射安全贴纸进行清晰的标签。</li>
	<li>工作流程完成后，将鼠标从成像托盘中取出，并允许鼠标在预制的笼子中恢复，扫描（扫描结束 1 至扫描 2 开始）之间持续 6 小时，并允许广告利比图姆获得食物和水。选择 6 小时，因为它对应于根据 cilia 功能进行线性间隙的时间段，而紫藤间隙很少。</li>
	<li>6小时后，重新麻醉鼠标并扫描，连同幻影，使用相同的工作流程来测量从气道中清除的同位素量。 注:让小鼠恢复至关重要，因为连续6小时用异氟兰麻醉会导致显著的胆汁抑制作用，导致近零粘膜间隙。</li>
</ol>4. 分析
<ol><li>成像后，执行后处理以重建完整的 3D 堆栈图像。<ol>
<li>使用 99mTc 的出厂标准设置对 SPECT 图像进行直方图，然后使用 MAP3D 算法和点点分布功能 （PSF） 重建进行重建。 注:重建使用8次迭代和6个子集进行。有效的重建需要将子集与 1:10 的预测进行比对，或均匀地划分为预测数，因此使用 60 个投影进行收购使用了 6 个子集。</li>
		<li>使用费尔德坎普算法和谢普-洛根过滤器重建CT图像。 注:重建是使用4次迭代进行的。</li>
	</ol>
</li>
	<li>使用残片工具处理 FIJI ImageJ21 中的 CT 和 SPECT 图像，从默认轴向图像生成日冕视图图像。然后在 SPECT 图像上执行 z 堆栈总和投影，以添加每个切片的计数数据，并生成单个图像以便于分析。</li>
	<li>调整大小并共同注册CT和SPECT图像使用幻影埃彭多夫管作为参考（图2A，B）。跟踪并使用所有样品的一致调整尺寸测量。</li>
	<li>使用自动阈值对 CT 图像进行二进制，然后倒置堆栈，并执行 z 堆栈总和投影，生成肺部轮廓以供分析（图 2C）。</li>
	<li>旋转 CT 和 SPECT 图像，并使用通道工具合并图像。通过在右肺周围绘制投资回报率并测量（图 2D）来计算 MCC。 注:此测量将计算右肺 0 和 6 小时时间点的总计数，使用公式更正 6 小时图像以进行放射性衰变:N（t）= N0e+t.99米Tc-Sc 的衰变常数为每秒 3.21e=5，半衰约 6 小时。然后，这些值可用于计算百分之百的间隙。 注:右肺被选为投资回报率图纸和测量计数，因为粘膜间隙将把放射性同位素从肺部输送到咽部，从那里吞咽，最后在胃中。很多时候，计数可以看到在胃，可能与左肺重叠，从而产生错误的计数。这种混淆可以通过测量右肺的计数来避免。</li>
</ol>

<ol>
	<li>Afzelius, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia-related+diseases.">Cilia-related diseases.</a> Journal of Pathology. 204 (4), 470-477 (2004).</li><li>Mitchell, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+eukaryotic+cilia+and+flagella+as+motile+and+sensory+organelles.">The evolution of eukaryotic cilia and flagella as motile and sensory organelles.</a> Advances in Experimental Medicine and Biology. 607, 130-140 (2007).</li><li>Carvalho-Santos, Z., Azimzadeh, J., Pereira-Leal, J. B., Bettencourt-Dias, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution:+Tracing+the+origins+of+centrioles,+cilia,+and+flagella.">Evolution: Tracing the origins of centrioles, cilia, and flagella.</a> Journal of Cell Biology. 194 (2), 165-175 (2011).</li><li>Randell, S. H., Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=University+of+North+Carolina+Virtual+Lung,+G.+Effective+mucus+clearance+is+essential+for+respiratory+health.">University of North Carolina Virtual Lung, G. Effective mucus clearance is essential for respiratory health.</a> American Journal of Respiratory Cell and Molecular Biology. 35 (1), 20-28 (2006).</li><li>Wanner, A., Salathe, M., O'Riordan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucociliary+clearance+in+the+airways.">Mucociliary clearance in the airways.</a> American Journal of Respiratory and Critical Care Medicine. 154, 1868-1902 (1996).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+genetic+analysis+in+mice+unveils+central+role+for+cilia+in+congenital+heart+disease.">Global genetic analysis in mice unveils central role for cilia in congenital heart disease.</a> Nature. 521 (7553), 520-524 (2015).</li><li>Zahid, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+ciliary+dysfunction+and+respiratory+symptoms+in+patients+with+transposition+of+the+great+arteries.">Airway ciliary dysfunction and respiratory symptoms in patients with transposition of the great arteries.</a> PLoS One. 13 (2), 0191605 (2018).</li><li>Stewart, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+ciliary+dysfunction:+Association+with+adverse+postoperative+outcomes+in+nonheterotaxy+congenital+heart+disease+patients.">Airway ciliary dysfunction: Association with adverse postoperative outcomes in nonheterotaxy congenital heart disease patients.</a> Journal of Thoracic and Cardiovascular Surgery. 155 (2), 755-763 (2018).</li><li>Garrod, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+ciliary+dysfunction+and+sinopulmonary+symptoms+in+patients+with+congenital+heart+disease.">Airway ciliary dysfunction and sinopulmonary symptoms in patients with congenital heart disease.</a> Annals of the American Thoracic Society. 11 (9), 1426-1432 (2014).</li><li>Harden, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+postoperative+respiratory+complications+in+heterotaxy+congenital+heart+disease+patients+with+respiratory+ciliary+dysfunction.">Increased postoperative respiratory complications in heterotaxy congenital heart disease patients with respiratory ciliary dysfunction.</a> Journal of Thoracic and Cardiovascular Surgery. 147 (4), 1291-1298 (2014).</li><li>Leigh, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+genetic+aspects+of+primary+ciliary+dyskinesia/Kartagener+syndrome.">Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome.</a> Genetics in Medicine. 11 (7), 473-487 (2009).</li><li>Donaldson, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ivacaftor+on+mucociliary+clearance+and+clinical+outcomes+in+cystic+fibrosis+patients+with+G551D-CFTR.">Effect of ivacaftor on mucociliary clearance and clinical outcomes in cystic fibrosis patients with G551D-CFTR.</a> JCI Insight. 3 (24), (2018).</li><li>Donaldson, S. H., Corcoran, T. E., Laube, B. L., Bennett, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucociliary+clearance+as+an+outcome+measure+for+cystic+fibrosis+clinical+research.">Mucociliary clearance as an outcome measure for cystic fibrosis clinical research.</a> Proceedings of the American Thoracic Society. 4 (4), 399-405 (2007).</li><li>Ledowski, T., Hilmi, S., Paech, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchial+mucus+transport+velocity+in+patients+receiving+anaesthesia+with+propofol+and+morphine+or+propofol+and+remifentanil.">Bronchial mucus transport velocity in patients receiving anaesthesia with propofol and morphine or propofol and remifentanil.</a> Anaesthesia. 61 (8), 747-751 (2006).</li><li>Donnelley, M., Morgan, K. S., Siu, K. K., Parsons, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+deposition+of+pollutant+and+marker+particles+onto+live+mouse+airway+surfaces+enhances+monitoring+of+individual+particle+mucociliary+transit+behaviour.">Dry deposition of pollutant and marker particles onto live mouse airway surfaces enhances monitoring of individual particle mucociliary transit behaviour.</a> Journal of Synchrotron Radiation. 19, 551-558 (2012).</li><li>Christopher, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+and+anesthetic+agents+on+ciliary+function+in+murine+respiratory+epithelia.">The effects of temperature and anesthetic agents on ciliary function in murine respiratory epithelia.</a> Frontiers in Pediatrics. 2, 111 (2014).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+quantitative+study+of+airway+functional+microanatomy+using+micro-optical+coherence+tomography.">Method for quantitative study of airway functional microanatomy using micro-optical coherence tomography.</a> PLoS One. 8 (1), 54473 (2013).</li><li>Lam, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+6-mediated+selective+autophagy+regulates+COPD-associated+cilia+dysfunction.">Histone deacetylase 6-mediated selective autophagy regulates COPD-associated cilia dysfunction.</a> Journal of Clinical Investigation. 123 (12), 5212-5230 (2013).</li><li>Bhashyam, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+study+to+examine+the+effect+of+chronic+treatment+with+immunosuppressive+drugs+on+mucociliary+clearance+in+a+vagotomized+murine+model.">A pilot study to examine the effect of chronic treatment with immunosuppressive drugs on mucociliary clearance in a vagotomized murine model.</a> PLoS One. 7 (9), 45312 (2012).</li><li>Ortiz, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mucociliary+Clearance+by+Three+Dimension+Micro-CT-SPECT+in+Guinea+Pig:+Role+of+Bitter+Taste+Agonists.">Evaluation of Mucociliary Clearance by Three Dimension Micro-CT-SPECT in Guinea Pig: Role of Bitter Taste Agonists.</a> PLoS One. 11 (10), 0164399 (2016).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Solomon, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+ciliary+phenotype+in+primary+ciliary+dyskinesia+by+micro-optical+coherence+tomography.">Assessment of ciliary phenotype in primary ciliary dyskinesia by micro-optical coherence tomography.</a> JCI Insight. 2 (5), 91702 (2017).</li><li>Nakhleh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+respiratory+ciliary+dysfunction+in+congenital+heart+disease+patients+with+heterotaxy.">High prevalence of respiratory ciliary dysfunction in congenital heart disease patients with heterotaxy.</a> Circulation. 125 (18), 2232-2242 (2012).</li><li>Whaley, S. L., Renken, S., Muggenburg, B. A., Wolff, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+aerosol+deposition+restricted+to+the+nose+in+beagle+dogs.">Technique for aerosol deposition restricted to the nose in beagle dogs.</a> Journal of Toxicology and Environmental Health. 23 (4), 519-525 (1988).</li><li>Foster, W. M., Walters, D. M., Longphre, M., Macri, K., Miller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+the+measurement+of+mucociliary+function+in+the+mouse+by+scintigraphy.">Methodology for the measurement of mucociliary function in the mouse by scintigraphy.</a> Journal of Applied Physiology. 90 (3), 1111-1117 (2001).</li><li>Bhashyam, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagal+control+of+mucociliary+clearance+in+murine+lungs:+a+study+in+a+chronic+preparation.">Vagal control of mucociliary clearance in murine lungs: a study in a chronic preparation.</a> Autonomic Neuroscience. 154 (1-2), 74-78 (2010).</li><li>Van Audenhaege, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+SPECT+collimator+selection,+optimization,+and+fabrication+for+clinical+and+preclinical+imaging.">Review of SPECT collimator selection, optimization, and fabrication for clinical and preclinical imaging.</a> Medical Physics. 42 (8), 4796-4813 (2015).</li></ol>

与此工作无关。

湿润的呼吸道在疾病和发展中的作用继续发展，并得到更好的赞赏。气管支气管树内衬细胞的apical表面的多个摩尔西利亚的同步、元时间跳动产生头皮流，产生粘膜间隙或 MCC。MCC在像PCD22这样的心肌病中受到损害，像COPD18这样的疾病，其重要性在CHD中得到了承认，传统上并不被认为是心肌病。最近的数据显示，在患有异位轴23和无异质轴7的CHD中，呼吸系统肝功能障碍。这种移动性胆汁功能障碍被证明转化为更大的呼吸症状9以及更大的术后发病率8。大多数，如果不是全部，这些疾病，有小鼠模型可用，我们的协议，测量MCC在小鼠是一个有价值的工具，可以用来测试潜在的治疗方法。
动物模型为了解疾病和治疗的发展提供了效用。体内动物成像提供了进一步的效用，能够从同一动物获得多个数据点，而无需牺牲动物，使调查人员能够跟踪疾病的纵向过程以及治疗效果的研究持续时间。MCC的老鼠模型是由多个研究者在几十年的时间里开发的，最初是使用平面扫描技术（二维核成像技术24）在小猎犬身上进行的。十年后，这项技术被改用在小鼠身上，随后在25、26年之后的10年适应了SPECT成像。这种技术在小鼠模型中的发展是该技术相关性的一个重大发展，因为有多种人类疾病小鼠模型，如PCD，其中硅功能发生显著变化。MCC已被评估在小鼠模型的肺衰减和免疫抑制，并有可能与其他模型19，26一起使用。在人类气道疾病（如CF、哮喘、PCD和与CHD相关的心肌病）患者中进行了MCC测量研究，并取得了该技术可同时帮助肺生理学和治疗疗效的研究。
此协议的一个重要部分是设置具有正确成像参数的获取，以获取用于量化的准确图像。在设计 SPECT 收购设置时，有许多因素是关键，包括使用哪些校准器、每次革命获得的预测数和旋转步数大小。合金选择是收购的灵敏度和分辨率的一个主要因素，收购设置可能需要根据正在使用的27的对准器进行定制。或者，当使用更大的动物，如老鼠，碰撞器将需要调整。例如，多个针孔校准器更敏感，但在选择步骤大小时应小心，以避免重叠的投影和造成不受欢迎的多路复用，这可以进一步提高收购的灵敏度，牺牲一些图像模糊性，可能会导致重建文物25。重建设置也是生成可量化图像的关键。MAP3D 是常用的迭再重建算法，PSF 是常见的重建模型。两者都可用于重建图像，但在设置迭代和子集数量时应小心谨慎。更多的迭代将增加重建所需的计算时间，并随着回报的进一步增加而提高重建的质量。
为了量化 ImageJ 中的图像，理想的测量工具是 RawIntDen，它在选择中输出像素的总值。当在不同大小的肺 ROI 上量化 SPECT 数据时，RawIntDen 的使用提供了绝对的计数量度，并避免将测量结果调整到 ROI 的区域，就像平均测量结果为 21一样。
此技术具有许多相关的错误来源，调查人员在应用此技术时应了解这些错误源。一个值得注意的混淆是使用麻醉剂。伊索夫卢兰是一种快速作用，吸入麻醉剂，小鼠在完成采集后迅速恢复。然而，应小心为小鼠提供充足的时间在笼子里恢复，并且不要超过必要的时间进行麻醉。根据我们个人的经验（未公布的数据），在0至6小时时间点之间连续使用吸入的异氟兰进行麻醉的小鼠的间隙可以忽略不计。同样，控制剂量的麻醉剂也是必要的，以确保快速恢复。当将动物固定到托盘进行成像时，用于共同登记的幻影管应保持在腹部低位，以避免文物与肺部重叠。同样，为了确保高质量的 CT 图像，请注意从鼠标中删除任何金属标签，以避免从 X 射线散射中分离出工件。
目前的MCC协议可以应用于无数的动物模型。这种技术对被扫描动物的健康影响微乎其微，老鼠可以很好地耐受，因此它可以与疾病模型一起使用，而不会危及已经很脆弱的小鼠的健康。这种方法的强度来自于它是一种体内技术，它允许获得一致和可重复的气道功能测量，而无需牺牲动物来切除气管进行视频显微镜检查，前体内模型需要26。这种技术在对同一动物的多次扫描中产生可重复测量的一致性，允许用不同的剂剂或潜在的治疗方法对同一动物进行治疗，并在同一动物之间进行统计比较，以减少任何动物模型固有的生物变异性，从而减少显示统计显著差异所需的样本量。
使用MCC技术对气道功能的评估可以调整到各种动物模型，并应用于许多不同的气道健康模型，以及测试新的疗法。可使用此技术以及 COPD 模型评估 PCD 小鼠模型的气道。我们的方法也可以用来研究各种麻醉剂对MCC的差异效应，这是常见的临床用途。最后，也可以使用此模型评估治疗剂对气道的影响。如前所述，但值得重复，因为它是一种体内测量，它允许在疾病过程中重复MCC评估，以及随着时间的推移治疗干预的测试益处。此外，小鼠是用于模仿/研究人类疾病的最常见实验室动物，在某些情况下，有多种转基因小鼠模型可供选择。

Cilia 是基于微管的细胞器，保存于从藻类到人类的进化历史中。它们来自细胞表面，具有多种功能1，从识别局部环境感官信号到运动性，其功能可以追溯到人类早期的单细胞真核生物2、3。Cilia 可以是非移动和单体，作为细胞处理环境信号的专用天线:或移动和多，在同步的，元时波跳动，以产生流体流动，如在输卵管的衬里和上下气道，除了终端支气管导致肺泡1，2。
呼吸道广泛的上皮表面不断受到各种潜在危险吸入污染物和病原体形式的污染，因此需要防御。一个关键的防御机制是气管支气管树的粘膜装置，通过敲击气管支气管上皮细胞的apical表面的多块粘液，机械地将连续的分泌粘液流出气道。这些功能诱捕吸入的污染物，并通过其连续，同步殴打，运输他们头孢4，5。
Cilia已被证明在胚胎发育中具有关键作用，如在胚胎发育中左右模式，其中胚胎节点的摩尔西利亚断裂对称性6。由于心脏的不对称结构，与西莉亚相关基因的突变与先天性心脏病（CHD）等疾病有关。最近的研究报告，有染病患者呼吸道功能障碍的高发率，以及术后呼吸道并发症和慢性呼吸道症状在上、下气道7、8、9、10的患病率上升。有或无异质性心肺功能障碍的病人，在术后5、8、10日，已证明有呼吸系统并发症及负面呼吸道结果的风险增加。除了在信号和开发中的作用之外，气道西莉亚的重要性也由心肌病所证明，其中一个典型的例子是主要的胆囊性发育不良（PCD）。PCD是一种先天性疾病，由一些影响湿润的呼吸道的突变引起，导致肺部反复感染，支气管切除术，并有可能需要肺移植11。此外，即使西莉亚在囊性纤维化（CF）中是正常的，这是高加索人群中最常见的先天性疾病，但MCC由于CFTR基因12的突变引起的粘稠粘液而受损。PCD 和 CF 的多个鼠标模型，以及不断增加的 CHD 模型。最终，西莉亚是多功能结构，具有许多关键作用，评估活体中移动呼吸系统西莉亚功能的方法对于临床前研究、评估突变以及药物对粘结清除 （MCC）13的影响是有价值的。这种方法对于评估这些小鼠模型中新药、基因疗法或干预措施对MCC的影响也很有价值。
有许多不同的模型已经用于评估MCC。一种值得注意的方法是使用已灌输到支气管中的甲基蓝色染料，通过纤维光学测量染料运动的间隙14。这种方法受制于观察染料运动的能力，这种染料在人类中比在临床前小鼠模型中更为常规。另一个值得注意的方法是同步加速器相对比X射线成像（PCXI），它可以用来跟踪气道中的单个粒子。这种方法是比较新的，并不广泛访问15。有许多外活体方法来评估气道通过切除气管的视频显微镜，但这些模型提供很少的效用，在人类患者16。Cilia成像的高分辨率技术，如光学相干断层扫描，以同样的方式受到限制。
本文介绍了一种可重复的方法，用于测量无数动物模型中的肺间隙，以及研究慢性阻塞性肺病的MCC，并评估免疫抑制药物18、19的影响。这种方法跟踪放射性制药9900万技术硫胶体（99mTc-Sc），一种不溶性颗粒放射性跟踪器，在注入肺部后清除。然后，放射性核素可以使用单光子发射计算断层扫描（SPECT）18，20进行跟踪。我们进一步完善了这种技术，通过使用双模频谱和计算断层扫描 （CT） 成像与放射性同位素计数的共同本地化到肺部，并测量这些计数在 6 小时内的减少。双模成像，CT 和 SPECT 图像的共同注册，可准确定位辐射计数到我们感兴趣的区域，肺部。虽然我们详细描述了小鼠的MCC测量方法，但可以调整协议以研究大鼠的MCC。校准器需要调整以及辐射剂量。我们认为，由于动物体积小，小鼠MCC扫描在技术上更具挑战性，但由于人类疾病的既定小鼠模型数量众多，因此比大鼠更有用。此外，由于动物群落的成本和维护成本较低，较大的样本量在小鼠中更为可行。

<table><tbody><tr><td>500 &amp;micro;m Unjacketed Fiber Optic Wire</td><td>Edmund Optics</td><td>02-532</td><td></td></tr><tr><td>99mTechnecium-Sulfur Colloid</td><td>Cardinal Health</td><td></td><td></td></tr><tr><td>Anesthesia Vaporizer</td><td>Vetland Medical</td><td>A13480</td><td></td></tr><tr><td>Durmont #5 Forceps</td><td>Fine Science Tools</td><td>99150-20</td><td></td></tr><tr><td>FIJI ImageJ 2.0.0-rc-65/1.52p Software</td><td></td><td></td><td></td></tr><tr><td>Introcan Safety Catheters 20G 1inch</td><td>Fisher Scientific</td><td>NC1534477</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>118-2097</td><td></td></tr><tr><td>Mouse Intubation Stand</td><td>Kent Scientific</td><td>ETI-MSE-01</td><td></td></tr><tr><td>Siemens Inveon dual-modality SPECT/CT</td><td>Siemens</td><td></td><td></td></tr><tr><td>Single Channel Anesthesia Stand</td><td>Summit Anesthesia Solutions</td><td>22860</td><td></td></tr></tbody></table>

in vivo evaluation of mucociliary clearance in mice

呼吸摩尔胆汁，细胞的专门细胞器，线的上皮细胞的上皮表面衬里呼吸道。通过以元时间、同步的方式跳动，这些多重、多动、基于行为素的细胞器产生一个头孢流体流，清除吸入的污染物和病原体的呼吸道。随着环境污染的加剧、新型病毒病原体和新兴的耐药细菌的出现，胆汁产生的粘液清除（MCC）对于维持肺部健康至关重要。MCC也患有多种先天性疾病，如原发性囊性发育不良、囊性纤维化以及慢性阻塞性肺病等后天性疾病。所有这些疾病已经建立了，在某些情况下，多个鼠标模型。在本出版物中，我们详细介绍了一种使用少量放射性和双模SPECT/CT成像技术来准确和可重复地测量体内小鼠的MCC的方法。该方法允许在成像后恢复小鼠，使连续测量成为可能，并随着时间的推移对潜在的治疗方法进行纵向测试。野生型小鼠的数据表明，只要充分注意细节，并严格遵守协议，MCC测量的可重复性。

使用此协议，我们在异氟室（图1A）中麻醉小鼠。在达到足够的麻醉水平后，小鼠被放置在垂直支架上（图1B），声带使用照明导线（图1C-1D）进行可视化。这些小鼠通过管子和允许自发吸入肺部的小鼠（图1E-1F）被灌输并灌输0.2mCi 99mTc-Sc，体积为10μL。在图像采集和处理后，CT 和 SPECT 图像以幻影管为地标（图 2B）进行本地化 （图 2A）。肺部的口罩来自CT图像（图2C），用于在右肺周围绘制ROI，以0（图2D）和6小时（图2E-2F）进行分析。为了测试协议的可重复性，在实验条件相同的不同天共扫描了 8 只小鼠两次，使用配对的 t 测试进行分析，显示重复扫描（p 值=0.9904）（图 3A）之间没有显著差异。另有2只小鼠在实验条件相同的不同天被扫描了三次，使用单向ANOVA进行分析后显示重复扫描（p值为0.0041）（图3B）之间有显著匹配。共扫描了8只老鼠，并展示了两张有代表性的图像（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61929/61929fig01.jpg"> 图1:鼠标灌输和同位素灌输。将同位素灌输到气道所需的步骤的图像。 A） 鼠标在室内麻醉。 B） 麻醉鼠标被放置在垂直支架上，由前切口悬挂。 C） 通过20G的管线运行，准备了照明的0.5毫米光纤线作为导线。 D） 鼠标嘴使用钳子打开，并使用照明导线照明以可视化声带。 E） 管子被推过声带，导线被移除。 F） 可溶性同位素被灌输到管子使用移液器和老鼠允许自发吸入同位素到肺部。 <a href="https://www.jove.com/files/ftp_upload/61929/61929fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61929/61929fig02v2.jpg"> 图2:MCC扫描的SPECT/CT图像。A） 已与 CT 图像共同本地化的 SPECT 图像。 B） 带有可视幻管的CT图像，用于共同定位。 C） 通过将 CT 图像比纳化并执行 z 堆栈投影而衍生的气道面罩。 D） CT 面膜与 SPECT 图像共同本地化。在右肺周围绘制了用于分析的 ROI。 E） 6 小时后气道的口罩。 F） 在 6 小时内与投资回报率共同定位气道的 CT 和 SPECT 图像，以供分析。 <a href="https://www.jove.com/files/ftp_upload/61929/61929fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61929/61929fig03.jpg"> 图3:通过多次扫描对同一鼠标进行清除测量。A） 为8只实验条件没有变化的小鼠测量了两个单独的重复间隙。配对 t 测试表明，p 值为 0.9904 的重复扫描之间没有显著差异。 B） 为两只实验条件没有变化的小鼠测量了三个单独的重复间隙。单向 ANOVA 显示重复扫描与 p 值 0.0041 之间有显著匹配。 <a href="https://www.jove.com/files/ftp_upload/61929/61929fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61929/61929fig04.jpg"> 图4:2只小鼠0小时和6小时气道的共同本地化SPECT/CT图像，0和6小时绘制的ROI概述右肺。<a href="https://www.jove.com/files/ftp_upload/61929/61929fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
补充图1:由光纤线照亮的声带视频，其呼吸效果可视化。<a href="https://cloudflare.jove.com/files/ftp_upload/61929/61929supfig01.jpg" target="_blank">请点击这里下载此数字。</a>
补充文件。<a href="https://cloudflare.jove.com/files/ftp_upload/61929/Supplemental_Files.zip" target="_blank">请点击这里下载这些文件。</a>

Watch this Scientific Journal Video about In vivo Evaluation of Mucociliary Clearance in Mice at JoVE.com

In vivo Evaluation of Mucociliary Clearance in Mice

在本出版物中，我们描述了利用双模放射性核素成像评估小鼠体内气道粘膜间隙 （MCC） 的协议。此协议是专为单光子发射计算断层扫描 （SPECT） 和计算断层扫描 （CT） 采集协议而设计的，使用双 SPECT/CT 系统中的鼠标全身 （MWB） 校准器。

在体内评估小鼠的粘液清除

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

in-vivo-evaluation-of-mucociliary-clearance-in-mice

Research

JoVE Journal

Biology

4.3K 次观看.  University of Pittsburgh School of Medicine. 在本出版物中，我们描述了利用双模放射性核素成像评估小鼠体内气道粘膜间隙 （MCC） 的协议。此协议是专为单光子发射计算断层扫描 （SPECT） 和计算断层扫描 （CT） 采集协议而设计的，使用双 SPECT/CT 系统中的鼠标全身 （MWB） 校准器。

视频: In vivo Evaluation of Mucociliary Clearance in Mice

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

科研

免疫与感染

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

医学

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

口咽部气管内PAMP管理和支气管肺泡灌洗的利用率来评估小鼠宿主的免疫反应

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

使用强迫振动技术在小鼠呼吸系统力学评价

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

鼻电位差量化小鼠跨上皮离子转运

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

无创实时全身体积脉搏波检测小鼠咳嗽敏感性

Methods for Detecting Cough and Airway Inflammation in Mice

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

作者聚焦:调查慢性咳嗽的病理生理学

Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

检测小鼠咳嗽和气道炎症的方法

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge ...