Wykoff Jason A, Shaffer Kendall M, Araba Kenza C, Markovetz Matthew R, Patarin Jérémy, Robert de Saint Vincent Matthieu, Donaldson Scott H, Ehre Camille
Marsico Lung Institute / CF Center, The University of North Carolina at Chapel Hill.
Rheonova.
J Vis Exp. 2022 Apr 21(182). doi: 10.3791/63876.
In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G', G", G, and tan δ) and gel point characteristics (γc and σc) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is <10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more "liquid-like" behavior overall (e.g., higher tan δ). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.
在黏液阻塞性肺部疾病（如哮喘、慢性阻塞性肺疾病、囊性纤维化）和其他呼吸道疾病（如病毒/细菌感染）中，杯状细胞分泌亢进、气道脱水、氧化应激和细胞外DNA的存在会改变黏液的生物物理特性。先前的研究表明，痰液的黏弹性与肺功能相关，影响痰液流变学的治疗方法（如黏液溶解剂）可带来显著的临床益处。一般来说，非牛顿流体的流变学测量采用复杂、耗时的方法（如平行板/锥板流变仪和/或微珠颗粒追踪），需要进行大量培训才能进行检测和解释数据。本研究测试了Rheomuco的可靠性、可重复性和灵敏度，这是一种用户友好的台式设备，旨在通过动态振荡和剪切应变扫描进行快速测量，以在5分钟内为临床样本提供线性黏弹性模量（G'、G''、G和tan δ）以及凝胶点特性（γc和σc）。使用不同浓度的黏液模拟物8 MDa聚环氧乙烷（PEO）并与传统的整体流变学测量方法进行对比，验证了该设备的性能。然后，对一名哮喘持续状态（SA）插管患者的临床分离株进行了三次重复测量，测量之间的变异系数<10%。在SA黏液上体外使用强效黏液减少剂三（2-羧乙基）膦（TCEP），导致弹性模量降低了五倍，并总体上向更“类似液体”的行为转变（如更高的tan δ）。总之，这些结果表明，经过测试的台式流变仪能够在临床和研究环境中可靠地测量黏液的黏弹性。综上所述，所描述的方案可用于现场探索黏液活性药物（如重组人脱氧核糖核酸酶、N-乙酰半胱氨酸）的效果，以便根据具体情况调整治疗方案，或用于新化合物的临床前研究。
