Method for Measuring Mucociliary Clearance and Cilia-generated Flow in Mice by ex vivo Imaging

Background

The composition of diverse biophysical properties, each aiding a specified function, make up the human body. A key example of this is the generation of flow in many different organ systems, including the central nervous system (Olstad et al., 2019), reproductive tracts (Afzelius et al., 1978; Yuan et al., 2019) and the pulmonary system (Satir and Sleigh, 1990), made by specialized ciliated cells. Understanding how the environment or external stimuli affect these processes can give us insights into disease. In our recent study (Kudo et al., 2019), we explored how humidity affects influenza infections in a mouse model. By using this method of ex-vivo measurement of cilia-generated flow in mice, we were able to visually measure how ambient humidity altered the rate of virus clearance (Figures 1 and 2). Previous studies have used radiolabeled particles to measure mucociliary clearance using a noninvasive method (Hua et al., 2010; Bustamante-Marin and Ostrowski, 2017). However, this requires the use of radioactive materials and SPECT or other machines to measure radioactivity that may not be readily available. Measurements like this can be made more accessible using ex vivo imaging of particles in tissue (Nance et al., 2012; Francis and Lo, 2013; Mastorakos et al., 2015 and 2016). Here, we describe a detailed method of measuring the flow generated by cilia in upper airway tissue, adapted from Francis and Lo (2013). Our method is easy to perform and allows for measurement of large numbers of mice at a relatively low cost. This protocol can also be performed in a short time, allowing for the preservation of biological differences and measurement in a large number of animals. Finally, it can be adopted for other organ systems or tissues, measuring various biophysical properties, as shown by Nance et al. (2012), in identifying what criteria drug loaded particles must meet to navigate through brain parenchyma. Altogether, this protocol will provide researchers with an easy to follow, step-by-step methodology to measure properties of biological tissues ex vivo (Figure 3).