In vivo Evaluation of Mucociliary Clearance in Mice

Our protocol allows the accurate measurement of mucociliary clearance in an animal model using radionuclides that are tracked via dual-modality SPECT and CT imaging to accurately localize and measure airway clearance. This technique is a reproducible method for measuring MCC that can be applied to different animal and disease models due to the utilization of dual SPECT/CT. After confirming a lack of response to pedal reflex, equip the stand with a nose cone to maintain anesthesia and suspend the anesthetized mouse by the front incisors on an intubation stand at a 45 degree angle.

Connects one end of a 50 micron fiber optic wire to a light source and thread a 20 gauge cannula over the wire. Use blunt forceps to pull out the tongue and illuminate the guide wire to visualize the vocal cords. Pass the guide wire through the vocal cords so that the wire is just beyond the vocal cords resting in the upper trachea and slide the one inch cannula forward along the wire until it is deep enough that the hub is against the animal's incisors.

Remove the wire while leaving the cannula in place and use a finger to briefly plug the cannula to check for changes in breathing. Then add 10 microliters of freshly prepared 0.2 millicurie 99m technetium sulfur colloid to the cannula and allow the mouse to spontaneously inhale the radionuclide into the lungs over a period of one to two minutes. For SPECT/CT imaging, after removing the cannula, use tape to secure the mouse to a 25 millimeter palette with a nose cone and tape a 200 microliter PCR tube containing 0.05 millicurie radioactive phantom under the lower abdomen of the mouse below the lungs.

Place the mouse into the SPECT/CT system. Select the imaging workflow and click Setup. Set up the positioning of the detectors on the mouse and run the imaging workflow.

Upon completion of the workflow, transfer the mouse into a new cage with monitoring and unrestricted access to food and water and radiation safety sticker for six hours between scans. After imaging, histogram the SPECT images using the factory standard settings for 99m technetium sulfur colloid and use a MAP3D algorithm and point spread function reconstruction to reconstruct 3D stack images. Reconstruct the CT images using the Feldkamp algorithm and a Shepp-Logan filter and open the CT and SPECT images in FIJI.

Use the reslice tool to generate coronal view images from the default axial images and perform a z-stack sum projection on the SPECT image to add the count data from each slice. Generate a single image for ease of analysis. And using the Phantom Eppendorf tube as a reference, resize and coregister the CT and SPECT images.

Use auto thresholding to binarize the CT image, invert the stack and perform a z-stack sum projection to generate an outline of the lungs for analysis. Rotate the CT and SPECT images and use the channel tools to merge them into one image. Then draw a region of interest around the right lung to determine the mucociliary clearance.

After image acquisition and processing, the CT and SPECT images can be co-localized using the phantom tube as a landmark. Masks of the lungs can be generated from the CT image and used to draw regions of interest around the lung for their analysis at different time points. In this analysis, a total of eight mice were scanned two times on different days under identical experimental conditions to test the reproducibility of the protocol.

Paired T-test analysis showed no significant difference between the repeat scans. An additional two mice were also scanned three times on different days under identical experimental conditions. One-way ANOVA analysis showed significant matching between the repeat scans.

Here, representative images from two of the scanned mice can be observed. It is essential to be able to visualize the airway. And to verify that intubation is successful, if necessary, remove the cannula and reintubate to avoid erroneous esophageal intubation.

This protocol can be applied to disease models to assess the effects of different drugs on airway clearances and to aid in the development of novel drug therapy.