The overall goal of this procedure is to visualize and quantify respiratory CLIA motility and CLIA generated flow using freshly isolated mouse trachea. This is accomplished by first preparing a shallow imaging chamber. The next steps of the procedure are to harvest and mount the tracheal tissue for imaging.
It is then shown how to visualize and record C motility and CLIA generated flow using an inverted microscope with high speed videography through a 100 XDIC objective. The final step is to analyze the video by quantifying CLIA beat frequency and CLIA generated flow using Image J software. Ultimately, the results can show changes in respiratory CLIA motility and CLIA generated flow through high speed video microscopy.
The main advantage of this technique over existing methods is that it allows reproducible imaging of the rodent tra epithelium for consistent quantitative assessments of ciliary motion. This can be accomplished within 20 to 30 minutes with minimal training. This method can help address key questions relating to the cell and molecular mechanisms regulating muci clearance in the eWAY, especially when it is used in combination with different genetically engineered and mutant mouse models.
Begin by building the chamber used to hold the trachea samples for a base or a chamber floor. Use a glass bottomed 35 millimeter culture dish for the top. Use 0.3 millimeter thick silicone sheeting.
Lay the sheeting over an 18 millimeter round glass cover slip and trim the edge. Next with a scalpel from the middle of the silicone, cut out a 10 millimeter square. The glass cover slip and silicone make the top and sides of the culture chamber.
They will be placed on top of the sample that is placed in the culture dish. After euthanizing a mouse in accordance with the institutional guidelines surgically isolate the trachea. Collect the trachea in a plastic 35 millimeter cultured dish with enough L 15 media to completely submerge it.
Using blunt dissection, remove all the non tracheal tissue around the trachea. Next, using microdissection scissors, remove the larynx with the transverse cut just below the cricoid cartilage. Now remove the left and right primary bronchial branches with a transverse cut just above the carina with fine forceps.
Gently hold the trachea. Then using a one milliliter pipette, flush the lumen two or three times with L 15 medium to clear any mucus and blood. Next, using micro dissection scissors, make a transverse cut to remove a three or four ring segment of trachea.
Then in the small tracheal segment, make a longitudinal cut through the middle of the traches muscle. Lastly, cut longitudinally across the middle of the tracheal cartilage rings using microdissection scissors, leaving two sections of trachea tissue suitable for imaging. After the dissection transfer the trachea tissue segments lumen side down to the center of the glass bottom dish with approximately 100 microliters of L 15.
To quantify the CLIA generated flow at 20 microliters of a 2.5%suspension of fluorescent microspheres per milliliter of L 15 medium. Now place the silicon and glass cover over the trachea and give it a gentle push to secure it in place. Then along the edges, remove any excess media with a one mill pipette.
With now transfer the chamber to an inverted microscope fitted with a 100 x subjective and DIC optics. Using a high-speed camera and movie acquisition software, collect movies of CLIA motility at 200 plus frames per second along the ciliated trache epi epithelia of the curled up edge of the traches muscle. Using FS e epi fluorescent imaging and a low light CCD camera, collect movies of the fluorescent microsphere moving across the surface of the ciliated trache epithelia.
Using these movies proceed with quantification of CLIA generated flow Samples can be imaged for up to an hour without any noticeable alteration in Celia Beat function. Begin by calculating the CLIA beats per minute. Load the DIC imaged movies into the Image J software package.
Follow the technique described by Aian Drummond. Use the line tool to draw a raster line. Crossing the beating clia.
Make a res slice of this line to generate a graph type image where CA movement across the line generates a wave pattern. Now count the number of pixels between each wave peak along this line. From this count.
Calculate the beats per minute knowing that each movie frame is represented by one pixel along the line. Next, measure the C Cate generated flow. Load the fluorescent bead movies into the Image J software package.
Using the MT track J plugin manually track the fluorescent beads across the surface of the trachea epithelia. The software will calculate the velocity and directionality for each tracked bead. Take care to notice that the distance from the beading cilia can strongly influence the measured flow magnitude.
In this example, bead movement is fast at the surface of the beading cilia and drops off for beads further away in the bathing media to control for this bead tracings should only be collected at a fixed distance above the ciliated epithelia in all samples to allow correct comparisons to be made. Finally, for fluid flow, track the beads as long as possible. It is preferable to use beads that pass more than 10 ciliated cells to make these calculations.
Using the described protocol control airway cilia should be clearly visible and seen to be in a coordinated manner. Likewise, there should be noticeable flow in the direction of the beat. Typically, collection of high speed DIC movies allows for the quantification of C beat frequency and gives good images.
For qualitative assessment of CLIA beat shape. The tracking of fluorescent beads provides a measurement of CLIA generated flow velocity. Directionality is calculated by dividing the total displacement by a fluorescent bead by the length of the trace of a fluorescent bead.
Thus, a straight trajectory line scores one. While a meandering path scores a much lower value. While attempting this procedure, it's important to remember to properly clear the traches muscle from all surrounding connective and adipose tissue before cutting.
As curling of the traches muscle after it has been cut is essential for positioning the respiratory CLIA for ideal visualization. Following this procedure, other methods like pharmacological studies can be performed in order to answer additional questions like how or why do pharmacological agents modulate respiratory C activity?
