Name: ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia
Uploaded: 2013-08-08T13:00:00
Description: Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

The project was supported by NIH grant U01HL098180 from the National Heart, Lung, and Blood Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.

1. Reagents Setup
1.1 Dissection and Imaging Medium
Leibovitz's L-15 medium (L15) is supplemented with FBS (10%) and Penicillin-Streptomycin (100 units/ml of penicillin G sodium and 100 &mu;g/ml of streptomycin sulfate) is used during both harvest and imaging of trachea samples.
2. Shallow Walled Culture Chamber Assembly
The chamber used to hold trachea tissue is shown in Figure 1. The floor of the ch...

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Measurement of cilia beat frequency (CBF) is relatively easy using high power microscope objectives and fast image acquisition hardware 13,15, and explains why CBF measurements form the basis of most studies investigating mucociliary clearance during health and disease. However, while CBF measurement is essential for understanding mucociliary clearance, measurement of CBF alone ignores the underlying importance of both ciliary generated flow and cilia beat waveform, both of which are more difficult to measure,...

Analysis of motile cilia function in the airway epithelia is experimentally important for elucidating the genetic and environmental factors that can affect mucociliary clearance and pulmonary health 1. The simple protocol developed for imaging the mouse airway epithelia provides an efficient method to interrogate airway cilia motility in mutant and knockout mouse models and require only basic skills in mouse tracheal tissue dissection and ex vivo imaging of airway cilia motility with high resolution videomicroscopy. This protocol was established and refined during a large-scale mouse mutagenesis screen to allow rapid evaluation of motile cilia function (cilia beat frequency, cilia beat shape, cilia generated flow) in mutants with congenital heart disease associated with heterotaxy 2-5.
Current techniques used to study airway cilia motility can be grouped into either the acute ex vivo type or longer term in vitro experimental approaches. Acute experiments include ex vivo visualization of human nasal/airway brush biopsies 6,7 and analysis of simple transverse airway sections 8. The in vitro approaches utilize various cell culture techniques to generate sheets of differentiated ciliated epithelia such as in air liquid interface cultures or airway suspension cultures9-11. However, these airway epithelia reciliation techniques require very significant investment in time and training before any useable ciliated epithelial cells are produced for experimentation (4-6 weeks 9,10). While acute ex vivo analysis of airway epithelial brush biopsies are commonly used for human clinical studies, this method is not usable in mouse studies due to exacerbated mechanical tissue injury 12.
 
The technique outlined in this protocol for analysis of mouse tracheal airway epithelia is not only simple to execute, but it requires no special dissection skills nor any specialized equipment besides those standard for imaging by videomicroscopy. There are many advantages to this simple protocol. First, as the mouse trachea tissue harvest is quick and easy to perform, it allows for rapid assessment of airway cilia function in a large number of mice. This can include acute analysis of the short term effects of different in vitro treatments. Second, being an ex vivo technique, the ciliated airway epithelium remains attached to it underlying supporting tissues and thus retain associated cell signaling pathways. Therefore in comparison to in vitro reciliated airway epithelia, this preparation is a better representation of the natural in vivo tissue environment. Third, this protocol allows the acquisition of a number of different quantitative parameters that can provide the objective assessment of motile cilia function. Finally, in contrast to other current methods for airway cilia visualization, this protocol allows for visualization of the cilia at right angles to the cilia beat direction, allowing profile view of the cilia that is optimal for high resolution imaging of cilia beat and metachronal wave generation.
This protocol can be modified in a number of ways to address a wide range of experimental needs such as the role of pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load on airway cilia function and generation/maintenance of airway cilia beat and metachronal wave propagation.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 Medium </td>
 <td>Invitrogen </td>
 <td>21083-027 </td>
 <td>No phenol red </td>
 </tr>
 <tr>
 <td>Fetal bovine serum </td>
 <td>Hyclone </td>
 <td>SH30088.03 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin </td>
 <td>Invitrogen </td>
 <td>15140-122 </td>
 <td></td>
 </tr>
 <tr>
 <td>2x fine forceps </td>
 <td>Roboz </td>
 <td>RS-4976 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5676 </td>
 <td></td>
 </tr>
 <tr>
 <td>Micro dissection scissors </td>
 <td>Roboz </td>
 <td>RS-5620 </td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel </td>
 <td>Roboz </td>
 <td>RS-9801-15 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 pipetman </td>
 <td>Gilson, Inc </td>
 <td>F123602 </td>
 <td></td>
 </tr>
 <tr>
 <td>P1000 tips </td>
 <td>Molecular BioProducts </td>
 <td>2079E </td>
 <td></td>
 </tr>
 <tr>
 <td>18 mm round glass cover slips </td>
 <td>Fisher Scientific </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic 35 mm culture dishes </td>
 <td>Corning </td>
 <td>430588 </td>
 <td></td>
 </tr>
 <tr>
 <td>Glass bottom 35 mm culture dishes </td>
 <td>Warner Instruments </td>
 <td>W3 64-0758 </td>
 <td></td>
 </tr>
 <tr>
 <td>Silicone sheet 0.012" (0.3 mm) thick </td>
 <td>AAA Acme Rubber Co </td>
 <td>CASS-.012X36-63908 </td>
 <td></td>
 </tr>
 <tr>
 <td>0.20 &mu;m diameter Fluoresbrite YG Carboxylate Microspheres </td>
 <td>Polysciences </td>
 <td>09834-10 </td>
 <td></td>
 </tr>
 <tr>
 <td>Inverted microscope, with 100x oil objective and DIC filters </td>
 <td>Lecia</td>
 <td>DMIRE2 </td>
 <td>Brand is not critical. </td>
 </tr>
 <tr>
 <td>100-watt mercury lamp, epifluorescent FITC excitation/emission filters</td>
 <td>Lecia </td>
 <td></td>
 <td>Brand is not critical.</td>
 </tr>
 <tr>
 <td>Microscope stage Incubator</td>
 <td>Lecia </td>
 <td>11521749 </td>
 <td>Not required if imaging cilia at room temperature</td>
 </tr>
 <tr>
 <td>High-speed camera bright field </td>
 <td>Vision Research </td>
 <td>Phantom v4.2 </td>
 <td>Brand is not critical. Must be faster than 125 fps</td>
 </tr>
 <tr>
 <td>High-speed fluorescent camera </td>
 <td>Hamamatsu </td>
 <td>C9100-12 </td>
 <td>Brand is not critical. Must be faster than 10 fps</td>
 </tr>
 <tr>
 <td>Movie analysis software</td>
 <td>National Institutes of Health</td>
 <td>ImageJ with MtrackJ plugin</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Control airway cilia should be clearly visible and seen to beat in a coordinated manner (Supplemental Movie 1; movie playback is slowed to 15% real time), with noticeable flow in the direction of cilia beat (Supplemental Movie 2; movie playback is 100% real time). Quantitation of cilia movies should yield results similar to that seen in Figure 3. Collection of high-speed DIC movies makes possible the quantification of cilia beat frequency (Figure 3C) and...

Watch this Scientific Journal Video about Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia at JoVE.com

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.

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  ...

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