Name: in vitro measurements of tracheal constriction using mice
Uploaded: 2012-06-25T12:00:00
Description: Watch this Scientific Journal Video about In vitro Measurements of Tracheal Constriction Using Mice at JoVE.com

This work was funded by a grant from the Center for Innovation in Prevention and Treatment of Airway Diseases, NINDS grant (NS052574), and from the Sandler Program for Asthma Research.

1. Equipment
The major components of a contraction measurement device are shown schematically in Figure 2A).
<ol>
 <li>A tissue bath. The tissue bath maintains an oxygenated physiological solution at warm temperature. For mice trachea rings, we use a 10 ml tissue bath that contains a water jacket for circulating a warming solution, a fritted glass inlet to bubble oxygen (95%/5% O2/CO2 mixture) and inlet and outlet ports for changing sol...

<ol>
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The protocol presented here provides a physiological preparation to assess airway muscle function. We generally operate 3-4 organ bath preparations simultaneously, however, prepackaged systems are available from a number of suppliers that allow simultaneous measurements of up to 8 preparations (ADInstruments, World Precision Instruments, and Harvard Apparatus). We have utilized a number of force transducers and tissue organ baths with equivalent results. However, we find that electric field stimulation provides som...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Analogue-Digital Converter</td>
 <td>ADInstruments</td>
 <td>PowerLab 4/35</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Carbachol
 (Carbamoylcholine Chloride)</td>
 <td>Sigma-Aldrich</td>
 <td>C4832
 </td>
 <td>10-2 M in water (aliquots can be stored at -20&deg;C)</td>
 </tr>
 <tr>
 <td>Charting Software </td>
 <td>ADInstrtuments</td>
 <td>LabChart</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heating Circulator</td>
 <td>Haake</td>
 <td>Mixer Mill MM400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Isometric Force Transducer</td>
 <td>Kent Scientific</td>
 <td>TRN001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Stimulator</td>
 <td>Grass Technologies</td>
 <td>S88 Dual Output Square Pulse Stimulator</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tissue Bath</td>
 <td>WPI</td>
 <td>47264</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

in vitro measurements of tracheal constriction using mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

Watch this Scientific Journal Video about In vitro Measurements of Tracheal Constriction Using Mice at JoVE.com

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

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