The overall goal of this procedure is to measure contractility of tracheal smooth muscle from mice. This is accomplished by first preparing the tissue baths and calibrating the isometric force transducer. Next, the mouse trachea is isolated and mounted on the force transducer rods.
Then the trachea is stimulated and the resulting tension is measured and analyzed. Ultimately, results can show contractility of tracheal muscle through isometric force measurements. The main advantage of this technique over existing methods, like in vivo measurements of airway resistance, is that in vitro tension measurements provide a more direct assay of smooth muscle contractility.
The disadvantage of this technique is that it is an organ bath preparation, and therefore circulating factors that may affect smooth muscle contractility in vivo may be absent in the in vitro preparation. Though this method can provide insight into mouse tracheal smooth muscle. It can also be applied to other smooth muscle tissues, such as large vessels, various gut and bladder smooth muscle where ring preparations of muscle are sufficiently large, that they can be mounted on forced transducer wires.
Two components of the contraction measurement device require some description there, the tissue bath and the force transducer. The tissue bath maintains an oxygenated physiological solution at a warm temperature for mice trachea rings. A 10 milliliter tissue bath is used, which is equipped with a water jacket for warmth glass inlet to bubble oxygen and inlet and outlet ports to change solutions stored away.
There is a reservoir of PSS solution bubbled with oxygen and maintained at 37 degrees Celsius by a water bath for solution exchanges. The PSS solution is pumped from the reservoir to the tissue bath inlet at approximately 100 milliliters per minute. Meanwhile, a heated circulator pump moves 37 degree Celsius water through the tissue bath jacket.
The second piece of equipment of interest is the force transducer. This is used to measure isometric tension in the trachea. The trachea tube is threaded over the L-shaped ends of two rods made of biologically compatible material such as stainless steel.
The top rod is connected via a clip to an isometric force transducer. The bottom rod holds the trachea at a fixed position and is mounted on a micrometer for adjustment of passive tension and or muscle length. Contraction of the trachea creates tension on the force transducer, which is converted to a voltage signal at the pre amplifier for electrical stimulation.
The bottom rod is configured with two rectangular platinum plates placed four millimeters apart. To flank the trachea. The platinum plates are wired to a conventional stimulator such as a grass S 88.
In this preparation, the open wires must be soldered and coated with so guard to prevent leaching of metals into the bath solution. Additional equipment includes an ad converter computer and acquisition software. Signals from the pre amplifiers are recorded on a Mac Lab eight ad system.
Before beginning the tissue isolation. The tissue bath must be filled with normal PSS and the air inlet must be adjusted to obtain a light stream of oxygen. Also, calibrate the force transducers mount known weights on the force, transducer wires, and calibrate the acquisition software accordingly.
Sacrifice mice that are at least two months old because the smaller tracheas of younger mice are more difficult to dissect and mount. We have observed that aine triol sedative commonly used in mice has a strong relaxant effects on higher waist smooth muscles, and therefore should not be used for trachea contraction studies. After isolating the trachea, place the isolated trachea in a s cigar coated dish with ice cold oxygenated PSS solution.
Pin the trachea to the dish below the bifurcation and above the pharynx under a dissection microscope. Remove the surrounding tissues with forceps. Hold the trachea at the pharynx or below the bifurcation, but never directly apply the forceps to the trachea itself.
Similarly, when cutting tissues away, only cut parallel to the trachea after removing surrounding tissue. Cut the trachea perpendicularly below the pharynx and above the bronchial bifurcation. The trachea may now be mounted to the device.
The mounting of the trachea should be done as quickly as possible to minimize the time that the trachea are held outside of PSS carefully, but quickly thread the trachea over the two L-shaped metal prongs. Then quickly raise the tissue bath to immerse the trachea in warm PSS. Now that the trachea is back in PSS slowly adjust the micrometer to obtain a passive tension of about five to 10 millinewtons or one half to one gram of force.
This is the optimal resting tension over the next five to 10 minutes. The tension will decline, so continue to make adjustments to maintain the desired tension until it has stabilized. Once stable, allow the trachea to equilibrate for at least one hour before experimentally evoking contractions.
Following equilibration, contractions can be evoked with potassium, electrical field stimulation, or drugs of interest. To use potassium stimulation, challenge the trachea with a high potassium PSS solution. Generally, after five to 10 minutes, the contractions reach a steady state at that time, rinse the bath several times with normal PSS to completely relax the trachea.
Then keep switching back to the high potassium PSS solution. Until reproducible contractions are obtained, usually no more than three baths are ever needed. Electrical field stimulation is delivered by platinum plates that flank the trachea.
The contractile response is affected by the area of the electrodes and the distance between them. Due to the power characteristics of the stimulator, the current outputs reach a maximum at higher voltages. A near maximum response can be found using 0.5 millisecond pulses or 44 volts delivered at 30 hertz intervals.
But this will vary between systems and should be determined empirically. The system also permits measuring the response of the trachea to a drug delivered exogenously via it. The bath solution, single doses of a drug or multiple additions of a drug in a cumulative dose fashion can be evaluated.
For example, carbahol can be used to activate cholinergic receptors. Unlike acetylcholine, it is not degraded by acetylcholinesterase and produces a more stable response. Fitting the results to a logarithmic response curve with a hill type equation allows an estimation of the half maximal effective concentration or C 50.
A tracheal preparation was stimulated with a high potassium bath. The contraction reached a maximum within approximately 10 minutes. During early washout of high potassium, the muscle showed a transient increase in contraction due to a drop in temperature.
As the small volume of unheated PSS solution. In the solution lines, transiently perfused the preparation. Next two tracheas with different muscle mass were challenged with high potassium and carbahol.
Although their cholinergic evoke contractions differed, they were similar after normalization to the response with high potassium solution. Next, carbahol was used to evoke contraction using single doses and a cumulative increase. OL solutions were added directly to the bath.
It is worth noting that addition of a single dose had a slightly larger response than the equivalent concentration during a cumulative dose response. Plotting the contractile force as a function of caracol concentrations showed that the effects of carbahol saturate at 10 to the minus five molar tracheas were subjected to EFS using 0.5 millisecond 40 volt pulses until contractions reached a plateau. An increase in stimulation frequency caused an increased contractile response.
This may be because EFS has been shown to evoke contractions predominantly by activating presynaptic nerves. For example, botulinum toxin blocks the majority of the EFS evoked contractions of trachea. Moreover, tetrodatoxin inhibits nerve activity and eliminates the response of trachea to EFS.
Once master, this technique can be done in roughly one and one half hour if it is performed properly. However, tracheas are viable in organ bus for many hours in log experiments.Unnecessary.
