The overall goal of this procedure is to demonstrate forced oscillation measurements of respiratory mechanics in mice using the flex event, a commercial computer controlled piston ventilator. This will be done through the assessment of airway responsiveness to inhaled methylcholine. The first step is to prepare the required reagents, the equipment, and the subject.
Then the subject is connected to the ventilator for mechanical ventilation and respiratory mechanics measurements. Using a predefined script, a collection of highly controlled automated measurements is made at baseline and following methylcholine challenge. Analysis of the results can show airway hyperresponsiveness as noted by the greater maximal responses of resistance and elastin in chlorine exposed mice compared to control subjects.
The main advantage of this technique over existing methods is that it provides an accurate tool by which to measure lung function in a comprehensive, detailed, and translational manner. This is accomplished through the analysis of pressure and volume signals acquired in reaction to predefined oscillatory airflow waveforms applied at the subject's airway opening. In addition, many factors that influence physiological response can be controlled and standardized.
Using this method, Turn on the Flex event FX system and start the software. The present protocol applies to either of the two flex event generations supported by flexi. Wear seven software at the first experimentation session or any time before it Open the study definition and planning module to predefine the study structure.
Click on the create a new study button and follow the wizard to create a study. Outline the protocol and define the experimental groups and subjects to be studied. Initiate an experimentation session by opening the experimentation session module and following the startup sequence.
For study and template selection. Assign a subject to the measurement site and confirm its weight. Proceed with the calibration of the system by following the steps described in the operating software.
There comes a prompt to attach the cannula to be used to the Y tubing for calibration. Continue with the calibration of the cannula by following the steps in the calibration wizard and verify that the calibration values obtained are within the specified range. If needed, repeat the calibration process unless ready, cancel the prompts to start ventilation and data recording.
These can be initiated at a later point. Anesthetize the subject using appropriate doses of anesthetic agents. Verify that the subject has reached a surgical level of anesthesia.
The subject should show no reaction to a toe pinch and its breathing should be regular and not labored. Position the animal supine with a heat source to maintain proper body temperature. A 60 watt bulb placed 45 centimeters from the mouse is adequate.
Then clean the throat area with alcohol to expose the trachea. Make an incision and gently separate the sub maxillary gland and the muscle layer covering it. Gently lift the trachea using a pair of micro forceps.
Then pass a suture underneath it. Cut between the two rings of cartilage closest to the larynx. This makes a small incision in the trachea without sectioning it.
Insert the calibrated cannula into the incision and gently advance it through five tracheal rings. In this example, a 1.2 centimeter metal 18 gauge cannula is in operation. It is critical to secure the cannula using the suture.
The attachment should form an airtight seal around the cannula. Bring the cannulated animal close to the ventilator. Start the mechanical ventilation by selecting a predefined or a customized ventilation profile in the ventilation docker.
Now connect the animal to the ventilator via the Y tubing. Carefully align the animal to the ventilator and ensure that the tracheal cannula is at the same level as the ventilator. This avoids a possible cannula, occlusion, or tracheal twist in the software.
Execute a deep inflation perturbation by double clicking on the perturbation name to verify the cannula insertion and attachment. Unless there is a leak, the system will hold a pressure of 30 centimeters of water over a three second period without excessive volume displacement. The recorded volume and pressure traces should be smooth with no signs of offset or deformation.
Otherwise, there may be a cannula, obstruction or misplacement at this point. Vital sign transducers for heart rate and body temperature monitoring can be connected if needed. Data recording of these signals can be initiated at any time, either manually or automatically via a script.
Measurements or commands for nebulizer activation, event markers and so forth can be automated using predefined or customized scripts for a highly controlled and repeatable experimental process. Six families of perturbations giving rise to a number of parameters can be used to describe the subject respiratory system mechanics at baseline and following a given challenge. When ready to start taking measurements, it is important to first run the deep inflation perturbation to recruit closed lung areas and standardize the lung volume history.
Next, verify the absence of spontaneous inspiratory efforts by running a test measurement. Observe the pressure signal traces in the selected dataset view. With stepwise PV curves.
Pressure plateaus should be well-defined with no downwards deflections. A downward swing in pressure would indicate an inspiratory effort from the animal. Now initiate the script for the assessment of airway responsiveness to inhaled methylcholine by double clicking on its title.
In this example, the script makes a sequence of baseline measurements in triplicate. Then there is a prompt to load the nebulizer with saline. Or in this example, a solution of methylcholine load approximately 100 microliters of solution into the nebulizer.
The nebulization will start automatically as soon as the information is entered in the software. Shortly after the end of nebulization, a sequence of closely spaced measurements is initiated for approximately three minutes. Next, a prompt to perform.
Another challenge is provided and the sequence of measurements can be repeated between challenges. Drying the inside of the nebulizer mount with a swab can help prevent droplets or condensation from building up in the inspiratory line. As data is collected, the software calculates parameters associated with perturbation and provides a coefficient of determination based on the fit of the data to the model.
Later, the experiment can be accessed via the review and reporting module of the software to export or further analyze data. At the end of the experiment, stop the ventilation and detach the subject. Once detached quickly euthanize the animal with an overdose of sodium pentobarbital, then a bronchoalveolar lavage may be performed or the lungs may be isolated for further analysis.
Before switching to the next subject, rinse and dry the nebulizer adapter y tubing and cannula. Switch to the next subject in the operating software and confirm its weight. Then proceed with the suggested calibration steps before continuing with the experiment.
At the end of the day, rinse and dry the equipment. Remember, before closing the experimental session to also clean the system expiratory valve according to the manufacturer's instructions in order to maintain the performance of your system. The following examples represent a selection of typical results from naive and chlorine exposed mice experiments equivalent results from naive aj.
Mice were obtained at baseline and following methylcholine induced broncho constriction using either of the two flex event generations supported by flexi wear seven. Because broadband FOT measurements permit a partitioning of the lung response into airway and parenchymal tissue parameters, they provide a mean to identify affected lung regions. For example, naive AJ mice showed an increase of baseline resistance when the end expiratory pressure was increased from three to nine centimeters of water.
The change in end expiratory pressure resulted in a decrease in the airway resistance consistent with the bronchodilating effects of a higher lung volume and the larger inflation pressure, and an increase in tissue damping, a parameter closely related to tissue resistance that reflects tissue viscoelasticity and possibly the resistance of the small airways. The latter is known to increase with increasing lung volume. This protocol is often used to assess airway responsiveness to inhaled methylcholine after exposure to chlorine gas.
B.SEA mice exhibited airway hyperresponsiveness compared to air exposed controls. In the present example, mice exposed to chlorine gas displayed greater maximal responses at all FOT parameters. These mice also showed a statistically significant leftward shift of the concentration response curve or hypersensitivity to inhaled methylcholine, which is exemplified by a reduction of the concentration of methylcholine required to cause a doubling in resistance and elastin.
In addition to FOT, the flex event system can record other measurements such as pressure volume curves. The upper portion of the deflation limb is fit to the Salazar Knolls equation, and the associated parameters are calculated by the software. In order to succeed with this technique, it's important to pay attention to each step of particular importance are the calibration of the system, the resistance of the endotracheal cannula, positioning of the animal, and the standardization of lung volumes.
If these steps are followed precisely, this method will provide you with an accurate characterization of mechanical properties of the mouse lung.
