The overall goal of this procedure is to demonstrate how to accurately evaluate diaphragm contractility in a mouse model. Using ultrasound imaging, the first steps are to anesthetize the mouse and remove its abdominal hair. The next steps are to apply ultrasound gel and resolve the diaphragm image using the ultrasound probes.
Ultimately, the results can show diaphragm excursion and respiratory rate through utilizing the electronic calipers of the software. The main advantage of this technique over existing methods like invasive myography, is that it is not invasive and reduces the number of animals sacrificed. The implications of this technique extend toward the diagnosis and potential therapy of respiratory muscle dysfunction.
Though this method can provide insight into diaphragm contractility in a mouse model, it could also be applied to other systems such as clinical models to evaluate disease progression and therapy efficacy. Set up a clean procedure table with a heated isothermal pad wrapped in a surgical towel. The heated pad should be used to stabilize the animal's core temperature while also reducing potential stress to the animal.
The mouse should be placed in the anesthesia induction chamber with an oxygen flow rate set to 1.5 liters per minute, and with 3.5%vaporized isof fluorine Sedation usually sets within two minutes and is noted by loss of voluntary motor function. Then maintain the anesthetized state by nose cone gas delivery. After anesthetizing the animal apply a small amount of ophthalmic ointment directly to the corneas to reduce eye dryness during the anesthesia.
The mouse should never show the pedal withdrawal reflex. The mucus membranes should always have a pink color and the animal should breathe steadily. Restrain each leg of the anesthetized mouse on the heated procedure table with a removable adhesive, such as surgical tape.
Using an electric razor, remove most of the hair on the ventral body surface between the abdomen and halfway up the thoracic cavity. Then apply hair re removal cream to remove the remaining hair. After two to three minutes, wipe off the cream with a damp gauze pad.
Also, remove the excess hair with water and gauze. Next, clean the shaved region with four scrubs of 70%alcohol or an equivalent antiseptic turn on the ultrasound device. A micro convex array or linear phased array transducer is ideal due to these probes, small footprint and excellent axial resolution.
Adjust the ultrasound frequency to a range of 6.5 to 12 megahertz. Now set the machine's mode, brightness and motion modes together, provide the best imaging of diaphragm contraction. Next, apply a small amount of ultrasound gel to the upper abdomen and massage the gel toward the thoracic cavity.
Place the ultrasound transducer on the gel and angle it upwards towards the heart. Adjust the probe to optimize the image resolution. This is challenging, but will become easier with practice.
It's very important to remember to monitor the animal's respiration while under anesthesia, just to make sure that the results of the examination are not compromised. Press the freeze button to temporarily save the diaphragm images and view the selected contractions. Save the recording as a cyl loop, which allows for later measurements of diaphragmatic excursion and respiration rate.
After imaging, the mouse should completely recover from the anesthesia within 30 minutes. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbent. To analyze the data in M mode, measure the depth and rate of diaphragm movement from relaxation to contraction using the electronic calipers that are part of the ultrasound software.
Alternatively, in B mode, convert the CINA loop file into an MP four file and determine the respiration rate by counting the number of diaphragmatic contractions during the recording period, the electronic calipers may also be used in B mode to determine the depth of diaphragm movement. A mouse's diaphragm, maximal vertical displacement was recorded and the distance was then calculated as described by the protocol using electronic calipers. Such measurements were made on three different mice.
To illustrate, its after converting the CIL loop file into an MP four file. The respiration rate was determined by counting the number of diaphragmatic contractions during a six second recording period. This analysis was performed using B mode.
Alternatively, M mode provided a visualized picture of diaphragm vertical motion as well as respiration rate. There are some potential imaging artifacts which may occur when performing diagnostic ultrasound imaging such as reverberation. Another artifact that can be encountered is the presence of comet tales Once mastered, this technique can take 20 to 30 minutes if performed properly.
After watching this video, you should have good understanding of how to accurately evaluate a diaphragm contractility in the mouse model using a diagnostic ultrasound imaging technique.
