Protocol echocardiology/cardiac ultrasound using isoflurane
Prepared by: Laura Lee Davis, BSDate Prepared: February 18, 2005
Abstract: Echocardiography is ultrasound imaging of the heart. This non-invasive procedure is used to identify phenotypic deviants and abnormalities surrounding cardiac function primarily on mutagenized ENU B6 mice that have been on a high fat diet. This procedure is used to obtain measurements of the different heart chambers, valves and aorta and to observe color flow patterns and Doppler imagery to locate valve changes such as mitral stenosis or regurgitation.
Instrument layout: The ultrasound machine used is the Agilent Sonos 5500 manufactured by Philips. The machine is located in a barrier facility. There is both a 15-16L linear transducer and an S12 sector transducer. The sector transducer is used when performing acoustic quantification or color kinesis. A water pad bed connected to a circulating water bath is positioned adjacent to the machine for placement of the anesthetized mouse.
Reagents and expendables:
Expendables: Aqua sonic ultrasound transmission gel manufactured by Parker Laboratories, Inc. is used on the mouse to increase acoustic transmission. Isoflurane is the anesthetic agent used to sedate the mice during the procedure. It is purchased with Webster Veternarian Supply. The Isoflurane set up is purchased at Vet Equip, Pleasanton, CA. 1” utility masking tape is used to secure the mouse to the platform of the water pad. Depilatory for thoracic hair removal may be purchased anywhere.
Reagents: Magneto-optical disks manufactured by Hewlett-Packard are used to record images and data reports. Floppy disks are used to download and save presets for the machine. Videotapes may be used to record any images or analysis reports these can be of any manufacturer. The paper used to print out images and reports is manufactured by Sony.
Setup: Turn on the circulating water bath that attaches to the water pad. The water pad is maintained at a constant temperature of 38 degrees. It is attached by tubing to a heated circulating water bath manufactured by Lauda Model E100 which maintains the temperature by heating and circulating water through the water pad.
Turn the ultrasound machine on and allow it to go through a self-test procedure. Using the touch screen on the monitor, press the preset button to be sure that the machine is back to its original settings. During the initial start-up diagnostics, the equipment does an internal digital calibration. While the machine is warming up, prepare the Isoflurane equipment:
(1) Weigh each carbon filter and record on the cylinder. The carbon traps are used to capture the Isoflurane and prevent it from being released into the work area. When the weight of the cylinder increases by 50 grams they are considered saturated with Isoflurane and are discarded and replace with a fresh carbon filter trap.
(2) Make sure the valve to the clear housing unit is open. This means the valve will be turned the same direction as the clear tube. Make sure the valve for the tubing portion of the set up is closed. This means the valve will be turned so that the tubing and valve will be in a cross position. Tubes run from the Isoflurane Vaporizer to a Carbon Filter. There are valves that either open the tube’s circulation or close it. One tube controls the flow of Isoflurane and Oxygen to the clear housing unit of the Isoflurane set up (this is where the animal is initially exposed to knock them out) and another tube controls the flow of Isoflurane and Oxygen to the mask portion of the isoflurane set up, (this tubing has a nose cone attached and will be fitted to the mouse).
(3) Weigh animal. Record the weight.
(4) Place animal into the clear mouse box, (located on at the top of the Isoflurane set up). This housing unit will be ventilated with Oxygen and Isoflurane. Turn the Isoflurane Vaporizer on and adjust level gage to 3 (this is the large dial). Turn the Oxygen on and adjust the oxygen gage to 1. This level of Oxygen will be maintained throughout the entire scan. Wait until the mouse is very drowsy or asleep.
(5) Turn the valve for the lower portion of the Isoflurane set up on (the valve will be parallel with the tubing). This provides Isoflurane to the mouse nose cone. Turn valve connected to the clear housing unit to the closed position. (so the valve and tube are in a cross position). This turns off the gas to the clear housing unit.
(6) A small table with a warm water pad covering is located next to the Isoflurane set up. Tubing connected to the Isoflurane vaporizer and Oxygen is taped to this table. The tubing has a nose cone attached as well. Remove the mouse from the clear mouse box and immediately place the animal’s muzzle into the nose cone. Turn the Isoflurane Level back to 1 (this is the adjustment on the large dial).
(7) Secure mouse on the water pad in dorsolateral recumbency with the mouse’s left side angled down. This allows for optimal viewing capabilities. Apply depilatory to the thoracic area from the neck caudally to the midline and laterally to the axillary areas of both forelimbs.
Turn Isoflurane to .5 until the depilatory is removed.
(8) Set up EKG electrodes. Using Neonatal type cloth electrodes (like Philips Medical Supplies number 13951C) with one electrode on each of the front paws (coded white and black) and one electrode on a back leg (coded red).
(9) The amount of Isoflurane may vary to desired BPM (beats per minute). Adjust the metering valve to maintain a pulse of from 400 to 500 BPM.
Run: Apply ultrasound transmission gel to both the transducer and the contact area on the mouse. The mouse should only be very lightly sedated. In order to obtain accurate measurements, the heart rate of the mouse should not be below 400 beats per minute (BPM). If the heart rate is below 400 BPM, you may have to wait for the anesthesia to subside before actually proceeding with taking measurements.
While in 2D mode, place the 15-16L transducer with the index pointing over the right shoulder of the mouse. This will obtain a parasternal long axis view. Press the zoom button on the screen to enlarge the image (for accurate imaging, be sure the posterior septal wall is aligned with the anterior aortic wall). You should see a nice “football” shape of the left ventricle so that the interior chamber is opened up to its greatest diameter and foreshortening is reduced completely. From this view, you can obtain the internal aortic diameter and the left atrium interior diameter. By placing the cursor in the aorta just behind the aortic valve, switch to the M-mode (time motion) to obtain a waveform image. From this image, one can also obtain the aortic diameter and the left atrium dimension. Touch the analysis setting on the screen, then touch aortic root and place the caliper on the anterior aortic wall; touch caliper again and place the second caliper on the posterior aortic wall. Measurements are taken from leading edge to leading edge. Do the same for the left atrium, measuring in diastole. Measure several beats in succession to get a more accurate average measurement. Once these measurements have been obtained, switch back to the 2D mode. Zoom in on the heart again. Rotate the transducer clockwise until the index is positioned toward the mouse’s left side near the axillary area for the short axis view showing the left ventricle and papillary muscles; the right ventricle should appear in the upper left position of the image. Acquire a loop of this image and use this to measure the intraventricular septum, left ventricular interior dimension and posterior wall in systole. Once the image has been acquired, freeze it and use the trackball to slowly view the contraction of the left ventricle until it reaches peak systole. Because the heart rate of the mouse is so rapid, accurate measurements in M-mode are not always possible. The 2D image provides a real picture of the complete contraction of the left ventricle.
4.0 From the 2D view, place the cursor in the left ventricle between the papillary muscles, but not through them and switch to the M-mode. From this waveform, measure the intraventricular septal wall thickness in diastole and systole (although the systolic measurements in M-mode are not very accurate because the Agilent system does not allow a more detailed image); left ventricular interior dimension in diastole and systole; and left ventricular posterior wall thickness in both diastole and systole. As before, measure several consecutive beats to obtain an accurate average measurement for each. By rotating the transducer clockwise just slightly and angling it anteriorly, find the apical 4 chamber view. Zoom in on the image and adjust the angle of the probe so that the atria are not foreshortened. From this view, obtain the Doppler and color flow images which are used to measure Aortic and Mitral blood flow velocities, Aortic R-R Interval, Peak E Wave, Peak A Wave and Deceleration Time. Place the cursor in the left ventricle just above the tips of the mitral valve leaflets. Touch Pulse Wave Doppler to get the mitral and aortic waveforms. The mitral blood flow appears above the baseline, and the aortic blood flow appears below the baseline. By using the trackball, one can move the cursor toward the left ventricular outflow track and obtain a stronger aortic flow. Switch back to the 2D image of the apical 4 chamber view and zoom in on the image. Switch to the S12 transducer by changing the probe and presets. Now place the cursor in the left ventricle just above the tips of the mitral valve leaflets. Touch continuous flow Doppler and switch to color to visualize mitral regurgitation. Adjust the size of the flow area by selecting size and position alternately and moving the trackball until the appropriate area is outlined. Remember that red color flow is toward the transducer and blue color flow is away from the transducer.
Clean up: Clean up of the machine is done by simply wiping the transducer heads with a water moistened soft cloth. The ultrasound transmission gel is water soluble so clean up is easy. The mouse can be gently wiped off with 4 x 4 8 ply gauze. The mouse is placed in a warmed cage to complete recovery.
Data reduction: Measurements are compiled in analysis reports and saved on the magneto-optical disc. Each patient is entered as a separate file and may be retrieved from the optical disc for viewing later. Measurements may also be made at a later time from images previously saved. Because the nature of mouse data acquisition in the high-throughput protocol we follow, and because the Agilent machine is designed for human patient care, the management of data for mice is not as straightforward for this screen. The JAX PGA has facilitated transfer of data from this machine in a more usable format through what we call Muta Jax. The data is also stored on a private archive for emergency reasons.
Safety: Wear surgical gloves while handling the mice. Since this machine is in a barrier facility all personal are required to wear gowns, bonnets, face masks, booties and gloves. Safety concerns surrounding the equipment and its usage are minimal. Be cautious about containing the water in the water bath and monitor the waterpad for leakage. Anestheic precautions are to be sure you are in a well ventilated area. Weigh each carbon filter at the end of each daily use and record the weight on the side of the filter in the place provided. Dispose of spent filters properly following the procedures set by the laboratory.
Time and capacity: It requires approximately 30 minutes from the time of weighing and anesthetizing the mouse, securing it to the platform to obtaining the necessary images and measurements and completing the study. Clean up time is about 2 minutes.
Protocols: The Agilent Sonos 5500 is a very user friendly system. With the touch screen, there is easy access to various settings. When first using the system, program all the presets for cardiac imaging. Save your presets to a floppy disk should the preset data stored on the machine be lost. The preset button is also the default and will return to the original settings when you start up your machine. On the touch screen, there are also probe selections to switch from one transducer to another. Use the sector transducer (S12 probe) to perform color kinesis or acoustic quantification. Most of the settings (gain, compression, LCG, TGC) on the machine are for maximizing and perfecting the image quality which the operator can adjust to their own specifications. There are a number of various fine-tuning settings which are all described in the operating manuals supplied by the manufacturer. See bench protocol for a simplified operator checklist.
