This video demonstrates a procedure for performing high frequency, high resolution echocardiography in ischemia. Reperfused mirroring hearts ischemia is surgically induced occluding, the left interior descending artery of an anesthetized mouse. Following recovery, the mouse is positioned on an ECG anesthesia, air coupled warm platform.
Movies are acquired in B mode to obtain long axis strain analysis and mode for heart function strain imaging and synchronicity and color doppler mode for velocity profile. Movies are then processed to obtain strain curves, strain based synchronicity, and to obtain the rate of change of LV pressure. Hi, I am Dr.Chandon Sen from the Davis Heart and Lung Research Institute and the Department of Surgery at the Ohio State University Medical Center.
High Frequency, high resolution echocardiography is a novel and powerful tool to obtain clinical relevant data by way of repeated measure from the same animal. Today we will show you how to use this technique to measure myocardial strain contractibility, as well as mitral regurgitation in the isch perfused mure heart. First, Jason Driggs from my laboratory will demonstrate surgical procedures for the induction of isch, perfusion of the murine heart.
Next, Dr.Surya Al, a biomedical engineering postdoctoral fellow in my laboratory will demonstrate the imaging procedures. Finally, Dr.Thomas Ryan, director of the heart Center at the Ohio State University Medical Center. Also, a clinical expert in e echocardiography will deliver the concluding remarks.
So let's get started. Begin this procedure by placing an anesthetized eight week old male C 57 black six mouse on a heated surgical table. The animal's body temperature should be monitored with a rectal thermal probe and should be maintained at 36.7 plus or minus 0.5 degrees Celsius throughout the surgery.
Disinfect the skin with a disinfectant followed by alcohol and repeated three times each intubate by making a 0.7 centimeter medial incision over the trachea. Visualize the trachea by bluntly dissecting nearby glands. Then pass a 20 gauge catheter through the mouth as an ET tube Using a catheter tip ventilate the mice with 0.5%isof fluorine.
To maintain anesthesia, close the skin around the neck with a five oh suture. Set up a three lead ECG by inserting subcutaneous needles to monitor cardiac electrophysiology throughout the surgery. Changes in cardiac electrophysiology should be recorded.
Using PC power lab software. Rotate the animal to expose the left side. Make an incision along the transverse plane.
Then bluntly dissect through the fourth and fifth intercostal spaces. Keep the chest open with copper rib spreaders while cutting the pericardium. Next, retract the left lung to allow access to the pericardium between the pulmonary artery and left atria.
Along the septal wall lies the left anterior descending artery past 7.0. Proline sutures under it. Then place PE ten two big on top of it and tie the tubing into place.
Leave the tubing for 60 minutes to induce ischemia. After 60 minutes, cut the ends of the suture in the loop to reperfuse the heart. Close the thorax with a seven oh proline suture.
Close the skin around the thoracic region with five oh proline sutures. Remove the gas source from the ventilator and allow the rodent to breathe room air. Once spontaneous breathing occurs and the animal response to toe pinching, put it back into a fresh cage on a 37 degree Celsius incubator.
Remove the tracheal tube when the animal is nearly conscious and no problems are evident from the animal. Once the animal has recovered, it can be returned to the vivarium until it is imaged. The imaging protocols described here should be performed both prior to three days and seven days following the induction of ischemia reperfusion.
To assess changes in cardiac function and wall motion abnormalities, begin the imaging procedure by positioning the isof fluorine anesthetized mouse in the prone position beneath the probe of a vivo 2100 imaging system. Place a nose cone to provide a continuous supply of 1.5%ISO fluorine air apply conducting gel to the mouse. Start the vivo 2100 and initialize the transducer MS 400.
Use the rolling track ball on the vivo 2100 control panel to select cardiology and acquire standard M mode image. Place the probe on the mouse, then click ctor to begin movie acquisition. To perform measurements, click measurement and trace at least three heart cycles on both upper and lower leaning of the LV chamber.
The following parameters will appear on the screen wall dimensions, volumes at systole and diastole stroke volume, ejection fractions or EF fractional shortening or FS cardiac output or co internal diameters. At systole and diastole, the parameters automatically appear on the screen once the tracing is complete. After the M mode imaging has been completed, place the mouse in a parasternal position and click on B mode.
To perform strain imaging, click sinister. Then using the probe, acquire short and long axis B mode movies. To analyze the movies, select B mode movie and click on the vivo strain icon to use the speckle tracking algorithm.
A new window will appear with B mode movie on the left and start analysis on the right. Trace the epi and endocardial borders. Click the start analysis icon to open another window.
Then select velocity displacement strain and strain rate. Choose a point of interest here. The injury site is chosen and the software plots curves corresponding to the injury point for all of the selected parameters.
Now click the right mouse button to copy the plots or export the data. For analysis of the myocardial synchronicity, go to the right of the new window and click on the time to peak analysis icon. Now a window will appear showing the LV segmental synchronicity page and the radial and longitudinal velocity displacement strain and strain rate selection options.
Select the strain button, then store the data. Repeat this process for displacement strain and strain rate. Keep the animal in the parasternal position to perform doppler flow measurements.
Begin using the tracking ball to place the sample volume in the mitral valve, aortic outflow and mitral jet. Click on PW to get flow profile. A new window will appear with the movie displayed at the top and velocity profiles at the bottom click image store.
To obtain a still image, proceed to acquire power doppler regurgitant flow velocity profiles of the mitral valve, regurgitant jet and aortic jet in the same way by placing the sample volume in the desired location, clicking on PW and then storing the image To acquire color doppler flow, go to the control panel and click on color. Then using the color coded blood flow image, find the orifice radius by clicking measure and selecting the length icon provided in the menu. Next, select MV flow from the dropdown menu.
Then place the cursor on the initial and final points of the steepest slope of the velocity profile. And click the left mouse button to see the velocity at the respective points from these velocities. Use a modified RNLI relation to find the pressure drop DP over DT in millimeters of mercury.
Next, subtract the aortic outflow from MV inflow in terms of blood volumes to determine the regurgitant volume, a marker of regurgit severity. Once all of the images have been acquired, remove the nose cone and place the mouse in its cage when the animal has recovered. Transfer it to the vivarium here.
Representative results of myocardial radial and circumferential strain, synchronicity, color flow and doppler flow measurements are shown. This movie of myocardial strain measurements performed three days following ischemia. Reperfusion demonstrates limited contraction at the injury site length and arrows of the vectors observed were abnormal at the injury site demonstrating deformed myocardium.
The color coded graphs represent the strain data corresponding to the color points in the movie. This image shows sectoral tissue synchronicity based on radial strain. Note that a synchronization time greater than 20 to 30 milliseconds is considered asynchronous.
These images taken three days post IR show the measurement of diameter of a mitral valve and demonstrates the mitral jet or face area. Here the measurement technique of functional parameters in pre IR mouse is used tracing the myocardial border using software enables evaluation of changes in parameters between pre IR and post ir. The red vertical line equals diastole the green vertical line systole.
This movie illustrates color doppler flow with mitral regurgitation. In post IR day three, this movie visualization of the blood flow in the left atrium, mitral valve, left ventricle aorta and right atrium is in turn used to find the mitral jet orifice area. As earlier, This is the first report demonstrating the potential of high frequency ultrasound to study ischemia reperfusion in the mouse model.
It is the first time that sophisticated quantitative parameters such as strain, synchronicity, and mitral regurgitation have been demonstrated in this model. As someone who's been involved in the field of echocardiography for almost 30 years and works regularly with patients with coronary artery disease, I can tell you that these findings are clinically relevant and this methodology has has importance to the study of ischemia and reperfusion. When doing these procedures, it's important to remember the following status and dose is crucial, minimal is recommended, acquiring para sternal long axis view needs, experience hands measurement of regurgitant peak velocity is to find DPDT should be done carefully to minimize errors.
So that's it. Thanks for watching and good luck with your experiments.
