The overall goal of this methodology is to acquire reliable and reproducible outcome measurements for the evaluation of new therapies in a pig model of acute myocardial infarction. This method can help answer key questions in the cardiovascular field, such as the effect of therapeutic interventions on the schema of a fusion injury and for modeling also an acute myocardial infarction. The main advantage of these techniques is that they allow for an accurate and a clinically relevant assessment of ischemia reperfusion injury and cardiac remodeling.
Through this method, can provide insight into myocardial ischemia reperfusion injury. It can also be applied to other studies involving the evaluation of cardiac function. The current protocol allows for the acquisition of primary outcome measurements prior to, or after an acute myocardial infarction.
The infarction is made using a 75-minute transluminal balloon occlusion of the left anterior descending artery, which is detailed by the cited video. This video demonstrates how to perform three dimensional transesophageal echocardiography, and the pressure of volume loop measurements for baseline, short-term, and long-term follow up measurements. The video also demonstrates how to measure infarct size and area at risk staining after termination.
Prepare the pig with anesthesia as described in the text protocol, and connect the animal to the echocardiography machine using the EKG leads. Now, prepare the echo probe. Put some ultrasound gel on the tip and unlock it so that it is flexible.
Adjust the imaging angle to 90 degrees using the handheld operating piece. With the pig in a right lateral position, open the mouth and carefully insert the probe into the esophagus. Be careful not to apply too much pressure, and to avoid the normal anatomic pharyngeal pouch, which resembles a Zenker's diverticulum.
Then, insert the probe 50 to 60 centimeters, measure from the tip of the snout, and slowly rotate and flex the head to a left anterolateral position to visualize the heart. Make sure all of the heart's walls are clearly visible. If necessary, lock the echo probe tip to stabilize the image acquisitions.
On the display, toggle the 3D Full Volume option to produce to perpendicular images of the left ventricle. Then, maximize the sector width that is being acquired by selecting FV Opt Volume. Now, pause the ventilation and press Acquire to obtain full volume measurements.
After obtaining a complete measurement, switch on the mechanical ventilation. After the echo acquisition, unlock the probe and slowly remove it from the animal. Then, monitor the pig until it has recovered from the surgery.
In preparation, soak the sensing tips of the catheter in 0.9%saline for at least 20 minutes. After preparing the pig for surgery, infuse 100 international units of heparin per kilogram, and place an 8F introducer sheath in the carotid, and a 9F sheath in the jugular vain. Next, insert a Swan Ganz catheter thought the 9F sheath, inflate the balloon at the tip of the catheter, and wedge it in a small pulmonary artery.
Once adequately placed in there peripheral part of the lung, deflate the balloon and connect the Swan Ganz catheter to an external cardiac output device. Upon connection, view the cardiac output menu on the external cardiac output device. Enter the weight of the animal into the system and calculate the length so the BMI is 22.5.
Proceed by loading a 20 milliliter syringe with room temperate 0.9%saline and attaching it to the injection port connected to the lumin with the most proximal debouchment. Switch off the ventilation and wait for five heartbeats. First, press Manual, then press Start on the output device.
Then, rapidly infuse five milliliters of saline to measure the cardiac output. Within 15 seconds, the device measurements will be shown. Then, switch on the ventilation.
Repeat this procedure three times and calculate the average stroke volume. Between infusions, wait a minute or two to allow the end-tidal carbon dioxide to return to preexisting values. Now, remove the Swan Ganz catheter and insert an 8F Fogarty catheter through the 9F sheath in the jugular vein, and position it in the inferior vena cava.
While the tip remains in 0.9%saline, calibrate the pressure signal of the PV loop catheter as close to 0 as possible, using the course and fine buttons. Then, input the measured stroke volume into the system. The next step is to advance the PV loop catheter through the 8F sheath in the carotid artery, and, while using fluoroscopy, advance and center the tip into the left ventricle.
Now, plot the raw conductance signal against the pressure signal, which should make a rectangular loop. Using this graph, select the largest adequately placed segment. Then, pause the mechanical ventilation and press enter on the pressure volume loop module to take a baseline scan to convert conductance to volume.
When the module shows the heart rate, systolic and diastolic conductance, turn the respiration back on. Before proceeding, the measured heart rate must be equal to the EKG, or pressure derived heart rate. There cannot be any arrhythmia, and the systolic and diastolic conductance must be adequately sensed.
If the baseline data is determined to be adequate, accept it by pressing enter and write down the heart rate and systolic and diastolic conductance shown on the PV loop module. Then, plot the volume signal against the pressure signal. The loops must still be rectangular.
Now, pause the ventilation and acquire baseline pressure volume by recording 10 to 12 consecutive heartbeats. Then, switch on the ventilation again and allow the end-tidal carbon dioxide level to return to preexisting values. Next, under fluoroscopic guidance, inflate the Fogarty catheter to reduce the preload.
Then, record 10 to 12 consecutive beats without ventilation as before. The systolic blood pressure must remain above 60 millimeters of mercury, and no arrhythmia can interfere with the measurements. After acquiring the data, deflate the Fogarty catheter, switch on the ventilation, and wait for the end-tidal carbon dioxide to return to preexisting levels.
Now, remove the Fogarty and PV loop catheters while continuing to record arterial pressure to correct for pressure drift. Finally, monitor the animal until it has recovered before returning it to the other animals. After preparing the animal for surgery, access both carotid arteries using a surgical exploration of the neck.
Then, insert a 7F and an 8F introducer sheath in the respective carotid artery. Now, administer heparin at 100 units per kilo. In order to perform a sternotomy, make a medium 30 to 40 centimeter incision from just below the suprasternal notch to a point just below the xiphoid process.
Then, advance through the linea alba down to the sternum. Use diathermy to carefully split the xiphoid and use Klinkenberg scissors to separate the posterior sternum from the pericardium. Continue the separation bluntly, moving cranially.
Make sure to point the scissor tips upwards towards the sternum to avoid cardiac damage. Perform a sternotomy by using a hammer and Lebsche knife. Bone marrow bleeding can be minimized by rubbing bone marrow wax on the marrow.
Open the thorax with a retractor. Now, open the pericardium using surgical scissors. Connect standard Y catheters to the guiding catheters.
Then, connect a three-way tap with a 10 centimeter extension to both Y connectors. Then, position one of the guiding catheters in the ostium of the left main coronary artery via one of the sheaths. Next, using a guide wire inserted into the left anterior descending artery, advance a coronary dilatation catheter through the left main coronary artery catheter, and position the balloon where the coronary occlusion was performed during the myocardial infarction induction.
Do not inflate yet. Continue by positioning the other guiding catheter in the ostium of the right coronary artery using the other sheath. Now, perform a coronary angiography.
Infuse contrast agent under fluoroscopy. First, confirm correct positioning of the guiding catheter in the left main coronary artery with an anteroposterior view. Thereafter, visualize the right coronary artery.
The contrast infusion may propel the balloon further into the coronary artery. Reposition it if necessary. Then, attach syringes with freshly made 2%Evans Blue dye to the three-way taps.
Next, using a coronary angiography, inflate the balloon and confirm that it completely occludes part of the coronary artery. The occlusion is critical. While the balloon is inflated, inject the dye through both guiding catheters at 5 milliliters per second.
The heart should not turn blue, except for the part of the left ventricle that is deprived of blood flow. If the heart does not go into ventricular fibrillation spontaneously, touch a nine volt battery to the non-infarcted part of the heart five beats after dye infusion. Then, incise the cable vein to release pressure and use suction to drain out the blood.
Now, deflate the balloon. Retract it along with both guiding catheters. Then, explant the heart by dissecting the surrounding membranes, and making a transverse cut through the large vessels.
Make sure to wash off all the blood and dye with 0.9%saline, both on the outside and on the inside. Then, carefully dissect the left ventricle. Then, make five equal 10 millimeter thick sections from apex to base, in a plane parallel to the atrial ventricular groove.
Photograph both sides of all five slices under ambient light conditions using a ruler in the image for calibration. Then, stain the slices in TTC and photograph them again. 3D transesophageal echocardiography was used for the assessment of global cardiac function.
One week after an acute myocardial infarction, global cardiac function deteriorated when compared to healthy baseline values, based on results from 10 animals. This was accounted for by reduced contraction of the anteroseptal wall. Pressure volume loops were also used, both to assess global cardiac function, and to calculate intrinsic myocardial muscle properties.
Outcome measurements of the former were easily calculated, and include EDV int he lower right corner, and ESV in the upper left corner. These values provide important information on left ventricular geometry. From these measures, the left ventricular rejection fraction, an important measure of left ventricular pump function, was calculated, along with several other parameters.
For intrinsic cardiac performance, different measurements were derived from the PV loops, such as the end systolic and end diastolic pressure volume relationship. After myocardial infarction, the end systolic volume pressure relationship slope may decrease, indicating decreased contractility. Ultimately, stains were used to identify the remote myocardium from the area at risk, and the infarcted myocardium.
About 22%of the left ventricle made up the area at risk, most of which was infarcted. After watching this video, you should have a good understanding of how to perform 3D transesophageal echocardiography, pressure volume loop data acquisition, and infarct size to area at risk staining. Once mastered, transesophageal echocardiography can be performed in 20 minutes.
Pressure volume loop data acquisition in 30 minutes. And infarct size and area at risk staining in 60 minutes. While attempting PV loops and infarct size area at risk staining, it is important to remember to be cautious not to induce and arrhythmias.
The techniques shown in this video can help researchers in the field of cardiovascular disease to explore ischemia and reperfusion injury in large animal models.
