The overall goal of this procedure is to create an isolated left working heart model that permits precise control of ventricular loading conditions from rodents. This method can help answer key questions in cardiovascular physiology, including the effects of medications on myocardial contractility, textile performance, and oxygen consumption. The main advantage of this technique is that the loading conditions of the heart can be precisely controlled.
Visual demonstration of this method is critical as dissection and isolation of the aorta, granulation of the left atrium, and isolation of the pulmonary veins requires skill and practice to master. Following appropriate sedation, analgesia, and anticoagulation with heparin, begin the surgery on the donor rat. First, pull the skin away from the abdominal cavity with forceps, and use scissors to incise the peritoneal cavity.
Following the curve of the diaphragm back to the posterior angle of the ribs. Once the diaphragm is visible, use scissors to cut along the anterior surface of the diaphragm in line with the prior cuts to access the thorax. Extend each cut along the axillary line bilaterally to the axilla.
Then retract the rib cage anteriorly from the xyphoid process using forceps. Next, incise the pericardium and pleura. Proceed efficiently since ventilation is compromised once the diaphragm is incised.
Identify the inferior vena cava and aorta just above the diaphragm, and use blunted forceps to retract them on block anteriorly. Next, using large curved scissors, rapidly cut across the inferior vena cava and the aorta. Without cutting the ascending aorta, cut the esophagus, trachea, brachiocephalic arteries, and veins cephalad.
Then excise these thymic tissues along with the heart and lungs on block. Immediately immerse the hear and lungs in ice cold KHB and move to the Langendorff apparatus. Place the heart lung complex in a flat dish and orient the heart with the thymus and great vessels facing the experimenter.
And the posterior aspect of the lungs facing the table. Next, pull apart the two lobes of the thymus, and identify the takeoff of the brachiocephalic arteries from the aorta. Now drape the aorta over the edge of the dish.
Then using small scissors, transect the aorta approximately five millimeters above the aortic valve just proximal to the takeoff of the right subclavian artery. A circular cross section is required. Keep repeating the cut until one is made.
The following video microscopy provides clarity on a critical portion of this procedure, the dissection and transection of the aorta in preparation for cannulation. As shown here, the thymus should be divided in the middle, and will pull apart easily. Use gentle strokes with curved forceps to divide the pericardium and sift tissues surround the aorta.
Avoid the use of excessive force. The innominate artery stump should be identified and grasped as a point of traction on the aorta. The aorta should then be transversely incised using small sharp scissors at the midpoint between the aortic valve and the takeoff of the subclavian artery.
If the cut is made too high, the aorta becomes redundant, and can cause torquing of the heart around the cannula. Alternatively, cramps buffer may leak from a brachiocephalic stump. If it is too short, the cannula tip may cross the aortic valve.
Next, with two pairs of curved forceps, guide the aorta over the aortic cannula, which should be slowly dripping KHB. The aortic valve should sit one to two millimeters below the tip of the cannula. Next, reposition the forceps perpendicularly across the aorta to hold the aorta in place.
A clamp could also be used. Then have an assistant use silk four oh suture to secure the cannula with multiple knots on both sides of the heart. Now, fully open the cannula to begin full retrograde aortic flow.
The heart should beat vigorously. One of the potential pitfalls is for the aortic cannula tip to cross the aortic valve into the left ventricle. As shown here, if that occurs, the heart will appear pale and the left atrium will be distended.
This must be immediately corrected by briefly stopping coronary flow, and repositioning the heart down on the aortic cannula. Not that aortic tissues is fragile in rodents and tears easily. Another potential pitfall is a leak in the aortic root, either from a tear or from an excessively high cut on the aorta, such that a brachiocephalic artery leaks KHB.
This can be addressed by stopping coronary flow, removing the heart from the aortic cannula, and repositioning. The purpose of this step is to create a closed system in which all pressure from the preload block is transmitted to left heart structures. Failure to completely occlude the pulmonary veins may lead to loss of preload, and may create an unstable heart preparation, or may falsify results.
Manually rotate the aortic cannula so that the posterior aspect of the heart is facing the operator. Now dissect out the vessels leading to the left lung. To help access the vessels, use forceps to suspend the left lung tissue.
Then use a single medium surgical clip to occlude the left pulmonary artery and vein, and the bronchus. Next, resect the left lung distal to the clip. Now repeat the process on the right lung.
When both pulmonary arteries are fully occluded, the right atrium will distend, and the heart may become bradycardic. As needed, add additional clips or sutures to plug leaks and work efficiently. Now perform the pulmonary arterial incision.
Rotate the aortic cannula to access the anterior aspect of the heart and identify the pulmonary artery. Using small scissors, make a transverse incision about three millimeters above the pulmonary valve. The pulmonary artery can now be cannulated in a later step.
Next, cannulate the left atrium. When viewing the left atrium, use small scissors to make a two to three millimeter incision in the upper body of the left atrium about three millimeters above the atrioventricular groove. Then position the left atrial cannula perpendicular to the plane of the mitral valve, pointed towards the atrial septum.
Open the left anterior cannula until warm KHB is visibly flowing. Make sure the KHB is warm to the touch, or hypothermia can set in. During the cannulation, transition the KHB flow to a drip rate.
Now while using forceps to hold counter traction, insert the atrial cannula into the body of the left atrium. Don't use excessive force, as the atrium is fragile. Position the cannula so that it sits in the middle of the atrium without any tension on the atrial wall.
Now secure the cannula to the atrium with a four oh silk suture placed around the body of the left atrium, including the posterior aspect of the left atrium. Add additional sutures as necessary. Once sealed, pull the cannula back one to two millimeters so that it sits in the middle of the atrium rather than against the atrial septum.
This could be checked by looking at the left atrial waveform. Now open the cannula valve entirely to administer the full preload to the left atrium. It is critical to monitor the drip rate from the heart.
It should be steady while the cannula is open. If not, retie the atrium around the cannula to seal off the leak that is probably causing the problem. To measure substances in the coronary effluent such as gases, drugs, or cytokines, insert flexible tubing into the incision in the pulmonary artery.
To permit continuous measurement of pressure and volume, insert a calibrated pressure volume conductance catheter into the left ventricle. This is best done in a retrograde fashion through the aortic valve. Although when this is technically challenging, it can also be performed via apical ventricular puncture.
The DPDT max should be at least 3500 to 4000 for an adequate preparation. Collect the coronary sinus effluent from the pulmonary artery and from the heart in a graduated cylinder for timed quantification of coronary flow. Now calculate the myocardial oxygen consumption.
Ultimately, turn off the retrograde aortic pump to transition to working heart mode. A retrograde perfusion was prepared as described. Flow was initially retrograde during set up.
When the retrograde pump was turned off, flow switched to antigrade, making a working heart. The typical end diastolic pressure is about three to five millimeters of mercury in this model. And the peak systolic pressure is around 100 millimeters of mercury.
When the left atrial cannula was moved away from the atrial septum, a change in the left atrial tracing can be detected. In these experiments, the aortic pressure was set at 90 millimeters of mercury, and the left atrial pressure was set to 10 millimeters of mercury. For an experiment, dopamine was infused at 15 micrograms per milligram per minute into the left atrial block.
Although the end diastolic pressure was unaltered, dopamine caused the left ventricular end diastolic volume to decrease by 2.5%and the left ventricular end systolic volume to decrease by 4.9%yielding an increased stroke volume. Compared with placebo infusions, the left ventricular stroke work identified as the area within the pressure volume curve increased by 32%during treatment with dopamine. This was also associated with a greater increase in myocardial oxygen consumption.
Once mastered, this technique can be completed in 15 minutes if it is performed properly.
