The overall goal of this procedure is to provide a novel approach to cardiopulmonary bypass in a mouse to improve investigations into the molecular mechanisms underlying heart damage. This method can help to answer key questions in cardiac surgery especially those related to extended cardiopulmonary bypass and prolonged cardioplegia time. The main advantage of this novel non-survival acute model is that it facilitates the study of molecular mechanisms in cardiac surgery and its relation to other comorbidities.
First, prepare a number two French polyurethane cannula for the surgery. Insert a 27 gauge metal needle into the cannula and under a microscope, use a surgical blade and micro scissors to make three or four fenestrations within the distal third of the cannula. This will assure optimal venous return flow.
Prepare the priming solution for the CPB circuit. First, add Sterofundin and then Tetraspan into the tube. For completion add Heparin and for buffering sodium bicarbonate.
Store the solution at four degrees Celsius until used. To prime the CPB circuit, add 850 microliters of priming solution via the venous cannula. Once primed, keep the solution circulating until the animal is ready for cannulation.
Cannulate the left femoral artery for blood analysis. First, expose the groin area and then gently separate the common femoral artery from the femoral vein and femoral nerve. After full anesthesia and intubation is achieved, perform a midline skin incision into the neck using sharp fine scissors.
Then separate the muscles in a blunt fashion to expose the right jugular vein and left carotid artery. As needed, coagulate the small vessels using a veterinary coagulator connected to bipolar forceps. After preparing the jugular vessels, cranially ligate the distal segment of the left common carotid artery to its bifurcation using 8-0 silk sutures.
Next, using micro surgical forceps and scissors, make a blunt preparation of tissue laterally to the sternocleidomastoid muscle to expose the right jugular vein close to the clavicle. Then ligate the distal and proximal ends of the jugular vein using 8-0 silk. Following the ligation, place the tip of the venous cannula into the right jugular and advance it into the right atrium then knot the suture to secure the cannula.
Next, place 8-0 silk suture slip knots at the proximal segment of the artery and insert the tip of the cannula into the carotid artery. For optimal venous return, it may be necessary to advance the venous tip into the right ventricle. Once the correct cannula position is achieved, carry out a systemic heparinization by adding 2.5 units of Heparin per gram of the animal body weight via direct intravenous injection into the jugular vein.
Next, ligate the distal part of the femoral vein and temporarily occlude the proximal part with a slip knot. Then make a small incision on the anterior wall using micro forceps to fit the cannula. Now, insert a number one French polyurethane cannula and secure it with a silk 10-0 suture.
To initiate the CPB, start the pump at about 0.5 milliliters per minute. Within two minutes, increase the flow to 46 milliliters per minute. Next, perform an upper sternotomy under full monitoring.
Use sharp scissors starting from the manubrium and go five millimeters in the caudal direction. Coagulate the sternal edges immediately and expose the aortic arch by pulling the right carotid artery in the cranial direction. For optimal control, circumferentially free the aortic arch from the thymus and surrounding tissue to facilitate clamping.
Then place an 8-0 silk loop around the ascending aorta and pull the silk loop in a cranial direction. There should be enough exposure of the ascending aorta to place the cross clamp for cardioplegia. For BGA samples, use a glass capillary tube to collect 60 to 90 microliters of arterial blood via the femoral artery catheter.
To fill a tube, slowly release the clamp on the silicone tubing then transfer blood from the syringe into the capillary. Under a stable CPB flow, initiate respiratory arrest by stopping the ventilation. Within the first 20 minutes of respiratory arrest, topically cool the animal using gauze soaked in cold saline.
Once the body temperature is at 28 degrees Celsius and after 30 minutes of respiratory arrest, administer 0.3 milliliters of 7.45%potassium chloride into the CPB circuit to initiate cardioplegia. To cross clamp the aorta, pull the previously placed loop in the cranial direction and place a micro-serrefine clamp on the ascending part of the aorta. Perform a total of 60 minutes of respiratory arrest and 30 minutes of cardiac arrest.
To initiate re-perfusion of the heart, remove the clamp and simultaneously re-ventilate and warm the animal to 37 degrees Celsius. After re-perfusion is completed and the animal has reached to normothermia, terminate the CPB by turning off the pump. Using the described protocol, reproducible results could be consistently obtained.
Physiological parameters were assessed using BGA at four different time points during a successful procedure. Hematocrit measurements showed hemodilution due to the addition of priming volume to the circuit. There was, however, no need for blood transfusion as hemoglobin levels were maintained at sufficient levels during the course of the experiment.
BGA also provided insight into the metabolic status of the animals. After initiation of CPB with ventilation, arterial pH was elevated. As expected, a gradual reduction in arterial pH was observed during the respiratory arrest.
Despite its complexity, once mastered, this method can be done with all of the steps within three to four hours. While performing this procedure, it's very important to practice cannulation technique and very important during the experiment to monitor hemodynamic and oxygenation parameters. Ultimately, this novel model can help to investigate the molecular mechanisms of acute organ injury during cardiopulmonary bypass.
Our next step is to establish a clinical surviving model of cardiopulmonary bypass in a mice that may foster a new microsurgical technique in murine models.
