Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.
The overall goal of this procedure is to investigate multiple organ complications related to cardiopulmonary bypass in a rodent model without the use of transfusion or inotropic agents. This method for CPB in a small animal can help improve key operations in the cardiovascular cellular feed. Ultimately, use of this technique will help develop strategies to protect against complications encountered in cardiopulmonary bypass surgeries.
First, set up the cardiopulmonary bypass circuit, connect the polyvinyl chloride tubes with the venous reservoir pre-designed for the CPB circuit and connect the tubes to a modified neonatal membrane oxygenator. Then, ensure that all of the connections are tight and do not leak. Next, on a height adjustable table, secure the circuit to a roller pump.
Then, set the table's height to 10 centimeters below the plane of the experiment. Now, prime the circuit with 11 milliliters of the priming solution and start the pump roller gently rotating. Then, put an 18 gauge venting needle into the reservoir and heat the circuit using a heat lamp positioned over the reservoir.
Next, tilt the membrane oxygenator and trigger it several times to de-air the line. This step is essential to preventing air embolisms and insufficient oxygenation. After anesthetizing and intubating the rat, transfer it to an operating table equipped with an electric heating pad.
A respiratory rate of 70 cycles per minute and with 30%of inspired oxygen. Monitor the oxygen level using the oxygen sensor. To maintain the anesthesia, provide 1.5 to 2%isoflurane and administer pentobarbital at CPB initiation.
Next, set up a rectal temperature probe and maintain a normal thermic body by adjusting the temperature of the heat pad and by heating the circuit with a lamp. Then, monitor the heart rate using an ECG attached at the bilateral shoulder and the left abdomen. Now, place moisture gauze on the rat's face to cover the eyes.
Next, shave the hair over the bilateral inguinal region and right cervical region. Then, disinfect the skin using three alternating scrubs of 70%ethanol and betadine or chlorhexidine. The rat is now ready to be cannulated.
Begin the surgery by making a five millimeter incision at the bilateral inguinal region and right cervical region using scissors. Then, bluntly dissect the tissues to expose the right main femoral artery. Next, carefully separate the artery from the vein and nerve.
Then, ligate the end of the common femoral artery using 4-0 silk suture. Then, make a one millimeter perpendicular incision into the right femoral artery and carefully cannulate it using a 24 gauge intravenous catheter to a depth of one centimeter. Use this catheter to monitor systemic arterial pressure and analyze gas partial pressure in arterial blood.
Next, administer heparin sodium using the catheter. Repeat this process to cannulate the left common femoral artery, and use this cannula as an arterial infusion line for the CPB circuit. Next, insert a 17 gauge multi-orifice angiocatheter into the right internal jugular vein, and advance it into the right atrium and inferior vena cava.
Be gentle, as the vessel can be easily ruptured. Then, connect the angiocatheter to the CPB circuit for venous drainage. Finally, cover each cannulated region with moist gauze to avoid contamination.
The animal is now ready for CPB. Before starting the CPB, begin delivering 100%oxygen gas to the oxygenator at 0.8 liters per minute and decrease the respiratory rate to 30 cycles per minute. Maintain the arterial oxygen partial pressure between 200 and 400 millimeters of mercury.
Next, raise the heat pad temperature to 42 degrees celsius to mitigate the immediate drop in the body temperature after CPB initiation. Now, carefully start the CPB flow and keep an eye on the volume of blood in the reservoir. An empty reservoir may cause an air embolism, so adjust the pump flow by adjusting the table height.
Do not reposition the venous drainage catheter to change the flow rate. Movement of this catheter might perforate the atrium or create an arrhythmia. Next, increase the pump flow up to 100 milliliters per kilogram per minute.
Now, maintain the mean blood pressure at 70 millimeters of mercury. When the blood pressure is stable, a small one milliliter volume of blood in the reservoir is acceptable. Any less risks an air embolism.
If the blood pressure does not stabilize, add two to three milliliters of priming solution to the circuit. When the body temperature returns to normal, reduce the heat pad temperature to 37 degrees celsius. After the CPB is completed, clamp the venous drainage tube and remove it from the circuit.
Then, gradually infuse the remaining blood in the circuit to the artery to maintain the blood pressure. Next, increase the respiratory rate to 70 cycles per minute. Then, remove the venous drainage catheter and the left arterial catheter.
Next, ligate the vessel in the proximal and distal site. Next, remove the arterial line from the right femoral artery. Then, clean each wound with saline and close them with sutures.
As soon as spontaneous breathing begins, end the anesthesia and extubate the intratracheal tube. Then, administer warmed sterile isotonic fluids and use a heat mat and electrical heat lamp to keep the animal warm until it recovers from the anesthesia. Watch the animal carefully and provide respiratory support as needed.
Arterial blood gas analyses during CPB include partial pressure of arterial oxygen, partial pressure of arterial carbon dioxide, hematocrit, base excess, serum expression of potassium, and potential of hydrogen. Throughout the procedure, the heart rate and mean arterial pressure were stable. The rectal temperature and hematocrit decreased at the beginning of CPB because of hemodilution caused by the priming volume.
The partial pressure of arterial oxygen significantly increased during CPB due to membrane oxygenation. Hematoxylin and eosin staining of the lungs after CPB show interstitial edema, inflammatory cell infiltration, and hemorrhage in the CPB group as compared to the control group. In addition, serum concentration of tumor necrosis factor alpha, interleukin six and high mobility group box one were each augmented after CPB.
After watching this video, you should have a good understanding of how to conduct a rough CPB procedure without transfusion or entropic agents. Once mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it's important to remember to frequently check the hemodynamics of the rat.
After its development, this technique paved the way for researchers in the field of cardiovascular surgery to explore inflammatory and other major organ complications in CPB.
