This model provides a standard protocol for establishing animal models of coronary graft disease. The procedure of animal model established by this method is standard, and the result is stable. To begin, divide three-month-old, male minipigs weighing 30 to 35 kilograms into the sham and venous graft disease groups of five animals each.
After anesthetizing the animal, establish ear access by inserting an indwelling venous catheter into the marginal ear vein. Now, transfer the pig onto the operation table in the supine position. Perform tracheal intubation with a 7.0 to 7.5 French gauge tube, and connect it to the anesthesia breathing circuit.
Immobilize the limbs with bandages, and elevate the head with a sterile drape. After injecting vecuronium bromide intravenously for muscle relaxation, monitor heart rate, blood oxygen levels, and body temperature using an electrocardiogram. After shaving and sterilizing the surgical area, place a sterile surgical drape around it.
After making the incision and exposing the internal mammary vein as described in the text, locate the internal mammary vein and the left internal mammary artery on the left side of the sternum, and perform blunt dissection of the internal mammary vein with vascular forceps. Perform hemostasis by electrocoagulation of the left internal mammary vein branches with an electric knife. If hemostasis is incomplete, using cotton thread, ligate and mark the two ends of the vein while it is being harvested.
Once the vein is removed, inject heparin, normal saline into the vein for pre-treatment. Then, put the vein into the normal saline, keep it for backup, and similarly incise the internal mammary vein of the sham group. Then, open the pericardium, close the chest wall, and use the internal mammary vein for pathological control without coronary artery bypass grafting.
Make an approximately seven-centimeter incision with an electric knife on the pericardium to expose the right coronary artery trunk. Suspend the pericardium, and sew the skin on the ipsilateral side with 1-0 surgical sutures. Now, separate the right coronary artery trunk from the surrounding tissues.
Next, using a wire hook, bypass the blocking band under the proximal end of the isolated right coronary artery near the aorta. Then, treat the myocardium with three cycles of two minutes ischemia and five minutes reperfusion by tightening and relaxing the blocking band. Also, monitor the heart's electrical activity with the electrocardiogram during the ischemia reperfusion pre-conditioning.
Next, cut the epicardium covering the blood vessels after tightening the band to block the right coronary blood flow. Exposing the coronary artery wall, cut longitudinally with the tip of a surgical blade against the center of the anterior wall of the blood vessels. After cutting the lumen and enlarging the incision and placing a coronary shunt, insert one end of the shunt with a coil into the distal coronary artery through the tear.
Now, shunt the blood in the coronary arteries into the hollow coronary shunt to ensure a clear operative field, and perform an end-to-side continuous suture between the internal mammary vein and the aortic wall with the 6-0 polypropylene suture. In the middle of the ascending aorta, occlude the left anterolateral wall of the ascending aorta with a semi-occlusion clamp. After making a small incision with the surgical blade in the aortic wall where the adventitia has been cut, insert the head end of the sliding shaft at the head end of the punch into the aortic cavity through this incision.
Then, contract the sliding shaft outward and the circular knife cuts off a piece of the arterial wall. Once the shunt is pulled out, perform an end-to-side continuous suture between the internal mammary vein and the right coronary artery with the 6-0 polypropylene suture. After opening the semi-occlusion clamp, record the bypass flow of the right coronary artery trunk proximal to the anastomosis site using ultrasound.
Prepare the animal for surgery as demonstrated earlier. And with an electric knife, make a 10-centimeter median sternal incision to split the sternum and harvest the vein 30 days post-surgery. While separation, avoid the main blood vessels, heart, and separate the grafted blood vessels layer by layer.
Quickly cut off the large blood vessels connecting to the heart, and place the heart and ascending aorta on ice chips. Once the graft vascular bridge, the connected aorta, and the right coronary artery are removed, rinse all the samples with normal saline at four degrees Celsius. After taking the entire graft vessel of about three to four centimeters and dividing it into four to five equal parts, transfer it to CryoTubes to quickly flash-freeze in liquid nitrogen.
For analysis, fix the ice-cold, saline-rinsed graft in 4%paraformaldehyde solution. After 12 hours of tissue fixation, stain the sections in 50 milliliters of an aqueous solution of hematoxylin for three minutes. Then, separate the sections by washing with 50 milliliters of 0.5%hydrochloric acid-ethanol and 0.2%ammonia water for 10 seconds each.
Once the sections are rinsed for one hour with running water, clean them by soaking them in distilled water for three minutes. After dehydrating the sections in 70%and 90%ethanol for 10 minutes, place them in 50 milliliters of 0.5%alcohol eosin staining solution for two to three minutes. Once the stained sections are dehydrated in pure ethanol, soak the samples in pure xylene for 10 minutes to make them transparent.
Finally, drip the transparent sections with neutral glue before covering them with a cover slip. Observe pathological sections under a light microscope at 40 times magnification. Ultrasonic examination showed that the overall blood flow direction was normal in the proximal end, vascular cavity, and the distal end of the graft vessel.
However, the proximal and distal ends of the graft vessel showed some retrograde flow of blood. The histological analysis of the internal mammary vein revealed that in the sham group the tunica intima, tunica media, and the venous wall of the venous graft appeared normal. However, 30 days after coronary artery transplantation, the intima of the vessel was thickened and the lumen was narrowed.
Among the sham and venous graft disease groups, four biochemical indexes, aspartate aminotransferase, serum bilirubin, total bilirubin, and creatinine, showed statistically significant changes. The surgeon should completely expose the internal mammary vein and make sure it is not attached to the surrounding tissue. The technique provides a standardized modeling process for studying venous graft diseases in animal models.
