The overall goal of this procedure is to study bypass graft patency in a rat vein interposition model. This method can help answer key questions in the bypass surgery field, such as underlying path of biological and physiological processes during bypass occlusion. The main advantage of this technique is that the surgical procedure is easy and fast, which can be used to study novel therapeutics and tracts in vivo.
Demonstrating the technique will be Grigol Tediashvili, a micro-surgeon from our laboratory. Before the surgery, check for a sufficient depth of anesthesia by pinching the rat's hind feet and verifying an absence of reflexes. Then, apply some vet ointment to the eyes to prevent dryness while under anesthesia.
Next, spread the hind legs and fix their position using tape. Then, shave the inguinal hair with a hair trimmer, followed by disinfecting the area widely using povidone-iodine followed by 80%alcohol. Repeat these alternating scrubs a total of three times.
Now under a microscope, perform a vertical incision along the linea inguinalis. Next, use two forceps to gently separate the subcutaneous tissues, and expose the superficial epigastric vein up to its origin on the femoral vein. Do this very carefully, as the vein is fragile.
Next, stop the blood flow through the superficial epigastric vein using two clamps. Now, harvest a segment of the vein. Carefully lift the isolated vein and cut through the vessel using micro-scissors.
Then, place the vein segment on sterile gauze. There, carefully glide a 30-gauge needle into one end and flush the vein with heparin. Then, transfer the vein into 1%lidocaine, and keep it on ice to prevent spasms.
Now, euthanize the donor rat by increasing the anesthesia to 5%isoflurane. Next, in preparation for the anastamoses, prepare the recipient rat in the same way as the donor rat up to the shaving step. After two to three minutes of 5%isoflurane, open the donor rat's abdomen along the linea alba, cut through the diaphragm, and remove the heart to stop circulation.
Continue preparing the recipient rat for surgery. Shave the medial side of the legs with a hair trimmer and disinfect the skin with three alternating scrubs of povidone-iodine and 80%ethanol. Before proceeding further, perform another toe pinch to monitor the anesthesia.
Now, perform a median femoral incision from the knee to the inguinal fold. Under the microscope, use two forceps to separate the femoral artery from its surroundings. Then, use two micro clamps to stop the blood flow, placing the proximal clamp first.
Next, cut out a short segment of the clamped femoral artery and discard it. Shorten the remaining arterial stump to leave a gap that is one to two millimeters larger than the vein graft. Then, flush the arterial stump with heparin from a 30-gauge needle.
If the adventitia protrudes slightly beyond the vessel stump, use forceps to pull the adventitia slightly over the end of the vessel, and then, remove a piece of adventitia. Now, place the harvested vein segment between the arterial stumps in the correct orientation, and adjust the gap size for a good fit. Now, perform the proximal anastomosis first using 10-0 prolene suture.
Start with suturing each lateral side, then add three more sutures to the ventral side. Finish with three stitches on the dorsal side. Next, connect the distal vessels using the same technique, starting with a suture on each lateral side, followed by three sutures on the ventral side, and then finishing on the dorsal side.
After completing the anastomoses, load two one-milliliter syringes with the fibrin glue components. Then, carefully lift the graft with forceps, and apply approximately 100 microliters of component one under the graft, followed by 100 microliters of component two. The two components must always be added in the same volumes.
Now, place the graft back in its position, and apply 100 microliters of each component over the graft to completely cover the graft and the anastomosis. This prevents anastomotic insufficiency and overdistention of the vein graft. Now, carefully open the clamps starting with the distal clamp.
Then, confirm a successful surgery by checking for a visible pulse in the transplanted vein and distal artery of the graft. Before closing the skin, remove any excessive glue that could impede closure. Then, close the skin layers using 5-0 prolene sutures.
Before the rat wakes up, inject four to five milligrams of carprofen per kilogram subcutaneously. Keep an eye on the animal until it has regained sufficient consciousness to maintain sternal recumbency. Then, give the rat a home cage without other animals.
For the next three days, provide metamizole in the drinking water, and check the rat's recovery daily. Once fully recovered, the rat can be housed with other rats. Animals recover well from the surgery and showed excellent physical condition post-operation.
Successful integration of the vein into the femoral artery and graft patency after transplantation was confirmed noninvasively using duplex sonography. By transplanting the vein of a luke-positive rat to a luke-negative rat, bioluminescence imaging was used to monitor the graft's presence. It was found that myointimal hyperplasia develops progressively in the graft over time.
Histological staining with Masson's trichrome demonstrates myointimal formation inside the internal elastic lamina. To confirm that the model reproduces the dynamics of myointimal hyperplasia, luminal obliteration was measured. A gradual loss of graft patency was noted from seven days to 28 days post-surgery, which fits the model nicely.
Once mastered, this technique can be done in 20 minutes if it is performed properly. Thank you for watching, and good luck with your experiments.
