This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure.
Kidney transplantation in mice is essential for study of mechanisms of acute and a chronic allograft rejection. Here we introduce a new surgical technique for vessel anastomosis and the ureteral implantation in the abdominal kidney transplantation model in mice. In traditional mouse kidney transplantation models, the upper part of the aorta is ligated approximal to the renal arteries.
The donor's left renal vein is transected at the vena cava and the donor aorta is divided equally below the renal artery to be later anastomose to the recipient's abdominal aorta. In this modified technique of the mouse kidney transplantation, the donor left renal vein is also transected at the vena cava, but the donor aorta is cut above the offspring of the renal artery and its anastomose to the recipient abdominal aorta. While the donor end of aorta is ligated, the ureteral artery is deliberately preserved.
Now we start the demonstration of the surgical procedure. For anesthesia, place the mouse into the box for inhalation of isoflurane about 40 to 60 seconds in order to induce unconsciousness. Once the mouse is anesthetized, weigh the mouse.
According to the mouse weight, intraperitoneal injection of ketamine, xylazine, acepromazine is given to the anesthetized mouse. When anesthesia has taken effect, clip the abdominal fur. Then, fix and disinfect the mouse on the operation table.
Cut the skin using the cross abdominal incision. Cut the muscles of the abdominal wall. Next, cover cautiously move away the viscera with a saline imbibed gauze.
Expose the left kidney, aorta, and inferior vena cava. Cauterize the left lumbar veins, including their underlying branches and other small vessels including the left adrenal vessel carefully. Mobilize the aortic region between the left and right renal arteries approximately two millimeters in length.
Separate the infrarenal inferior vena cava and aorta. And pass curved forceps under the aorta to place a loose tie of 7.06 suture around this vessel. Cross clamp the area of the aorta below the right renal artery and the inferior vena cava using two five millimeter microvascular clamps.
Transect the left renal vein from the vena cava. Flush the aorta with one milliliter of heparin solution. Tighten the ligature and cut the aorta below the ligature as well as below the proximal clamp.
Prepare carefully, so that the delicate ureteral artery is completely preserved. free the left kidney and associated vessels completely by cautiously cauterizing all vessels surrounding tissue. Remove the kidney and store it in cell line solution at four degrees.
The initial operation steps are similar to those previously shown for the donor mouse, including anesthesia and disinfection. Open the abdomen via median incision, then cover the abdominal organs with a wet gauze using saline solution. Carefully preserve the infrarenal aorta and make sure every large vessel branch is cauterized.
Use the electric cautery to dissect the left ureter carefully at a position proximal to the kidney pelvis. Then, remove the left kidney. Expose the inferior vena cava and abdominal aorta and detach them from the surrounding adipose tissue approximately over four millimeters in length.
Then cross clamp each with two microvascular clamps approximately and distally of them. Use a micro needle holder to guide a 10/0 monofilament suture needle, which is placed through the aorta wall from proximal to distal manner. An elliptical arteriotomy of approximately one millimeter is achieved by gentle upward traction of the suture, while cutting directly below the lower face of the needle with fine, curved scissors.
Perform the donor-recipient aorta anastomosis in an end-to-side manner. Stitch the proximal and distal ends of the anastomosis. Last stitch using two separate 10.0 sutures.
After tying, leave the two long sutures, including the needle, in place. Sew the left side of the aortal wall of the anastomosis continuously with two evenly spaced stitches in a distal-proximal direction. After the last stitch, guide the suture through a partial thickness of the vessel wall above the upper stay suture tie and simultaneously exert gentle traction to the short end of the lower suture tie.
Note, in this new knotless technique, the last stitch is not tied to the short end of the upper tie. Turn over the transplanted kidney to its normal position. Now continuously sew the right wall of the aortal anastomosis in a proximal to distal manner using three stitches in a proximal to distal manner.
Note, compared to the conventional surgical technique, the last suture is merged with the distal tie nearby. Do not tie it to the end of the lower suture, cut it to leave a free length of around two to three millimeter instead. Cut the inferior vena cava longitudinal with sufficient length of approximately 1.5 millimeters.
Position this incision slightly below its aortic counterpart. Perform the venous anastomosis using the same suturing procedure as previously described with the difference that four to five stitches are needed for each side of the anastomosis. The final stitch is left as a free end of similar length similar to the aortal anastomosis described above.
And the new knotless anastomosis technique, the last stitch is not tied but left free. This shortens the operation time and it lets the surgeon to increase or decrease the size of the anastomosis. This improvement reduces the difficulty and post operation complications of the vascular anastomosis.
After completing both anastomoses, use a dry swab to exert gentle pressure toward the sutured area for about 10 to 20 seconds. After that, both clamps can be removed. Penetrate through the recipient's urine bladder using a straight needle with 10.0 suture and insert it into a 21 G needle lumen for guidance.
Now guide the 21 G needle to stitch a hole at the place of the previous needle application and pull out the needle. Stitch the trimmed ureter end and perforate the bladder with this 10.0 suture again at the place of its entry. Tow the 10.0 filament and the ureter into the urine bladder through the constructed hole.
Anastomose the donor's ureter to the recipient's urine bladder. Here, connect the outer membrane of the ureter to the outer membrane of the bladder wall and perform intermittent sutures with three to four stitches. Pull out the traction suture.
Place the intestines back into the abdominal cavity. Perform two-layer sutures, first the abdominal muscles followed by the skin to close the abdominal wound. Place the transplanted mice into an oxygen and temperature-controlled chamber for recovering after surgery.
Give adequate postoperative analgesia four consecutive days after operation. Perform contralateral nephrectomy of the recipient mouse five days after transplantation anesthesia. Raise and record the state of the mouse.
We compared the conventional and modified transplantation techniques by assessing histological changes of the transplanted isograft kidneys versus the native recipient contralateral kidneys. Both the degree of the trophy of the renal tubules and renal interstitial fibrosis was not found to be significantly different four and 12 weeks after transplantation respectively. We previously investigated the outcome of this new knotless technique and compared it to the classical approach in terms of technical aspects of the procedure interoperative and post-operative complications.
Modified technique that is shown here was associated with a lower occurrence of intragraft arterial venous thrombotic events. The time to perform the stenosis was significantly less and an excellent kidney isograft, long-term survival was achieved. The kidney transplantation in mice is a complicated and cutting edge procedure.
This is a new surgical technique for a vessel anastomosis and renal implantation committed to improve the overall success rate, making it a reliable tool for studying the autoimmune response after kidney transplantation.
