The mouse kidney transplantation procedure begins with a donor organ harvest in which the left kidney is removed from the donor mouse and stored on ice. The right kidney is removed from the recipient mouse to make room for the donor kidney. The donor kidney is then removed from ice and transplanted into the recipient mouse.
The mouse is allowed to recover for four days before the remaining native kidney is removed. After seven days, creatinine levels are monitored using an I STAT portable clinical analyzer to assess graft function. Hello, I'm Sarah Heimer from the laboratory of Dr.Greg Hadley and the Department of Transplant Surgery at the Ohio State University.
Today we are going to show you a procedure for renal transplantation in the mouse. The renal transplantation surgery that you'll see today will be performed by our resident expert neurosurgeon Jing Wing. Please use this procedure in our laboratory to study the immunological events of transplant rejection as well as strategies to prevent rejection from occurring.
So let's get started. To begin the donor organ harvest, a tow pinch is performed to ensure that the animal is fully anesthetized working within a sterile field and using sterilized instruments, make a midline incision in the anesthetized mouse from sternum to pubis and expose the left kidney by moving the intestine laterally to the right side in order to perform donor kidney perfusion. TY four eight oh silk sutures around the aorta and inferior vena cava below and above the renal artery and vein.
Perfused slowly in situ with cold heparinized ringers lactate solution via the infrarenal aorta. Isolate the left kidney by ligating and dividing the adrenal and testicular vessels of a few lumbar branches with an eight oh silk suture. Next, mobilize the junction of the aorta and inferior vena cava with the left renal artery and vein tie four eight oh silk sutures around the aorta and inferior vena cava below and above the renal artery and vein.
To minimize bleeding, ligate the aorta first below the renal vessel. Then dissect the left ureter free from the renal hilum to the bladder. Expose the ureter of vesicle junction using coda retraction on the bladder dome.
Being sure to avoid the right ureter excises small elliptical patch of bladder containing the left ureteral vesicle junction. This patch will be used for urinary reconstruction via a bladder to bladder anastomosis. Then ligate the aorta above the renal artery and vein.
Slowly perfused the raft in situ with cold heparinized ringers lactate solution via the infrarenal aorta. Divide the aorta obliquely approximately two millimeters below the renal artery. Finally, remove the kidney and its vascular supply with ureter attached to the bladder patch on block.
The kidney should be stored in ringer's lactate solution on ice until it is ready to be grafted into the host recipient and euthanize the donor mouse by cervical dislocation under anesthesia. As demonstrated previously, the recipient transplantation is initiated with a to pinch to ensure that the animal is fully anesthetized. To begin the surgery, make a midline incision, cover the intestine with wet gauze and carefully retract it to the left side.
In order to successfully complete this kidney transplant, anastomosis must be performed between the donor aortic cuff and the recipient's aorta. The donor renal vein to the recipient's inferior vena cava and the donor bladder patch to the recipient's bladder ligate. The right ureter renal artery and vein.
Remove the kidney After ligating. The lumbar branches carefully isolate and cross clamp the infrarenal aorta and inferior vena cava with two four millimeter microvascular clamps. Place a 10 oh nylon suture through a full thickness of aorta and retract to make an elliptical aorta otomy by a single cut about one fifth the diameter of the vessel.
Next, make a longitudinal ven otomy by piercing the inferior vena cava with a 30 gauge needle and extend appropriately using micro scissors. Irrigate both the aorta and inferior vena cava with heparinized saline to clear intraluminal blood or clots. Remove the donor kidney from the ice and place it in the right flank of the mouse.
Place two stay sutures at the proximal and distal apex of the recipient's aorta otomy with the donor's aortic cuff. Make an end to side anastomosis between the donor aortic cuff and the anterior wall of the recipient's aorta otomy using two to three continuous 10 oh nylon sutures in the outside of the aorta. After turning the donor kidney graft over to the left flank of the recipient.
Repeat the previous procedure between the donor aortic cuff and the posterior wall of the recipient's aorta.Otomy. It is important to remember that the anastomosis of both vein and artery must be finished within 20 minutes. The graft should also be rinsed with cold saline several times during anastomosis.
Perform an end to side anastomosis between the donor renal vein and the posterior wall of the recipient's Inferior vena cava using four to five continuous 10 oh nylon sutures in the inside of the inferior vena cava. Close the anterior wall of the inferior vena cava and the donor's renal vein with four to five continuous sutures outside of the inferior vena cava. The next step is to divide the dome of the recipient bladder.
Stop the bleeding with cautery. Once tation is complete, make a small cut on the dome place two stay sutures of 10 oh nylon to anastomose the donor's small bladder patch with the recipient's bladder dome suture each side of the anastomosis with eight to 10 continuous stitches. Finally close the abdomen in two layers with continuous four oh sterile synthetic absorbable vicryl sutures.
Postoperative care begins by administering a single intramuscular injection of penicillin. The transplant recipient is also given 0.05 milligrams per kilogram buprenorphine intramuscularly on day zero and day one post-surgery for pain as well as a continuous supply of water containing sulfa trim. The first 24 hours post-operation miser kept in a temperature controlled incubator.
Skin sutures are removed two weeks after the surgery. The removal of the remaining native kidney is essential so that decline in kidney graft function can be monitored by serum creatinine measurements. It is typically performed four to seven days after the initial grafting of the donor kidney and involves the major steps employed when excising the kidney from the donor mouse.
ISO fluorine anesthesia is used to sedate the host animal so that blood samples can be collected. Retroorbital quantitative whole blood creatinine levels are used as a readout to assess graft function and are determined using an I stat portable clinical analyzer. Conventional units are converted to SI units by multiplying the conventional units by 88.4.
The concentration of creatinine is expressed in micromoles per liter. Animals will typically recover two to three hours following surgery and will display normal mouse behavior following removal of the remaining native kidney. Survival of the animal is dependent on renal graft function, which can be assessed by daily examinations of overall animal health and creatinine.
Measurement of whole blood using a portable i-STAT clinical analyzer for reference. A non-transplant normal mouse has a creatinine level near 20 micromolar. Renal graft dysfunction is considered when the mouse shows signs of illness and the creatinine level is elevated near 100 micromolar.
We've just shown you the procedure for renal transplantation in the mouse. Although this procedure is technically challenging, our lab has had great success in performing it. So that's it.
Thanks for watching and good luck with your experiments.
