Mouse Kidney Transplantation: Models of Allograft Rejection

This video presents a mouse model of kidney transplantation. By utilizing the multiple inbred strains, which have known major histocompatibility classes we're able to recreate different aspects of rejection of the renal allograft. The presented data illustrates how this is a useful model In the study of these mechanisms, This Schematic cartoon demonstrates the major steps of this model.

A left nephrectomy is performed on a donor mouse and the donor kidney is then prepared on a sterile swab. In a culture dish, the recipient mouse undergoes a right nephrectomy before The donor kidney is transplanted. The procedure is performed using sterile autoclaved instruments and consumables positioned within a sterile surgical field.

If multiple animals are operated upon either a new set of sterile instruments is used for each procedure or instruments are sterilized between procedures. Using a bead sterilizer, the operator will wear a sterile gown, mask, hat, and gloves. A single operator can perform this procedure by preparing the donor up to the point of kidney retrieval.

The recipient is then prepared for transplant before returning to the donor to remove the kidney. This reduces the cold ischemia time. Alternatively, two operators can prepare the donor and recipient simultaneously.

This allows multiple animals to be operated on in one day. Here is a male eight week old C 57 black six mouse that has been anesthetized by an intraperitoneal injection of ketamine and hydrochloride and meine. The surgical area has been shaved and sanitized using a dilute iodine solution.

The mouse has been placed on a sterile draped heated surgical pad with the extremities affixed to the pad. Eye ointment has Been applied. A laparotomy is performed by a midline skin incision.

Then the peritoneal cavity is opened via the linear Albert using tissue separating scissors. The mouse is then draped and a calibra retractor is inserted into the incision. The intestines are displaced to the left or right of the surgical field to expose the left kidney and ureter to prepare the donor kidney.

For transplantation, the ureter is ligated and divided as close to the bladder as possible. The lower gonadal and upper adrenal veins are ligated and divided close to the renal vein. The aorta is ligated, superior and inferior to the renal artery and cold University of Wisconsin solution is injected into the aorta to perfuse the kidney.

The ureter can be easily dissected from the retroperitoneum and traced to its termination of the bladder. It should be ligated as close to the bladder as possible with 7.0 suture and then divided the left gonadal vein normally empties into the renal vein. It should be doubly ligated using 9.0 suture and divided between the sutures close to the renal vein.

As shown in this clip in a similar fashion, the left adrenal vein empties into the renal vein and should be ligated and divided. Attention should then turn to dissecting the donor aorta from the surrounding lymphatic tissue and the advent to allow placement of first and encompassing loose inferior suture. Then a superior suture to the renal artery.

Once the donor mouse is ready, five units of intravenous heparin is administered by the dorsal penal vein. First, the inferior aortic suture is tied tightly followed by the superior ligature. This isolates the kidney from the arterial circulation.

Approximately 500 microliters of cold university Wisconsin solution is administered by the aorta. Successful profusion is identified by blanching of the donor kidney. The aorta Is divided inferiorly first and then superiorly to the renal artery.

Next, the renal vein is divided at its junction with the vena cava. The donor kidney is then removed and placed on cold sterile gauze. In a culture dish, the aorta is incised longitudinally directly opposite the renal aorta.

To create an aortic patch for anastomosis, identify the lumen of the renal artery and ensure there are no extra polar arteries. The renal vein is then prepared by placing first a 10 O polyamide suture at the inferior apex of the renal vein, and then similarly, a suture at the superior apex. The ligatures that divided the adrenal vein and gonadal vein are useful markers to orientate the vein.

At this point, following the donor kidney harvest, a recipient mouse is prepared as described previously, the recipient mouse is draped and a calibra retractor is inserted. The intestines are displaced to the right or the left of the surgical field and covered with saline moisten, sterile drapes. First, A right nephrectomy is performed by ligating, the renal artery and vein with seven oh suture, and then the ureter is isolated and ligated before division.

Finally, the renal artery and vein are divided after which the kidney can be removed. This creates space within the peritoneal cavity for the transplant kidney recipient aorta and vena Caver then need to be prepared for vascular anastomosis. First, the lymphatic bundle is mobilized from the aorta and vena cava and the retroperitoneum is opened.

Bluntly dissect the lumbar vessels from the left and right aspects to allow placement of a suture around the lumbar vessels. The vessels can then be ligated in continuation without dividing. This prevents back bleeding when performing the vascular anastomosis.

Further dissection is then performed to create a window between the aorta and vena cava and from surrounding advent tissue. Identify a position for superior and inferior microvascular clamp, ensuring it is free of excess tissue and ready for anastomosis. At this point, heparin can be administered by intravenous injection as previously shown, The donor kidney is placed in the peritoneal cavity of the recipient.

Mouse care should be taken to ensure the sutures placed in the vein do not become entangled. This schematic cartoon shows the donor kidney together with the recipient's IVC aorta and bladder following the right nephrectomy to transplant a donor kidney two. A traumatic clamps are applied to the IVC and aorta above and below the site of vascular anastomosis.

The donor kidney's, renal vein, and artery are anastomosis into the recipient's IVC and aorta respectively. Following this, the atraumatic clamps are removed and the donor kidney is reperfused. The ureter is then anastomose into the recipient's bladder First.

The inferior microvascular clamp is applied encompassing both the aorta and vena cava. A superior clamp is then placed. Atomy is made with a 30 gauge needle and the vena cava flushed with saline.

The ven otomy is widened with forceps. The suture previously placed at the superior apex of the donor renal vein is then passed through the recipient's vena cava at the superior apex of the ven otomy. The suture is then tied and a running suture anastomosis of the posterior wall of the renal vein to the vena caver is made once the inferior apex of the pH otomy is reached.

The second suture previously placed in the renal vein is passed through the inferior vena cava and tied. This can then be tied to the first suture and a running vascular anastomosis performed to complete the front wall. In the accompanying protocol, a diagrammatic representation of each step of the vascular anastomosis is provided.

It is critically important to avoid inadvertently suturing the front wall to the back wall and also from involving the real artery or aortic patch in the sutures of the Venus anastomosis. The donor aor autotomy can then be created by picking up a small ellipse of the aorta in the forceps and cutting precisely with scissors. A 10 oh suture is then placed at the superior point of the aortic patch.

Take care to perform minimal handling of any of the arterial endothelial surfaces. As this increases the risk of thrombosis, the suture is then passed through the superior apex of the A autotomy and tide. The aortic patch can then be anastomose in a running suture to the recipient's aorta and tied to itself.

Take care not to tie too tightly as this may cause a per string effect and jeopardize inflow. The inferior microvascular clamp is removed first, often reperfusion occurs due to backflow into the renal artery from the distal aorta. Then the superior clamp is removed.

The ureteric anastomosis is then performed by passing a 21 gauge needle in and out of the bladder. Forceps are then passed back through the tract and then the ureter drawn through via the suture. The advent tissue of the ureter is then sutured to the advent tissue of the bladder with nine oh polyamide at three points to fix the ureter in place.

The ureter is then cut flushed to the bladder and often urine can be seen being produced by the donor kidney. Note also bleeding from the per ureteric vessels. The ureter is allowed to lie loosely in the bladder and the defect closed after replacing the intestines.

The peritoneum is sutured and the skin is closed using metallic clips. Iodine is then applied to the surgical area and anesthesia is partially reversed by a subcutaneous injection of he tapazole Hydrochloride. Analgesics Are administered by a injection of buprenorphine hydrochloride.

Fluids are administered by a subcutaneous injection of one mil warmed saline. Mice are carefully monitored until you have recovered consciousness appear alert and unable to write themselves. Mice are allowed to recover any heated box kept to 29 degrees for 24 hours for long-term experiments.

Ongoing analgesics are administered and the skin clips are removed. Seven days following surgery. Here is a mouse 24 hours following surgery noted is fully mobile and is past bloodstained urine onto the bedding tissue.

It is important to monitor for handling paralysis in this model as this may indicate vascular complications presented. Next is data that indicates how this model and different mouse strains can be used to study the development of renal transplant rejection to model acute transplant rejection. The mutant mouse strain BM 12 is used as a donor.

These mutant BM 12 mice differ from the parental C 57 black six strain by a single MHC class two difference transplanting a BM 12 kidney into a bowel C recipient. Results in acute rejection due to a complete class one and class two MHC mismatch. A C 57 black six allograft leads to the development of chronic rejection due to the isolated MHC mismatch BM 12 ISO grafts have a complete class one and class two MHC match and therefore exhibit no signs of rejection.

Renal structure is examined by histopathology on paraffin embedded kidney sections stained with hematin and eoin. As this model is not dependent on the transplant for survival, the native kidney can be used as a control Following ISO graft renal transplantation, the kidney undergoes ischemia reperfusion injury, but by four weeks tubules have recovered following BM 12 into biopsy allograft transplantation. Acute rejection is apparent with diffuse mononuclear cell infiltrates denoted by the single asterisk necrotic tubules with double asterisk and tubul BM 12 kidneys transplanted into C 57 black six mice.

Results in chronic allograft damage characterized by perivascular, lymphatic infiltrate denoted by the block arrows with interstitial fibrosis and tubular atrophy indicated by hollow arrows. Quantification of tubules per high powered field can be used to assess the functional nephron mass. There was no difference between ISO graft and native kidney tubules counts of four weeks post transplantation.

Acute rejection results in a significant loss of tubules by four weeks. In comparison, chronic rejection shows a more varied but gradual loss. Piro serious red is a pan collagen stain and can be performed on paraffin embedded kidney sections To quantify interstitial fibrosis.

ISO graft renal transplantation is associated with insignificant fibrosis compared to the native kidney BM 12 inter C 57. Block six results in fibrosis at 12 weeks. Following transplantation, we have described a model of renal transplantation with emphasis on demonstrating the surgical techniques.

This model is associated with a significant learning curve, which can be overcome to achieve a reproducible functioning mouse kidney transplantation Model. The strain Combination described develops key features of chronic renal transplant damage and can be used to investigate the resulting pathology of interstitial fibrosis and tubular atrophy. In addition, acute rejection can also be modeled, allowing the investigation of important pathophysiological mechanisms of this entity.

This video provides key surgical points to aid the viewer in faithfully replicating this model.