Funding from Kidney Research UK, The Royal College of Surgeons of Edinburgh and The European Society of Organ Transplantation supported this study.

Appropriate national and local institutional ethics should be in place before performing animal experiments. Specifically in the United Kingdom the following experiments were undertaken under the Animals (Scientific Procedures) Act 1986. Where two microsurgeons are available to operate simultaneously the donor surgeon should perform steps 1.1 to 1.16 then 3.1 to 3.5, whilst the recipient surgeon performs 2.1 to 2.8. For a single operator the steps can be followed sequentially.
1. Donor Preparation
Note: The procedure presented here is for a donor C57BL/6BM12 and recipient C57BL/6 male mice aged between 8 to 16 weeks old with a body weight greater than 20 g. However this procedure can be reproducibly performed on a variety of mouse strains. The data presented in the representative results section was obtained from C57BL/6, C57BL/6BM12 and BALB/c mice.
<ol>
	<li>Conduct procedures using sterile surgical instruments and consumables (autoclaved), with endeavors to keep the operating area as sterile as possible. Perform the donor preparation simultaneously with the recipient preparation if two surgeons are available.</li>
	<li>Anesthetize the mouse with an intraperitoneal injection (31G needle) of medetomidine (0.5 mg/kg) and ketamine hydrochloride (200 mg/kg). This results in an anesthetic plane that is maintained for 4 hr providing enough time for the whole procedure to be performed. Supplemental anesthesia is not necessary.</li>
	<li>Confirm that the mouse is anaesthetized (no response to toe pinch).</li>
	<li>Shave the mouse&#8217;s abdomen and remove any loose hair with sticky tape.</li>
	<li>Place the mouse on its back on a sterilely draped heated mat and loosely immobilize the limbs with sterile masking tape.</li>
	<li>Monitor the mouse throughout the procedure for thermal burns. If possible use a non-electric heat source.</li>
	<li>Apply an eye lubricant and sanitize the abdominal wall with a dilute iodine solution.</li>
	<li>Make a midline incision in the abdomen to enter the peritoneal cavity and insert a 3 cm Calibri abdominal retractor.</li>
	<li>Apply warmed saline to keep the intestines and surgical area moist and avoid unnecessary drying of the viscera.</li>
	<li>Cover the mouse with sterile drapes and move the intestines to the operator&#8217;s left (mouse&#8217;s right) to expose the aorta, vena cava and left kidney.</li>
	<li>Apply a ratcheted forcep to the stomach and pull superiorly to expose the major vessels and left kidney fully. Pack damp sterile swabs (2 mm x 2 mm) into the abdomen to retract tissues away from the surgical area, such as the liver lobes, seminal vesicles, and bowel.</li>
	<li>Isolate the left kidney from surrounding adventia, fat and the left adrenal gland in the peritoneal cavity by bluntly dissecting the connective tissue using fine tip forceps. Place the closed tips of the forceps between the areas that need to be separated and slowly allow the forcep tips to open to dissect the space.</li>
	<li>Isolate the left renal vein by ligating and then dividing the left adrenal vein and left gonadal vein with 9/O nylon. Place the suture close to the renal vein.</li>
	<li>Ligate and divide the ureter with 7/O silk suture close to the bladder leaving the suture ends long. These long suture ends will be used when the kidney is harvested and are necessary for the ureteric anastomosis.</li>
	<li>To mobilize and fully dissect the aorta and vena cava superiorly and inferior to the renal artery and vein use fine tip forceps to bluntly dissect lymphatic vessels and fat from the front and sides of the vessels.</li>
	<li>Find the tissue plane between the aorta and vena cava and slowly spread the forceps. Slowly open the tips of the fine tipped forceps to spread the tissue with minimal trauma.</li>
	<li>Tie a loose 7/O silk around the aorta superior and inferior to the renal artery by passing an angled fine tip forceps around the back of the vessels and drawing a suture through. These sutures will be tightened prior to kidney retrieval to allow retrograde perfusion of University of Wisconsin solution.</li>
</ol>
2. Recipient Preparation
As per the donor preparation follow steps 1.2 to 1.8.
<ol>
	<li>Move the intestines to the operator&#8217;s right (mouse&#8217;s left) to expose the aorta, vena cava. Cover the intestines with a sterile saline soaked swab.</li>
	<li>Perform a right nephrectomy by ligating the right renal artery and vein together with 7/0 silk suture and then divide.</li>
	<li>Ligate the right ureter with 7/O silk suture and divide.</li>
	<li>Mobilize and fully dissect the aorta and vena cava inferior to the renal artery and vein as described in step 1.14 and 1.15. Ensure complete dissection between the aorta and vena cava. Take care to preserve the internal spermatic artery which runs anterior to the vena cava and aorta along with the lymphatic bundle.</li>
	<li>Identify lumbar vessels running in the retroperitoneum from the vena cava and vein to the spine, ligate with 9/O silk in continuity, there is no need to divide the vessels.</li>
	<li>Identify sufficient space to place microvascular clamps with space between for vascular anastomoses.</li>
	<li>Administer intravenous heparin (5 units) via dorsal penile vein.</li>
</ol>
3. Donor Kidney Retrieval
Tighten the 7/O silk ties that have been placed around the inferior and superior aorta to isolate the kidney from the arterial circulation.
<ol>
	<li>Infuse retrograde cold 0.2 &#8211; 0.5 ml of University of Wisconsin solution with a needle (31G) into the aorta.</li>
	<li>Divide the aorta within the sutures and divide the renal vein at its junction with the vena cava to remove the kidney and the renal artery with a length of aorta. Divide lumbar vessels arising from the aorta, if they are present, without ligation.</li>
	<li>Place the kidney in cold saline on a sterile swab in a culture dish.</li>
	<li>Euthanize the donor mouse by cervical dislocation.</li>
</ol>
4. Kidney Transplantation &#8211; Kidney Preparation
<ol>
	<li>Create an aortic patch by dividing the aorta wall longitudinally directly opposite the renal artery. Identify any vessel lumens in the patch that need to be ligated or avoided when performing the arterial anastomosis.</li>
	<li>Place a 10/O suture from outside to inside the renal vein lumen superiorly and a second separate suture inferiorly. The sutures used to divide the adrenal vein and gonadal vein can be used to orientate the vessel.</li>
	<li>Place the kidney in the right flank of the recipient (Figure 1.1) and ensure the sutures are well placed to ensure vascular anastomoses can be completed without the sutures becoming entwined in swabs or other instruments.</li>
</ol>
5. Kidney Transplantation &#8211; Vascular Anastomosis
Apply microvascular clamps first inferiorly then superiorly encompassing the vena cava and aorta.
<ol>
	<li>Make a ventomy with a needle (31G) by puncturing the anterior wall of the vena cava. Flush blood from the vena cava by injecting approximately 50 &#956;l of 0.9% NaCl.</li>
	<li>Widen the venotomy using fine tip forceps by opening them within the venotomy so that the length is equivalent to the diameter of the donor renal vein.</li>
	<li>Place the 10/O superior suture (which is already in the lumen of the renal vein) first at the apex of the venotomy (Figure 1.2) and join the back wall of the anastomosis in a running suture until the inferior apex is reached (Figure 1.3).</li>
	<li>Place the 10/O inferior suture and tie first with a single knot (Figure 1.4) then tie to the running suture from the back wall.</li>
	<li>Using the inferior suture create the front wall of the anastomosis (Figure 1.5) with a running suture and tie to the superior suture end at the superior apex (Figure 1.6).</li>
	<li>Create an aortotomy by picking up the aorta wall with forceps and cutting an elliptical patch with scissors (approximately the aortotomy should be a fifth of the circumference of the aorta and three times the renal artery lumen in length).</li>
	<li>Place a 10/O suture at the superior point of the aortic patch from outside to inside (Figure 1.7) and pass through the aorta to tie outside the vessels at the superior apex of the aortotomy.</li>
	<li>Create the arterial anastomosis with a running suture starting superiorly (Figure 1.8) anastomosing the donor patch to the recipient aorta, take care not to constrict the anastomosis by tying the suture too tight (Figure 1.9).</li>
	<li>Remove the inferior vascular clamps first then the superior clamp to reperfuse the kidney (Figure 1.10 and 1.11). Visible peristalsis of the ureter maybe seen if adequate blood supply to the ureter is achieved.</li>
</ol>
6. Kidney Transplantation &#8211; Ureteric Anastomosis
<ol>
	<li>Divide any attachments of the bladder from the abdominal wall.</li>
	<li>Pass a needle (21G) from the left to right of the bladder through both walls.</li>
	<li>Place straight forcep tips in the needle lumen and pass both back through the bladder such that the forceps are now passing from right to left out of the left side of the bladder.</li>
	<li>Draw the suture on the donor ureter through the bladder such that the ureter passes in the left bladder defect then out of the right side.</li>
	<li>Suture the adventitia of the ureter to the adventitia of the bladder with three single interrupted sutures around the entry point with 9/O nylon suture on a round-bodied needle.</li>
	<li>Cut the ureter proximal to the ligature, thus opening the ureter to allow urine to flow and allow the ureter end to retract into the body of the bladder. Visible production of urine may be observed along with bleeding from peri-ureteric vessels.</li>
	<li>Close the right side bladder defect with a single interrupted 9/O silk suture.</li>
</ol>
7. Recovery and Post-operative Care
Replace the gastrointestinal viscera into the abdomen in their original orientation and close the abdominal wall by approximating the rectus muscles with 6/O absorbable suture.
<ol>
	<li>Approximate the skin with metal skin clips.</li>
	<li>Partially reverse the anesthesia with a subcutaneous injection of atipamazole hydrochloride (10 &#956;l/g).</li>
	<li>Administer analgesia by subcutaneous buprenorphine hydrochloride (0.05 mg/kg) and for fluid support injection 1 ml of subcutaneous 0.9% NaCl.</li>
	<li>Recover the mouse in a warming cabinet at 28 &#176;C for 24 to 48 hr. Observe the mouse for illness up to the completion of the experiment.</li>
	<li>Remove metal skin clips 7 &#8211; 10 days post operatively.</li>
	<li>Once the experiment is complete euthanize the mouse by cervical dislocation.</li>
	<li>Remove and collect the contralateral kidney and allograft for histological analysis.</li>
</ol>

<ol>
	<li>Guild, W. R., Harrison, J. H., Merrill, J. P., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+homotransplantation+of+the+kidney+in+an+identical+twin.">Successful homotransplantation of the kidney in an identical twin.</a> Trans. Am. Clin. Climatol Assoc. 67, 167-173 (1955).</li><li>Wolfe, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mortality+in+all+patients+on+dialysis,+patients+on+dialysis+awaiting+transplantation,+and+recipients+of+a+first+cadaveric+transplant.">Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant.</a> N. Engl. J. Med. 341, 1725-1730 (1999).</li><li>Nankivell, B. J., Alexander, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rejection of the Kidney Allograft. N. Engl. J. Med. 363, 1451-1462 (2010).</li><li>Tse, G. H., Hughes, J., Marson, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+mouse+kidney+transplantation.">Systematic review of mouse kidney transplantation.</a> Transplant International. 26, 1149-1160 (2013).</li><li>Skoskiewicz, M., Chase, C., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+transplants+between+mice+of+graded+immunogenetic+diversity.+Transplant.">Kidney transplants between mice of graded immunogenetic diversity. Transplant.</a> Proc. 5, 721-725 (1973).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+liver,+kidney,+heart,+and+intestine+allograft+rejection+in+different+mouse+strain+combinations.">Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combinations.</a> Transplantation. 62, 1267-1272 (1996).</li><li>Qi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+cells+of+monocyte+lineage+prevents+loss+of+renal+microvasculature+in+murine+kidney+transplantation.">Depletion of cells of monocyte lineage prevents loss of renal microvasculature in murine kidney transplantation.</a> Transplantation. 86, 1267-1274 (2008).</li><li>Dang, Z., Mackinnon, A., Marson, L. P., Sethi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+atrophy+and+interstitial+fibrosis+after+renal+transplantation+is+dependent+on+galectin-3.">Tubular atrophy and interstitial fibrosis after renal transplantation is dependent on galectin-3.</a> Transplantation. 93, 477-484 (2012).</li><li>Jabs, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+the+Evolution+and+Mechanisms+of+the+Lesions+of+Kidney+Allograft+Rejection+in+Mice.">Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice.</a> Am. J. Transplant. 3, 1501-1509 (2003).</li><li>Lin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+C4+from+Donor+or+Recipient+Mouse+Fails+to+Prevent+Renal+Allograft+Rejection.">Deficiency of C4 from Donor or Recipient Mouse Fails to Prevent Renal Allograft Rejection.</a> Am. J. Pathol. 168, 1241-1248 (2006).</li></ol>

The authors have nothing to disclose.

The most well described manner to perform the arterial anastomosis is to use the distal aorta of the donor, with the renal artery in continuation, in an end-to-side manner to the recipient aorta. We describe the use of an aortic patch, similar to the &#8216;Carrell patch&#8217; mirroring that performed in human kidney transplantation which we believe to be more convenient. Though reports in the literature of donor and recipient operative time are sparse we believe that utilizing an aortic patch to recipient aorta instead...

Successful renal transplantation for the treatment of renal failure was first described in 1955 between monozygotic twins1, since then it has become a revolutionary treatment for patients with end-stage renal failure throughout the world, offering both improvement in length and quality of life2. However long term graft survival has been hampered by a multitude of pathological processes resulting in chronic allograft damage3.
Rejection of the transplanted kidney in humans remains a major cause of morbidity, despite significant improvements in immunosupporessive regimens. The aim of developing a mouse model of renal transplantation is to closely replicate the process and pathology found in human renal transplantation4. Skoskiewicz et al. first described the mouse model of renal transplantation in 19735. Although advanced microsurgical skills are required, it is a valuable tool for several reasons: the mouse genome has been well characterized and there is a great variety of experimental methods and techniques available for mouse studies.
Many groups using the mouse model of renal transplantation have used the transplanted kidney as a life-supporting organ, however in other studies and in our described methodology one of the recipient mouse&#8217;s native kidneys is left in situ for the duration of the experiment4. The benefit is that the mouse undergoes a single anesthesia and operation thereby reducing the morbidity to the mouse and the risk of death from a second procedure. Additionally the mouse does not suffer from the adverse effects of gradual renal failure.
Although models of allogeneic rejection exist in other organs such as the heart and skin, these are not always directly relevant to renal transplantation. There is evidence that these models elicit different modes and dynamics of rejection, for instance the time course of rejection in cardiac allograft and renal allograft differs significantly in certain strain combinations6. We have described acute renal allograft rejection patterns in BALB/c donors into non-transgenic FVB/nj mice, this model showed cellular mediated injury with accumulation of T cells and macrophages7. Alternatively we have also described a model of chronic allograft damage that exhibits interstitial fibrosis and tubular atrophy, this results from transplanting a kidney from C57BL/6BM12 donors into C57BL/6 recipients, as these mice are characterized by a single MHC class II loci mis-match8.
Multiple aspects of transplantation have been studied using the mouse model of renal transplantation including acute rejection, cellular and humoral rejection, ischemia reperfusion injury, and trialing novel therapeutic agents. We have modified the surgical technique to reduce operating time and improve the ease of surgery. Particularly we have described simultaneous donor and recipient preparation and a simplified vascular anastomosis technique by utilizing a continuous aortic patch anastomosis. This video and manuscript will provide key points to aid in the establishment of this technique.

<table><tbody><tr><td colspan="4">Surgical Instruments</td></tr><tr><td>Blunt Dissecting Scissors</td><td>Fine Science Tools</td><td>&amp;nbsp;14072-10</td><td>For skin cutting</td></tr><tr><td>Curved Castoviejo scissors</td><td>Fine Science Tools</td><td>15017-10</td><td>For tissue cutting</td></tr><tr><td>Spring Scissors &amp;ndash; straight</td><td>Fine Science Tools</td><td>15000-08</td><td>For suture cutting</td></tr><tr><td>Toothed forceps 1x2 teeth</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>2 x Fine Tip forceps (Dumont No.5)</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Angled Fine Tip forceps (Dumont No. 5/45)</td><td>Fine Science Tools</td><td>11253-25</td><td>For blunt dissecting</td></tr><tr><td>Curved Fine Tip forcep (Dumont No.7)</td><td>Fine Science Tools</td><td>11273-22</td><td>Useful to pass around vessels</td></tr><tr><td>Curved Crile Haemostat</td><td>Fine Science Tools</td><td>1300-04</td><td></td></tr><tr><td>Micro clip applicator with lock</td><td>Fine Science Tools</td><td>18056-14</td><td></td></tr><tr><td>2 x Micro serrefines spring width 2mm, jaw length 4mm</td><td>Fine Science Tools</td><td>18055-04</td><td>Microvascular clamps</td></tr><tr><td>2 x Colibri 3cm wire retractor</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Castroviejo needle holder with lock</td><td>Fine Science Tools</td><td>120660-01</td><td></td></tr><tr><td>Wound clip applicator</td><td>Fine Science Tools</td><td>12031-07</td><td></td></tr><tr><td>7mm wound clips</td><td>Fine Science Tools</td><td>12032-07</td><td>Remove 7 to 10 days after surgery</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>OPMI pico microscope</td><td>Carl Zeiss</td><td>S100</td><td></td></tr><tr><td>Thermal cautery unit with fine tip</td><td>Geiger</td><td>150A</td><td></td></tr><tr><td>Heat electronic pad</td><td>Cozee Cumfort</td><td>n/a</td><td></td></tr><tr><td>Euroklav 23-S</td><td>Melag</td><td>n/a</td><td>Autoclave</td></tr><tr><td colspan="4">Disposable equipment</td></tr><tr><td>7/O Silk braided suture</td><td>Pearsall</td><td>30514</td><td></td></tr><tr><td>10/O Dafilon (polyamide) suture</td><td>B-Braun</td><td>&amp;nbsp;G1118099</td><td></td></tr><tr><td>6/O Vicryl (plygalectin)</td><td>Ethicon</td><td>W9537</td><td></td></tr><tr><td>Regular bevel needle, 1 inch, 21G</td><td>Bection, Dickinson and Company</td><td>305175</td><td>For ureteric anastamosis</td></tr><tr><td>Regular bevel needle, 5/8 inch, 25G</td><td>Bection, Dickinson and Company</td><td>305122</td><td></td></tr><tr><td>Regular bevel needle, 1/2 inch, 30G</td><td>Bection, Dickinson and Company</td><td>304000</td><td></td></tr><tr><td>Insulin needle 1ml, 29G</td><td>Bection, Dickinson and Company</td><td>324827</td><td></td></tr><tr><td>Insulin needle 0.3ml, 30G</td><td>Bection, Dickinson and Company</td><td>324826</td><td></td></tr><tr><td>1 ml syringe slip tip</td><td>Bection, Dickinson and Company</td><td>300184</td><td></td></tr><tr><td>5 ml syringe slip tip</td><td>Bection, Dickinson and Company</td><td>302187</td><td></td></tr><tr><td>Wypall paper swabs</td><td>Kimberley-Clark</td><td>L40</td><td>sterilised by autoclave</td></tr><tr><td>Cotton wool buds</td><td>Johnson and Johnson</td><td>n/a</td><td>sterilised by autoclave</td></tr><tr><td>Plain drapes</td><td>Guardian</td><td>CB03</td><td>sterilised by autoclave</td></tr><tr><td>Cell culture dish 60mm x 15mm</td><td>Corning Incorporated</td><td>430166</td><td></td></tr><tr><td>Dispensing Pin</td><td>B-Braun</td><td>DP3500L / 413501</td><td>Used with NaCl 0.9%</td></tr><tr><td>Re-agents and Drugs</td><td></td><td></td><td></td></tr><tr><td>(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2%</td><td>Allergan Ltd</td><td>21956GB10X</td><td></td></tr><tr><td>(Videne) Povidone-iodine 10%</td><td>Ecolab Ltd</td><td>PL 04509/0041</td><td></td></tr><tr><td>(Vetalar V) Ketamine hydrochloride</td><td>Pfizer Animal Health</td><td>Vm 42058/4165</td><td>100mg/ml solution (dose 200mg/kg)</td></tr><tr><td>(Domitor) Medetomidine hydrochloride&amp;nbsp;</td><td>Orion Pharma</td><td>Vm 06043/4003</td><td>1mg/ml (dose 0.5mg/kg)</td></tr><tr><td>(Vetergesic) Bupernorphine hydrochloride&amp;nbsp;</td><td>Alsto Animal Health</td><td>Vm 00063/4002</td><td>0.3mg/ml (dose 0.05mg/kg)</td></tr><tr><td>(Antisedan) Atipamezole hydrochoride</td><td>Orion Pharma</td><td>Vm 06043/4004</td><td>5mg/ml (dose 2mg/kg)</td></tr><tr><td>University of Wisconsin Solution</td><td>Belzer Bridge to Life</td><td>n/a</td><td>dose approximately 500 microlitres/mouse</td></tr><tr><td>NaCl 0.9%</td><td>Baxter</td><td>FKE1323</td><td></td></tr><tr><td>Heparin Sulphate</td><td>non-proprietary</td><td>n/a</td><td>5000units/ml (dose 5units/mouse)</td></tr></tbody></table>

mouse kidney transplantation: models of allograft rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Renal allograft rejection can be assessed by histological analysis of methacarn-fixed paraffin-embedded tissue sections of the transplanted kidney (Figure 2). Isograft transplantation of kidneys between syngeneic mice results in renal ischemic reperfusion injury, however by 4 weeks the tubules have recovered and are histologically comparable to native kidneys. Acute rejection can be modeled by C57BL/6 kidney transplantation into BALB/c recipients, within 1 week there is diffuse mononuclear cell infiltrat...

Watch this Scientific Journal Video about Mouse Kidney Transplantation: Models of Allograft Rejection at JoVE.com

Mouse Kidney Transplantation: Models of Allograft Rejection

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

mouse-kidney-transplantation-models-of-allograft-rejection

Universidad de Cartagena UDEC

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JoVE Journal

Medicine

20.5K Views.  The University of Edinburgh. Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Video: Mouse Kidney Transplantation: Models of Allograft Rejection

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Immunology and Infection

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

A Modified Surgical Technique for Kidney Transplantation in Mice

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

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 a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Summary

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Chapters in this video

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using ¹⁸F-FDG PET

Mouse Models for Graft Arteriosclerosis

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Murine Kidney Transplant Technique

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Orthotopic Hind Limb Transplantation in the Mouse

A Modified Surgical Technique for Kidney Transplantation in Mice

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection