Name: a modified surgical technique for kidney transplantation in mice
Uploaded: 2022-07-22T13:15:00
Description: Watch this Scientific Journal Video about A Modified Surgical Technique for Kidney Transplantation in Mice at JoVE.com

We thank Dr. Tiantian Bai team for help with voice over, Miss Mian Pao for her help in medical illustration. This work was supported in part by the German Research Foundation (DFG) to promote international collaborations (HO2581/4-1 to AH) and the National Science Foundation of China (NSFC; #81760291 to FJ).

All animal experiments were conducted according to the guidelines from the directive 2010/63/EU of the European Parliament on protection of animals used for scientific purposes (Animal ethics card: Lower Saxony Ministry of Food and Drug Safety, #33.9-42502-04-11/0492). Conduct procedures using sterile surgical instruments and consumables (autoclaved) and try to keep the operating area as sterile as possible.
NOTE: C57BL/6J male mice served as donors and recipients (syngeneic transplant model) ...

<ol>
	<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.">Kidney transplants between mice of graded immunogenetic diversity.</a> Transplantation Proceedings. 5 (1), 721-725 (1973).</li><li>Jiang, K., 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=Noninvasive+assessment+of+renal+fibrosis+with+magnetization+transfer+MR+imaging:+Validation+and+evaluation+in+murine+renal+artery+stenosis.">Noninvasive assessment of renal fibrosis with magnetization transfer MR imaging: Validation and evaluation in murine renal artery stenosis.</a> Radiology. 283 (1), 77-86 (2017).</li><li>Tse, G. H., 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=Mouse+kidney+transplantation:+Models+of+allograft+rejection.">Mouse kidney transplantation: Models of allograft rejection.</a> Journal of Visualized Experiments. (92), e52163 (2014).</li><li>Okazaki, M., 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=et+al.Costimulatory+blockade-mediated+lung+allograft+acceptance+is+abrogated+by+overexpression+of+Bcl-2+in+the+recipient.">et al.Costimulatory blockade-mediated lung allograft acceptance is abrogated by overexpression of Bcl-2 in the recipient.</a> Transplantation Proceedings. 41 (1), 385-387 (2009).</li><li>Chuck, N. C., 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=et+al.Ultra-short+echo-time+magnetic+resonance+imaging+distinguishes+ischemia/reperfusion+injury+from+acute+rejection+in+a+mouse+lung+transplantation+model.">et al.Ultra-short echo-time magnetic resonance imaging distinguishes ischemia/reperfusion injury from acute rejection in a mouse lung transplantation model.</a> Transplant International. 29 (1), 108-118 (2016).</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 (9), 1267-1272 (1996).</li><li>Wang, J., Hockenheimer, S., Bickerstaff, A. A., Hadley, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+renal+transplantation+procedure.">Murine renal transplantation procedure.</a> Journal of Visualized Experiments. (29), e1150 (2009).</li><li>Plenter, R., Jain, S., Ruller, C. M., Nydam, T. L., Jani, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+kidney+transplant+technique.">Murine kidney transplant technique.</a> Journal of Visualized Experiments. (104), e52848 (2015).</li><li>Plenter, R. J., Jain, S., Nydam, T. L., Jani, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+arterial+anastomosis+for+improving+murine+kidney+transplant+outcomes.">Revised arterial anastomosis for improving murine kidney transplant outcomes.</a> Journal of Investigative Surgery. 28 (4), 208-214 (2015).</li><li>Rong, S., Lewis, A. G., Kunter, U., Haller, H., Gueler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+knotless+technique+for+kidney+transplantation+in+the+mouse.">A knotless technique for kidney transplantation in the mouse.</a> Journal of Transplantation. , 127215 (2012).</li><li>Kreimann, K., 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=Ischemia+reperfusion+injury+triggers+CXCL13+release+and+B-cell+recruitment+after+allogenic+kidney+transplantation.">Ischemia reperfusion injury triggers CXCL13 release and B-cell recruitment after allogenic kidney transplantation.</a> Frontiers in Immunology. 11, 1204 (2020).</li><li>Schmidbauer, M., 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=Diffusion-Weighted+imaging+and+mapping+of+T1+and+T2+relaxation+time+for+evaluation+of+chronic+renal+allograft+rejection+in+a+translational+mouse+model.">Diffusion-Weighted imaging and mapping of T1 and T2 relaxation time for evaluation of chronic renal allograft rejection in a translational mouse model.</a> Journal of Clinical Medicine. 10 (19), 4318 (2021).</li><li>Wu, K., 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=Novel+technique+for+blood+circuit+reconstruction+in+mouse+heart+transplantation+model.">Novel technique for blood circuit reconstruction in mouse heart transplantation model.</a> Microsurgery. 26, 594-598 (2006).</li><li>Haas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+allograft+nephropathy+or+interstitial+fibrosis+and+tubular+atrophy:+what+is+in+a+name.">Chronic allograft nephropathy or interstitial fibrosis and tubular atrophy: what is in a name.</a> Current Opinion in Nephrology and Hypertension. 23 (3), 245-250 (2014).</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 (5), 477-484 (2012).</li><li>Yin, D., 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=Blood+circuit+reconstruction+in+an+abdominal+mouse+heart+transplantation+model.">Blood circuit reconstruction in an abdominal mouse heart transplantation model.</a> Journal of Visualized Experiments. (172), e62007 (2021).</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=Improved+techniques+for+kidney+transplantation+in+mice.">Improved techniques for kidney transplantation in mice.</a> Microsurgery. 16 (2), 103-109 (1995).</li><li>Mannon, R. B., 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=Chronic+rejection+of+mouse+kidney+allografts.">Chronic rejection of mouse kidney allografts.</a> Kidney International. 55 (5), 1935-1944 (1999).</li><li>Coffman, 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=Improved+renal+function+in+mouse+kidney+allografts+lacking+MHC+class+I+antigens.">Improved renal function in mouse kidney allografts lacking MHC class I antigens.</a> Journal of Immunology. 151 (1), 425-435 (1993).</li><li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+curve,+surgical+results+and+operative+complications+for+kidney+transplantation+in+mice.">Learning curve, surgical results and operative complications for kidney transplantation in mice.</a> Microsurgery. 26 (8), 590-593 (2006).</li></ol>

While the skin transplantation model in mice is simple and easy to perform to study alloimmune rejection events, the surgical techniques for investigating more specifically the alloimmune-related inflammatory alterations after heart16 and kidney transplantation10 has been proven to be complex and very demanding. From the transplant nephrologist&#39;s point of view, the establishment of an effective and long-term stable mouse renal transplantation model has still an irreplac...

Since Sakowitz et al. developed mouse models of kidney transplantation in 1973 for the first time1, it has proven as an important experimental tool to study the mechanisms of transplant ischemic injury and alloimmune rejection as well as for developing new treatments aimed to prolong allograft survival and possibly to achieve immunological tolerance. However, the surgical technique has proven to be complex and very demanding, sometimes having complications such as vascular anastomotic strictures leading to prerenal non-immunological kidney transplant failure2, postrenal failure caused by ischemia and subsequent necrosis of the transplanted ureter, strictures of the anastomosis of the transplanted ureter and/or the recipient&#39;s urine bladder leading to a disruption of the urinary outflow. All of these are reasons why renal transplantation in mice has not been further developed and is therefore not widely used. Establishing an effective and long-term stable mouse kidney transplantation model without vascular and urinary tract complications still has irreplaceable significance for many studies in the transplant field with focus on the renal immune mediated but also infectious diseases3. In addition, compared with other organ transplants in murine models such as lung, heart, and intestinal transplantation4,5, the mouse kidney transplantation model offers a chance for studying long-term survival even in the setting of major histocompatibility antigen disparity3,6. It has also been shown that in the same setting of donor-recipient strain combinations different organ transplants such as heart or kidney are characterized by different dynamics and onsets of allograft rejection3. Furthermore, from the nephrological point of view, it is a more suitable model for studying parenchymal mediated immune regulatory mechanisms in the context of acute and chronic rejection events than simple skin transplant experiments.
On the basis of previous reports on the surgical technique of kidney transplantation in mice3,7,8,9, we here demonstrate the following reliable improvements that have been successfully applied during the past 10 years within our group10,11,12: Firstly, the ureteral artery is safely conserved as the renal artery is resected en bloc together with the respective part of the abdominal aorta. Second, a new, simple, and rapid technique of a knotless vascular anastomosis in which the final stitch of anastomosis is not tied with the end of the upper tie like the traditional approach but remains free. This technique enables to increase or decrease the size of the anastomosis after renal reperfusion to avoid vessel stricture and intraabdominal bleeding. Third, 21 G and 30 G syringe needles were used as an auxiliary puncture guiding tool in order to implant the donor ureter into the recipient&#39;s bladder wall reducing the damage to the recipient&#39;s bladder and facilitating the formation of stricture free anastomosis.
In this report, we also compared the traditional, widely used technique with the modified one that is established in our laboratory and found no significant difference in the degree of renal tubular atrophy and kidney transplant interstitial tissue fibrosis. In previous studies, we additionally compared the results of this new technique with the conventional method in terms of local bleeding, thrombosis, time for performing the vessel anastomosis and survival rate. We found improvements such as significant reductions of local thrombosis events (1.1% versus 6.6%), a reduced time for the anastomosis procedure, and a highly reproducible kidney syngeneic graft long-term survival (95% versus 84% with the classical approach)10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30G-needles</td>
			<td>Braun</td>
			<td>456300</td>
			<td>-</td>
		</tr>
		<tr>
			<td>acepromazine</td>
			<td>CP Pharma</td>
			<td>Tranquisol P</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Bepanthen eye ointment</td>
			<td>Haus-Apotheke</td>
			<td>PZN 01578675</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Bonn Micro Forceps</td>
			<td>FST</td>
			<td>11083-07</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Box for insulation and oxygen supply device</td>
			<td>RUSKINN</td>
			<td>INVIV</td>
			<td>-</td>
		</tr>
		<tr>
			<td>C57BL/6J&#160; mice</td>
			<td>Charles River. Germany</td>
			<td>no catalog number</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Zoetis</td>
			<td>Rimadyl 50 mg/ml</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CATHETER-FEP 26G</td>
			<td>TERUMO</td>
			<td>Surflo-W</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Clip Applicator Forceps Style</td>
			<td>FST</td>
			<td>18057-14</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>WPI</td>
			<td>14114-G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cutasept skin disinfection</td>
			<td>VWR</td>
			<td>BODL980365</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dehydrator</td>
			<td>DIAPATH</td>
			<td>Donatello</td>
			<td>-</td>
		</tr>
		<tr>
			<td>electrosurgical pen</td>
			<td>Bovie</td>
			<td>CHANGE-A-TIP</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Embedding machine</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-P5</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Group Chemical Reagent Co. LtD</td>
			<td>100092683</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Frozen platform</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-L5</td>
			<td>-</td>
		</tr>
		<tr>
			<td>gauze pads, cotton swabs</td>
			<td>Lohmann-Rauscher</td>
			<td>13353</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Servicebio</td>
			<td>G6004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HE dye solution set</td>
			<td>Servicebio</td>
			<td>G1003</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating mat</td>
			<td>THERMO MAT PRO 30W</td>
			<td>HTP-30</td>
			<td>-</td>
		</tr>
		<tr>
			<td>hemostatic sponge</td>
			<td>CuraSpon</td>
			<td>J1276A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>heparine-solution</td>
			<td>Haus-Apotheke</td>
			<td>PZN 03029820</td>
			<td>-</td>
		</tr>
		<tr>
			<td>ice box</td>
			<td>PETZ</td>
			<td>No Catalog Number available</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Imaging system</td>
			<td>Nikon</td>
			<td>Nikon DS-U3</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Inhalation anesthesia device</td>
			<td>GROPPLER</td>
			<td>BKGM 0616</td>
			<td>-</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>CP Pharma</td>
			<td>Isofluran CP 1 ml/ml</td>
			<td>-</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>Zoetis</td>
			<td>no catalog numer</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Masson dye solution set</td>
			<td>Servicebio</td>
			<td>G1006</td>
			<td>-</td>
		</tr>
		<tr>
			<td>metamizole</td>
			<td>WDT</td>
			<td>no catalog numer</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>FST</td>
			<td>15000-00,15000-10</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Micro Serrefine ( Clamp ) Angled / 16 mm</td>
			<td>FST</td>
			<td>18055-06</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>LEICAMZ6</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microscope light</td>
			<td>SCHOTT</td>
			<td>KL2500LED</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Neutral gum</td>
			<td>SCRC</td>
			<td>10004160</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Tianjin Laibo Rui Instrument Equipment Co., Ltd</td>
			<td>GFL-230</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pathology slicer</td>
			<td>Shanghai Leica Instrument Co., Ltd</td>
			<td>RM2016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Saline solution (NaCl 0.9 %)</td>
			<td>Haus-Apotheke</td>
			<td>PZN 06178437</td>
			<td>-</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>Peha Instruments</td>
			<td>991083/4</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Servicebio</td>
			<td></td>
			<td>-</td>
		</tr>
		<tr>
			<td>small Petri dish</td>
			<td>Sarstedt</td>
			<td>8,33,900</td>
			<td>-</td>
		</tr>
		<tr>
			<td>straight forceps</td>
			<td>WPI</td>
			<td>14113-G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>surgical tape</td>
			<td>BSN</td>
			<td>4120</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Suture Tying Forceps - 10 cm</td>
			<td>FST</td>
			<td>18025-10</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sutures(10-0)</td>
			<td>Medtronic</td>
			<td>N2540</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sutures(4-0)</td>
			<td>ETHILON</td>
			<td>V4940H</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sutures(7-0)</td>
			<td>ETHILON</td>
			<td>1647H</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Syringe (0,3 mL)</td>
			<td>BD</td>
			<td>324826</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>BD</td>
			<td>320801</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tissue spreader</td>
			<td>Zhejiang Kehua Instrument Co., Ltd</td>
			<td>KD-P</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Upright optical microscope</td>
			<td>Nikon</td>
			<td>Nikon Eclipse E100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>Bayer</td>
			<td>Rompun</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sinopharm Group Chemical Reagent Co. LtD</td>
			<td>10023418</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

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.

Four weeks after transplantation, both the modified technique as well as the conventional technique displayed moderate signs of renal tubular atrophy14,15 when compared to the native recipient contralateral kidneys (Figure 1). The degree of the renal tubules atrophy demonstrated no significant difference between the two different techniques. Masson Goldner&#39;s trichrome staining14,<sup class="xref...

Watch this Scientific Journal Video about A Modified Surgical Technique for Kidney Transplantation in Mice at JoVE.com

A Modified Surgical Technique for Kidney Transplantation in 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 ...

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.

Research

JoVE Journal

Immunology and Infection

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