Name: a rat orthotopic renal transplantation model for renal allograft rejection
Uploaded: 2022-02-02T15:00:00
Description: Watch this Scientific Journal Video about A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection at JoVE.com

This work was supported by the National Natural Science Foundation of China (81870304) to Jun Li and by the Else Kr&#246;ner-Fresenius-Stiftung (Nr. 2017_A28) to Marcus Groettrup.

Inbred 8-10 weeks old male F344 and Lewis rats (200 g to 250 g) were commercially obtained. Allogeneic left kidney transplantation was performed between male F344 and Lewis rats. F344 rats were used as donors and syngeneic recipients, and Lewis rats served as allogeneic recipients. All animal handling procedures were conducted in compliance with guidelines for the Care and Use of Laboratory Animals published by NIH, and all animal experimental protocols were approved by the Animal Care and Use Committee of Chongqing Univ...

<ol>
	<li>Cooper, J. E., Wiseman, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+immunosuppressive+agents+in+kidney+transplantation.">Novel immunosuppressive agents in kidney transplantation.</a> Clinical Nephrology. 73 (5), 333-343 (2010).</li><li>Colaneri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+transplant+immunosuppression--history,+principles,+and+current+practices+in+kidney+transplantation.">An overview of transplant immunosuppression--history, principles, and current practices in kidney transplantation.</a> Nephrology Nursing Journal. 41 (6), 549-560 (2014).</li><li>Colvin, R. B., Smith, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-mediated+organ-allograft+rejection.">Antibody-mediated organ-allograft rejection.</a> Nature Reviews Immunology. 5 (10), 807-817 (2005).</li><li>Joosten, S. 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=Antibody+response+against+perlecan+and+collagen+types+IV+and+VI+in+chronic+renal+allograft+rejection+in+the+rat.">Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat.</a> American Journal of Pathology. 160 (4), 1301-1310 (2002).</li><li>Joosten, S. A., van Ham, V., Borrias, M. C., van Kooten, C., Paul, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+against+mesangial+cells+in+a+rat+model+of+chronic+renal+allograft+rejection.">Antibodies against mesangial cells in a rat model of chronic renal allograft rejection.</a> Nephrology Dialysis Transplantation. 20 (4), 692-698 (2005).</li><li>Yamanaka, 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=Depression+of+complement+regulatory+factors+in+rat+and+human+renal+grafts+is+associated+with+the+progress+of+acute+T-cell+mediated+rejection.">Depression of complement regulatory factors in rat and human renal grafts is associated with the progress of acute T-cell mediated rejection.</a> PLoS One. 11 (2), 0148881 (2016).</li><li>Li, 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=Immunoproteasome+inhibition+prevents+chronic+antibody-mediated+allograft+rejection+in+renal+transplantation.">Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.</a> Kidney International. 93 (3), 670-680 (2018).</li><li>Li, 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=Immunoproteasome+inhibition+induces+plasma+cell+apoptosis+and+preserves+kidney+allografts+by+activating+the+unfolded+protein+response+and+suppressing+plasma+cell+survival+factors.">Immunoproteasome inhibition induces plasma cell apoptosis and preserves kidney allografts by activating the unfolded protein response and suppressing plasma cell survival factors.</a> Kidney International. 95 (3), 611-623 (2019).</li><li>Miller, B. F., Gonzalez, E., Wilchins, L. J., Nathan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+transplantation+in+the+rat.">Kidney transplantation in the rat.</a> Nature. 194, 309-310 (1962).</li><li>Tillou, X., Howden, B. O., Kanellis, J., Nikolic-Paterson, D. J., Ma, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+renal+research:+kidney+transplantation+in+the+rat.">Methods in renal research: kidney transplantation in the rat.</a> Nephrology. 21 (6), 451-456 (2016).</li><li>Daniller, A., Buchholz, R., Chase, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+transplantation+in+rats+with+the+use+of+microsurgical+techniques:+a+new+method.">Renal transplantation in rats with the use of microsurgical techniques: a new method.</a> Surgery. 63 (6), 956-961 (1968).</li><li>Ahmadi, A. R., 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=Orthotopic+rat+kidney+transplantation:+A+novel+and+simplified+surgical+approach.">Orthotopic rat kidney transplantation: A novel and simplified surgical approach.</a> Journal of Visualized Experiments: JoVE. (147), e59403 (2019).</li><li>G&#252;nther, E., Walter, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+histocompatibility+complex+of+the+rat+(Rattus+norvegicus).">The major histocompatibility complex of the rat (Rattus norvegicus).</a> Immunogenetics. 53 (7), 520-542 (2001).</li><li>Loupy, 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=The+Banff+2015+Kidney+Meeting+Report:+Current+challenges+in+rejection+classification+and+prospects+for+adopting+molecular+pathology.">The Banff 2015 Kidney Meeting Report: Current challenges in rejection classification and prospects for adopting molecular pathology.</a> American Journal of Transplantation. 17 (1), 28-41 (2017).</li><li>Fisher, B., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+surgical+techniques+in+research,+with+special+reference+to+renal+transplantation+in+the+rat.">Microvascular surgical techniques in research, with special reference to renal transplantation in the rat.</a> Surgery. 58 (5), 904-914 (1965).</li><li>Kamada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+description+of+cuff+techniques+for+renal+transplantation+in+the+rat.+Use+in+studies+of+tolerance+induction+during+combined+liver+grafting.">A description of cuff techniques for renal transplantation in the rat. Use in studies of tolerance induction during combined liver grafting.</a> Transplantation. 39 (1), 93-95 (1985).</li><li>Grabner, 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=Non-invasive+imaging+of+acute+allograft+rejection+after+rat+renal+transplantation+using+18F-FDG+PET.">Non-invasive imaging of acute allograft rejection after rat renal transplantation using 18F-FDG PET.</a> Journal of Visualized Experiments: JoVE. (74), e4240 (2013).</li></ol>

Renal transplantation in the rat is a challenging work requiring a high level of micro-surgery techniques and the operation techniques have been optimized several times. From the beginning, Gonzalez et al. implanted the donor kidney into the neck of the recipient and anastomosed the donor ureter to the skin9. However, because of the high incidence of urinary infection and stenosis of the donor ureter, the operation was abandoned in a short time. Subsequently, the implant operation of the donor&#39...

Renal transplantation has become the most effective therapeutic approach for patients with end-stage renal function failure. However, T cell-mediated acute rejection and alloantibody-mediated humoral immune rejection result in the pathologic injury of renal allografts and limit the short-term and long-term survival of patients after kidney transplantation1,2,3. Unfortunately, the effective pharmaceuticals that prevent the rejection of renal allografts are still lacking, because the exact mechanisms of immune and pathologic rejection of renal allografts are not clear. Consequently, the preclinical studies that elucidate the mechanisms of immune and pathologic rejection of renal allografts contribute to finding novel targets and developing relevant effective pharmaceuticals to prevent the rejection of renal allografts and eventually prolong the survival of patients.
Many potential immunological and pathophysiological mechanisms of renal allograft rejection have been proposed recently in rat model studies of orthotopic renal transplantation4,5,6,7,8. These findings propose several novel targets and relevant interfering approaches as promising therapeutics to suppress renal allograft rejection, such as complement regulatory factors and anti-CD59 antibodies6, immunoproteasome, and epoxyketone inhibitors7,8. Thus, rat orthotopic renal transplantation is an ideal preclinical model to investigate the mechanisms of immune rejection and pathologic injury of renal allografts after kidney transplantation.
Rat kidney transplantation has gradually shifted from heterotopic implantation of donor&#39;s kidneys9 to orthotopic renal implantation using end-to-side anastomosis of vessels or using end-to-end anastomosis of the ureter using a cuff method10,11,12. The present study describes detailed protocols of the orthotopic renal transplantation between rats using end-to-end anastomosis to recipients&#39; renal artery and renal vein, and an end-to-side &#34;tunnel&#34; method of ureter-bladder anastomosis, which avoids the ischemia of the lower body and the thrombosis of inferior vena cava and reduces postoperative urine leakage and the twist of the ureter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;10-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>G0090781</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;4-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>C1048451</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;8-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>C2090880</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>US Biological life Sciences</td>
			<td>352004</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocoagulator</td>
			<td>Electrocoagulator</td>
			<td>ZJ1099</td>
			<td></td>
		</tr>
		<tr>
			<td>F344 and Lewis rats</td>
			<td>Center of Experimental Animals (Tongji Medical College, Huazhong University of Science and Technology, China)</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Henan piaoan group Co., LTD</td>
			<td>10210402</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Guangzhou Dewei Biological Technology Co., LTD</td>
			<td>DK0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>North China Pharmaceutical Co., LTD</td>
			<td>2101131-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe (1 ml and 10 ml)</td>
			<td>Shandong weigao group medical polymer Co., LTD</td>
			<td>20211001</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co., LTD</td>
			<td>21070201</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G Sodium</td>
			<td>Wuhan HongDe Yuexin pharmatech co.,Ltd</td>
			<td>69-57-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalp needle (24 G)</td>
			<td>Hongyu Medical Group</td>
			<td>20183150210</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Beyotime</td>
			<td>FS600</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>RWD Life Science</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Surgery Kit</td>
			<td>Beyotime</td>
			<td>FS500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride injection</td>
			<td>Southwest pharmaceutical Co., LTD</td>
			<td>H50021610</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical operation microscope</td>
			<td>Tiannuoxiang Scientific Instrument Co. , Ltd, Beijing, China</td>
			<td>SZX-6745</td>
			<td></td>
		</tr>
		<tr>
			<td>Swab</td>
			<td>Yubei Medical Materials Co., LTD</td>
			<td>21080274</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Minnesota Mining Manufacturing Medical Equipment (Shanghai) Co., LTD</td>
			<td>1911N68</td>
			<td></td>
		</tr>
		<tr>
			<td>UW solution</td>
			<td>Bristol-Myers Squibb Company</td>
			<td>17HB0002</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this rat orthotopic renal transplantation model, the recipient rats move normally after operation. To observe the chronic rejection of renal allograft, recipient rats are raised for 10 weeks after transplantation, and the total survival rate of recipient rats at this time point is approximately 90%. The major causes of death are bleeding and leakage of urine post-operation. The other major complications include bleeding during the operation, thrombosis in renal vessels, and hydronephrosis, whose respective incidence i...

Watch this Scientific Journal Video about A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection at JoVE.com

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

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 ...

Rat orthotopic renal transplantation is an important model to investigate the mechanism of renal allograft rejection in preclinical studies and could aid in developing novel approach to improving the long-term survival of renal allografts. End-to-end anastomosis of renal vessels in this surgery avoids ischemia of the lower body and the thrombosis of inferior vena cava, while anastomosis of ureter in an end-to-side tunnel method reduces postoperative urine leakage and the twist of the ureter. This method provides insight into investigating the mechanisms of immune rejection and injury of renal allografts after kidney transplantation.Begin by incising the abdomen using a surgical scissor along the midline from the symphysis pubis to the subxiphoid. Then pull open the incision using a retractor. Take out the intestines from the right side of the incision and wrap them with moistened gauze to prevent them from drying out.Then expose the left kidney. Dissociate the left renal artery and vein using micro-forceps and cotton swabs under 20 times magnification. Ligate and transect the adrenal vein and genital vein if necessary.Dissociate the ureter with the help of micro-forceps, preserving the peripheral connective tissues to ensure blood supply to the ureter. Dissociate and clip or ligate the aorta approximately five millimeters above the left renal artery with a vascular clamp or 4-0 polyamide monofilament suture. Then transect the left renal vein distal to the conjunction of the left genital vein and adrenal vein.Using a 24 gauge scalpel needle, flush the kidney from the aorta below the left renal artery with ice cold UW solution supplemented with 100 units per milliliter Heparin until blood fades in color. Transect the left renal artery next to the aorta, then transect the ureter next to the bladder and dissociate the kidney with the help of micro-forceps and scissors. Preserve the donor left kidney in ice cold UW solution.Under 20 times magnification, dissociate the left renal artery and renal vein of the recipient rat using cotton swabs and micro-forceps. Ligate the adrenal vein and genital vein if necessary. Clip the left renal artery and renal vein at the root using microvascular clamps, then transect the renal artery and renal vein three millimeters away from the microvascular clamp.Dissociate and ligate the left ureter approximately two to three centimeters below the kidney with an 8-0 polyamide monofilament suture and transect it at the ligation. Resect the recipients native left kidney by dissociating the kidney with the help of micro-forceps and scissors. Implant the donor kidney into the left renal fossa of the recipient rat and put ice around the implanted donor kidney.Switch to 45 times magnification, then anastomose the donors renal artery and renal vein to the recipients renal artery and renal vein in an end-to-end pattern using 10-0 polyamide monofilament sutures. To anastomose the renal artery with interrupted sutures, place the stay sutures at 12 and six o'clock positions of anastomosis. Equidistantly suture one side of the anastomosis between the two stay sutures with two to three stitches.Turn over the stay sutures and similarly suture the other side of the anastomosis between the two stay sutures with two to three stitches. To anastomose the renal vein with continuous sutures, place the stay sutures at the six and 12 o'clock positions of the anastomosis. Suture one side of the anastomosis from the 12 o'clock position with four to five stitches using the running sutures.Turn over the stay sutures and similarly, suture the other side of the anastomosis from the six o'clock position. Reperfuse the donor kidney by releasing the non-invasive microvascular clamp of the renal vein, identify the bleeding sites and make additional stitches if necessary. Next, release the non-invasive microvascular clamp of the renal artery, identify the bleeding sites and make additional stitches if necessary.Under 20 times magnification, ligate or stitch the end of the donor ureter with a 4-0 polyamide monofilament suture as a tow to drag the end through the tunnel of the recipients bladder. Fix the donor ureter with the recipients bladder by stitching the adventitia of the donor ureter with the muscle layer of the recipients bladder outside at four equidistant positions using an 8-0 polyamide monofilament suture. Afterward, transect the end of the donor ureter with the suture outside the recipients bladder and allow the donor ureter to shrink back into the recipients bladder.Place the intestines back into the abdominal cavity and close the abdominal incision with continuous sutures in the muscle layer and then the skin layer with a 4-0 polyamide monofilament suture. At ten weeks after transplantation, hematoxylin and eosin staining and periodic acid shift staining revealed glomerular sclerosis, interstitial fibrosis, tubular atrophy and interstitial arterial sclerosis in the renal allograft in contrast to the isograft kidney. Silver staining showed the thickening of the glomerular basement membrane in the allograft kidney when compared with the isograft kidney, indicating the success of the rat orthotopic renal transplantation model.Because the vascular hole of the renal is very thin, to avoid the wrong suturing, remember to perform the anastomosis on the high magnification field.

a-rat-orthotopic-renal-transplantation-model-for-renal-allograft

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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 ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

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.

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 ...

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 ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

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 ...