Name: Murine Renal Transplantation Procedure (Video) | JoVE Journal
Uploaded: 2009-07-10T00:00:00.000+00:00
Duration: 18 min 48 s
Description: Learn the murine renal transplantation procedure for studying kidney function and transplant rejection using mouse models. This method demonstrates surgical techniques and post-operative monitoring essential for successful graft assessment.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

These studies were supported in part by funding by NIH R01 AI36532 to GAH. We would like to acknowledge the outstanding support in transplant pathology provided by Dr. Tibor Nadasdy, and the invaluable guidance and foresight provided by the late Dr. Charles G. Orosz in initiating this research effort.

Donor Organ Harvest (2):
<ol>
 <li>Anesthetize the donor using 65mg/kg pentobarbital IP; anesthesia is maintained by Isoflurane (2-3%). </li>
 <li>Make a midline incision from sternum to pubis to enter the donor abdomen and expose the left kidney by moving the intestine laterally to the right side. </li>
 <li>Isolate the left kidney by ligating and dividing the adrenal and testicular vessels with an 8-0 silk suture. </li>
 <li>Mobilize the aorta above its junction with the renal artery and vein. </li>
 <li>Mobilize the inferior vena cava (IVC) above and below its junction with the renal artery and vein. </li>
 <li>Ligate the aorta below the renal artery and vein with an 8-0 silk suture to minimize bleeding. </li>
 <li>Dissect the left ureter from the renal hilum to the bladder. </li>
 <li>Expose the ureterovesical junction using caudad retraction on the bladder dome. </li>
 <li>Excise a small, elliptical patch of bladder containing the left ureterovesical junction, used for urinary reconstruction via a bladder-to-bladder anastomosis. The right ureter is not included with the bladder. </li>
 <li>Ligate the IVC below the renal artery and vein with an 8-0 silk suture. </li>
 <li>Ligate the aorta above the renal artery and vein, and slowly perfuse the graft in situ with 0.2-0.4ml cold, heparinized Ringer's lactate solution via the infrarenal aorta. </li>
 <li>Ligate the IVC above the renal artery and vein with an 8-0 silk suture.</li>
 <li>Transect the renal vein at its junction with the IVC. </li>
 <li>Divide the aorta obliquely, approximately 2mm below the renal artery. </li>
 <li>Remove the kidney and its vascular supply with ureter attached to the bladder patch en bloc. Store in Ringer's lactate solution on ice. </li>
 <li>Euthanize the donor mouse by cervical dislocation under anesthesia. </li>
</ol>
Recipient Transplantation (2):
<ol>
 <li>Anesthetize the recipient with 65 mg/kg pentobarbital IP and maintain the anesthesia by Isoflurane (2-3%). </li>
 <li>Make a midline incision, cover the intestine with wet gauze and carefully retract it to the left side. </li>
 <li>Ligate the right ureter, renal artery and vein, and remove the right kidney. </li>
 <li>Carefully isolate and cross-clamp the infrarenal aorta and IVC with two 4-mm microvasular clamps after ligating the lumbar branches. </li>
 <li>Place a 10-0 nylon suture through a full thickness of aorta and retract to make an elliptical aortaotomy by a single cut (about one-fifth of the diameter of the vessel). </li>
 <li>Make a longitudinal venotomy by piercing the IVC with a 30 gauge needle and extend appropriately using microscissors. </li>
 <li>Irrigate both the aorta and IVC with heparinized saline to clear intraluminal blood or clots. </li>
 <li>Remove the donor kidney from the ice and place it intra-abdominally in the right flank of the mouse. The donor kidney is kept moist with a cold saline-soaked gauze pad. </li>
 <li>Place two stay sutures at the proximal and distal apex of the recipient&#8217;s aortaotomy with the donor&#8217;s aortic cuff. </li>
 <li>Make an end-to-side anastomosis between the donor aortic cuff and the anterior wall of the recipient&#8217;s aortaotomy, using 2 ~ 3 continuous 10-0 nylon sutures in the outside of the aorta. </li>
 <li>Turn the donor kidney graft over to the left flank of the recipient. repeat the previous procedure between the donor aortic cuff and the posterior wall of the recipient&#8217;s aortaotomy </li>
 <li>Perform an end-to-side anastomosis between the donor renal vein and the posterior wall of the recipient&#8217;s IVC, using 4 to 5 continuous 10-0 nylon sutures in the inside of the IVC. and then the anterior wall of IVC and donor&#8217;s renal vein are closed with 4 ~ 5 continuous sutures outside the IVC.</li>
 <li>The anastomosis of both vein and artery must be finished within 20 minutes. </li>
 <li>Rinse the graft with cold saline several times during anastomosis. </li>
 <li>Divide the dome of the recipient bladder, and stop bleeding with cautery. </li>
 <li>Make a small cut on the dome. </li>
 <li>Place two stay sutures of 10-0 nylon to anastomize the donor&#8217;s small bladder patch with the recipient&#8217;s bladder dome. Each side of anastomosis is sutured with 8 ~ 10 continuous stitches. </li>
 <li>Close the abdomen in two layers with continuous 4-0 sterile, synthetic absorbable vicryl suture. </li>
 <li>Give recipient mice 0.4ml warm saline during surgery by intermittent injection IV.</li>
 </ol>
Post-operative Care of Recipient
<ol>
<li>After closing the abdomen, a single intramuscular injection of penicillin (500U/10g) is administered (2). </li>
<li>In addition, recipients receive 0.05 mg/kg buprenorphine intramuscularly on day 0 and day 1 post surgery for pain. Following surgery, transplant recipients receive water containing sulfatrim (100mg/kg) until the experiment is terminated. </li>
<li>For the first 24h postoperatively mice are kept in a temperature controlled incubator. </li>
<li>Mice usually recover from anesthesia within 1 hour of the operation and are given regular food and water ad libitum. </li>
<li>Skin sutures are removed 2 weeks after the surgery. </li>
</ol>
Recipient Nephrectomy
<ol>
<li>Anesthetize the recipient 4 ~ 7 days post kidney transplantation with 65 mg/kg pentobarbital IP; anesthesia is maintained by Isoflurane (2-3%). </li>
<li>Open the abdomen via a midline incision. </li>
<li>Cover the intestine with saline-soaked gauze and carefully retract it to the right side. </li>
<li>Remove the left kidney after ligating the left ureter, kidney vein and artery. </li>
<li>Close the abdomen in two layers with continuous 4-0 synthetic absorbable vicryl suture. </li>
</ol>
Whole Blood Creatinine Measurements
<ol>
<li>Weekly to biweekly blood collection of less than 0.2 cc is performed either submandibularly without anesthesia or retro-orbitally under Isoflurane anesthesia.</li>
<li>Quantitative whole blood creatinine levels are determined using an I-Stat Portable Clinical Analyzer (Heska Corp., Fort Collins, CO). </li>
<li>Conventional units (mg/dl) are converted to SI units of micromol/L by multiplying the conventional units by 88.4.</li>
</ol>

<ol><li>Russell, P. S., Chase, C. M., Colvin, R. B., Plate, J. M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kidney+transplants+in+mice.+An+analysis+of+the+immune+status+of+mice+bearing+long-term%2C+H-2+incompatible+transplants%5BTitle%5D)+AND+%22JExpMed%22%5BJournal%5D)">Kidney transplants in mice. An analysis of the immune status of mice bearing long-term, H-2 incompatible transplants.</a> JExpMed. 147, 1449-1468 (1978).</li>
<li>Zhang, Z., Schlachta, C., Duff, J., Stiller, C., Grant, D., Zhong, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+techniques+for+kidney+transplantation+in+mice%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Improved techniques for kidney transplantation in mice.</a> Microsurgery. 16 (2), 103-109 (1995).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by The Ohio State University IACUC.

We have performed approximately 1200 cases of renal transplantation in mice with a technical success rate of &gt;90%. Compared to heart transplantation, the success rate of kidney transplantation is usually low. However, our laboratory has maintained a high success rate (&gt; 90%) of kidney transplantation. The key points for success are to reduce reperfusion injuries to both donor kidney and recipients, and minimize bleeding and thrombosis throughout the procedure and post-operatively. The following factors may be most ...

<table><tbody><tr><td>i-STAT</td><td>Other</td><td>HESKA</td><td>Cat 5101</td><td></td></tr><tr><td>i-STAT Cartridge</td><td>Other</td><td>HESKA</td><td>CAT 5110-CREA</td><td></td></tr><tr><td>Sulfatrim</td><td>Other</td><td>Actavis</td><td>R02109</td><td></td></tr><tr><td>Isoflurane</td><td>Other</td><td>NOVAPLUS</td><td>CA-0474</td><td></td></tr><tr><td>10-0 Suture</td><td>Surgery</td><td>Sharpoint</td><td>AK0100</td><td></td></tr></tbody></table>

murine renal transplantation procedure

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

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

Research

JoVE Journal

Biology

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes.  Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

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.

Mouse Kidney Transplantation: Models of Allograft Rejection

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

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.

Murine Kidney Transplant Technique

We provide a simple and efficient method to transplant 2’-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude 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.

A Modified Surgical Technique for Kidney Transplantation in Mice

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.

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

This protocol describes a survival procedure for safe and well-tolerated murine bilateral renal lymphadenectomy.

Murine Bilateral Renal Lymphadenectomy

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response.  However, the techniques involved are still challenging.  By modifying standard techniques we have had success with more than 1000 transplants.

Heterotopic Heart Transplantation in Mice

Learn the murine renal transplantation procedure for studying kidney function and transplant rejection using mouse models. This method demonstrates surgical techniques and post-operative monitoring essential for successful graft assessment.

Murine Renal Transplantation Procedure

Explore More Videos