Name: Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy (Video) | JoVE Journal
Uploaded: 2010-12-04T17:00:00.000+00:00
Duration: 8 min 32 s
Description: Learn orthotopic aortic transplantation in rats to study transplant vasculopathy development using the PVG to ACI strain combination. This model enables investigation of neointima formation and chronic rejection mechanisms.

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5 minutes

The authors thank Christiane Pahrmann for her histological work.
Funding
Sonja Schrepfer has received a research grant from the Deutsche Forschungsgemeinschaft (DFG) (SCHR992/3 1 and SCHR992/4-1).]

Rats weighing approximately 250-300g are purchased from Charles River (Sandhofer Weg 7, D-97633 Sulzfeld). 
 Transplants are performed using PVG rats as donors, and ACI rats as recipients.
Rats are housed under conventional conditions, fed standard rat chow and water ad libidum.
All surgical instruments are sterilized prior to use.
Donor preparation:
Anesthetize rat with isoflurane (2,5-3%) using an induction chamber.
<ol>
 <li> Shave the abdominal and thoracic hair and place the rat on its back and place a facemask over its nose and mouth to keep up the anesthesia.</li>
 <li> Disinfect the abdominal and thoracic area widely using Provo-Iodine, next use 80% ethanol, repeat this step three times.</li>
 <li> Check reflexes pinching the hind feet to be sure that the rat is sufficient anesthesized.</li>
 <li> Exsanguinate the rat by opening the abdomen and cutting a hole into the abdominal aorta.</li>
 <li> Carefully open the thorax to find the descending thoracic aorta.</li>
 <li> Dissect the thoracic aorta from surrounding tissues like fat, nerves and the oesophagus.</li>
 <li> Withdraw a 1.5cm piece of the aorta using a couter, without tissue damage of the aorta.</li>
 <li> Perfuse the aortic graft throughly with cold sterile saline and store the graft at 4&deg;C.</li>
</ol>
Recipient:
Anesthetize rat with isoflurane (2,5-3%) using an induction chamber. Body temperature is maintained during the surgical procedure.
<ol>
 <li> Shave abdominal area and apply eye ointment to prevent the eyes from drying during anesthesia. </li>
 <li> Place the rat on its back and place a facemask over its nose and mouth to keep up the anesthesia.</li>
 <li> Disinfect the abdominal area using Provo-Iodine, next use 80% ethanol, repeat this step three times.</li>
 <li> Check reflexes pinching the hind feet to be sure that the rat is sufficient anesthesized.</li>
 <li> Perform a midline abdominal incision separating the skin and muscle in two steps to open the abdomen.</li>
 <li> Place the intestines in a warmed saline moisturized powder free glove. Fold the glove around the intestines to prevent loss of moisture.</li>
 <li> Remove the fatty tissue covering the abdominal aorta.</li>
 <li> Dissect the aorta form the infrarenal region to the bifurcation, careful not to cause damage on the branches of the vessels.</li>
 <li> If necessary ligate branches of the aorta.</li>
 <li> First use a microsurgical clamp on the infrarenal part of the dissected aorta to stop the blood flow.</li>
 <li> Next, place a second clamp close to the bifurcation of the aorta.</li>
 <li> Now that the blood flow is stopped, remove a short segment of the aorta.</li>
 <li> Take the donor aortic graft, that has been kept in 0,9% sterile saline on ice, shorten it to the adequate length and position it in the gap.</li>
 <li> Connect the donor aorta to the recipient aorta, performing running sutures using 8-0 prolene suture (Ethicon, Norderstedt, Germany). Start with the cranial end-to-end anastomosis.</li>
 <li> When the distal end-to-end anastomosis is finished, carefully open the distal clamp. </li>
 <li> When there is no bleeding after opening the distal clamp, carefully open the cranial clamp. In case of bleeding at the sutures, close the clamp again, locate the bleeding and stop the bleeding using one single stitch.</li>
 <li> There should be a visible pulse at the distal end of the aorta. </li>
 <li> Next move the intestines back into the abdomen.</li>
 <li> Flush the abdomen with prewarmed sterile saline.</li>
 <li> Close the muscle layer of the abdominal wall using 6-0 prolene running sutures (Ethicon, Norderstedt, Germany).</li>
 <li> Use 5-0 prolene (Ethicon, Norderstedt, Germany) running sutures to close the skin.</li>
 <li> While the rat is still in anesthesia, inject 4-5mg/kg Carprofen subcutaneously.</li>
 <li> To provide sufficient analgesia for this type of procedure, metamizol is added to the drinking water (50mg Metamizol per 100ml) for pain medication for 3 days post transplantation. </li>
</ol>

<ol><li>Deuse, T., Hoyt, G., Koyanagi, T., Robbins, R. C., Schrepfer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevention+and+Inhibition+But+Not+Reversion+of+Chronic+Allograft+Vasculopathy+by+FK778%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Prevention and Inhibition But Not Reversion of Chronic Allograft Vasculopathy by FK778.</a> Transplantation. 85, 870-877 (2008).</li>
<li>Mennander, A., Tiisala, S., Halttunen, J., Yilmaz, S., Paavonen, T., Hayry, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+rejection+in+rat+allografts.+An+experimental+model+for+transplant+arteriosclerosis%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">Chronic rejection in rat allografts. An experimental model for transplant arteriosclerosis.</a> Arterioscler Thromb Vasc Biol. 11, 671-680 (1991).</li>
<li>Ono, K., Lindsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+technique+of+heart+transplantation+in+rats%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Improved technique of heart transplantation in rats.</a> J Thorac Cardiovasc Surg. 57, 225-225 (1969).</li>
<li>Poston, R. S., Billingham, M., Hoyt, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapamycin+reverses+chronic+graft+vascular+disease+in+a+novel+cardiac+allograft+model%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Rapamycin reverses chronic graft vascular disease in a novel cardiac allograft model.</a> Circulation. 100, 67-100 (1999).</li>
<li>Schrepfer, S., Deuse, T., Koch-Nolte, F., Krieger, T., Haddad, M., Schäfer, H., Pelletier, M. P., Robbins, R. C., Reichenspurner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FK778+in+Experimental+Xenotransplantation%3A+A+Detailed+Analysis+of+Drug+Efficacy%5BTitle%5D)+AND+%22J+Heart+Lung+Transplant%22%5BJournal%5D)">FK778 in Experimental Xenotransplantation: A Detailed Analysis of Drug Efficacy.</a> J Heart Lung Transplant. 26, 70-77 (2007).</li>
</ol>

No conflicts of interest declared.

For several reasons we found the orthotopic aortic transplantation model a more accurate way to investigate the development of TVP compared to the heterotopic heart transplantation model: 
Regarding the surgical technique, the aortic transplant model embodies an easily feasible method, requiring solely the accomplishment of end-to-end-anastomosis, whereas the heterotopic heart transplant model may be considered a more complex and error-prone challenge to the surgeon s skills, requiring end-to-side anastomosis on tw...

<table><tbody><tr><td>Surgical microscope (magnification 25x)</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>shaver</td><td></td><td></td><td></td></tr><tr><td>Provo-Iodine</td><td></td><td></td><td></td></tr><tr><td>ice cold 0.9% saline</td><td></td><td></td><td></td></tr><tr><td>prewarmed 0.9% sterile saleine</td><td></td><td></td><td></td></tr><tr><td>eye ointment</td><td></td><td></td><td></td></tr><tr><td>glove</td><td></td><td></td><td></td></tr><tr><td>microsurgical scissirs</td><td></td><td></td><td></td></tr><tr><td>forceps</td><td></td><td></td><td></td></tr><tr><td>clamps</td><td></td><td></td><td></td></tr><tr><td>8-0 suture (prolene)</td><td></td><td></td><td></td></tr><tr><td>6-0 suture (prolene)</td><td></td><td></td><td></td></tr><tr><td>5-0 suture (prolene)</td><td></td><td></td><td></td></tr><tr><td>Carprofen</td><td></td><td></td><td></td></tr><tr><td>Metamizol</td><td></td><td></td><td></td></tr><tr><td>Q-tips</td><td></td><td></td><td></td></tr></tbody></table>

orthotopic aortic transplantation: a rat model to study the development of chronic vasculopathy

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 ...

The overall goal of this procedure is to establish an accurate model and easily feasible method to investigate the development of transplant vasculopathy. This is accomplished by using the PVG to a CI strain combination. The second step of the procedure is to harvest the donor aorta.The third step of the procedure is to prepare the recipient. The final step of the procedure is to anastomose the donor aorta with the recipient's aorta. Ultimately, results can be obtained that show neointima formation using patho histology.Hi, I'm Sonya Schwefel, professor in transplant immunology in Thet Eye Lab at the University Heart Center in Hamburg, Germany. And I am Manish Duboff. Also from the TS Eye Lab.My laboratory is interested in transplant immunology. In this video we'll introduce you to the Orthotopic aortic transplantation model, an easy and valid model to study the development of transplant vasculopathy In Wes. We use this procedure in our laboratory to investigate chronic rejection in cardiovascular questionings.In the Orthotopic aortic transplant model, the aorta can easily be evaluated histologically. In addition, the PVG to a CI model is especially useful for transplant vasculopathy studies. Since acute vascular rejection is not a major confounding factor and the development of vasculopathy is similar to the findings in the clinic.So let's get started. The donor used in this ORTHOTOPIC aortic transplantation is A PVG rat weighing approximately 250 to 300 grams. After the animal is anesthetized shave its abdominal and thoracic hair.Place the rat on its back and place a face mask over its nose and mouth. To maintain the anesthesia, disinfect the abdominal and thoracic area widely using Provo iodine, followed by 80%ethanol. Next, check the reflexes of the animal by pinching its hind feet.To confirm that it is sufficiently anesthetized. Exsanguinate the rat by opening the abdomen and cutting a hole into the abdo aorta. Carefully open the thorax to find the descending thoracic aorta.Dissect the thoracic aorta from surrounding tissues, such as fat nerves and the esophagus. Withdraw a 1.5 centimeter piece of the aorta using acuta without tissue damage of the aorta. Perfuse the aortic graft thoroughly with cold saline and store the graft at four degrees Celsius.The recipient for this orthotopic aortic transplantation is an A CI rat weighing approximately 250 to 300 grams. To begin this procedure, shave the abdominal area of the anesthetized rat and apply eye ointment to prevent the eyes from drying during anesthesia. Place the rat on its back and place a face mask over its nose and mouth to maintain the anesthesia.Next, disinfect the abdominal area using provo iodine, followed by 80%ethanol. After pinching the hind feet to confirm that the rat is sufficiently anesthetized, open the abdomen by performing a midline abdominal incision and separating the skin and muscle. Two steps.Once the abdomen is open, place the intestines in the saline ized glove. Fold the glove around the intestines to prevent loss of moisture. Remove the fatty tissue covering the abdominal aorta.Dissect the aorta from the infrarenal region to the bifurcation, being careful not to cause damage to the branches of the vessels. If necessary, ligate branches of the aorta after dissecting the aorta. First, use a microsurgical clamp on the infrarenal part of the aorta to stop the blood flow.Next place a second clamp close to the bifurcation of the aorta. Now that the blood flow has been stopped, remove a short segment of the aorta. Then take the donor aortic graft that has been kept in 0.9%saline on ice.Shorten it to the appropriate length and position it in the gap in the aorta. Connect the donor aorta to the recipient aorta by performing running eight zero prolene sutures, starting with the cranial end-to-end anastomosis. When the distal end-to-end anastomosis is finished, carefully open the distal clamp.Next, carefully open the cranial clamp. There should be a visible pulse at the distal end of the aorta. Now move the intestines back into the abdomen and flush the abdomen with prewarm sterile saline.Close the muscle layer of the abdominal wall using running six zero proline sutures. Then use running five zero proline sutures to close the skin while the rat is still under an anesthesia. Inject four to five milligrams per kilogram of carprofen subcutaneously.Return the animal to its cage. Add methimazole to the drinking water for pain medication for three days. Post transplantation.The histopathology results after 120 days will be demonstrated on our histology microscope. The pictures will clearly show the development of transplant vasculopathy. The developed luminal narrowing will be highlighted by computer animation.We have just shown you how to perform an auto topic aortic transplantation in rats. We've also demonstrated the successful development of transplant vasculopathy after 120 days using histopathology. This method enables the investigation of initial effects of drugs on chronic allograft vasculopathy.

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Learn orthotopic aortic transplantation in rats to study transplant vasculopathy development using the PVG to ACI strain combination. This model enables investigation of neointima formation and chronic rejection mechanisms.

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

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