The authors wish to thank Dr. Biagio Pietra for his previous work in our lab.

All animals were housed under pathogen-free conditions at the University of Colorado Barbara Davis Center Animal Facility under IACUC approval and cared for according to NIH guidelines.
Depth of anesthesia is judged by toe pinch initially and by observance of respiration rate once the procedure has begun.
1. Donor Heart Harvest
<ol>
	<li>Anesthetize the donor mouse by injecting pentobarbital (60 mg/kg IP).</li>
	<li>Immobilize the mouse by 4-way restraints and clip the fur. Wipe the skin with alcohol.</li>
	<li>After dissecting away the skin, open the abdominal cavity by making a transverse incision just inferior to the diaphragm.</li>
	<li>Cut the diaphragm posterior to costal insertion and lift the anterior chest wall anteriorly and superiorly exposing the heart.</li>
	<li>Extend a full thickness cut up the posterior lateral thoracic cavity on the left and right sides of the chest.</li>
	<li>Isolate the inferior vena cava (IVC) and place a loose 5-0 silk suture around the IVC adjacent to its insertion into the right atrium.</li>
	<li>Inject 1.0 cc of 4 &#176;C heparinized saline (200 u/cc) into the IVC, then ligate the vessel with the 5-0 silk suture and divide.</li>
	<li>Isolate the right superior vena cava (rSVC) in similar fashion and ligate with 5-0 silk suture and divide.</li>
	<li>Gently roll the heart towards the animals&#39; right side and isolate the left superior vena cava (lSVC), ligate with a 5-0 silk suture and divide exposing the left pulmonary artery.</li>
	<li>Secure the heart under a wet gauze while the thymus is blunt dissected away from the main pulmonary artery (PA) and the left and right PA branches and ascending aortic arch.</li>
	<li>Blunt dissect the aortic arch free of the surrounding tissues. Micro-scissors are used to divide the aorta proximal to the right brachial-cephalic artery; i.e. there should be no branches between the heart and the division point of the aorta.</li>
	<li>This section of the aortic arch forms the arterial cuff for the implant process.</li>
	<li>Reflect the aortic cuff inferiorly to expose the PA trunk and the left and right branches of the PA.</li>
	<li>Blunt dissect the left and right PA branches away from the surrounding tissues as far from the heart as possible. This allows for easy dissection of the PA trunk from surrounding tissues.</li>
	<li>Divide the PA trunk as distally as possible, just proximal to its bifurcation. This section of the pulmonary artery forms the venous cuff for the implant process.</li>
	<li>Place a 5-0 silk suture around the base of the heart and tie. The heart is then cut free at the base, and placed in 4 &#176;C saline. Total harvest time is approx. 10-15 min.</li>
</ol>
2. Heart Implant Technique
<ol>
	<li>Anesthetize the recipient mice with pentobarbital (60 mg/kg IP initial dose, 25 mg/kg IP supplemental dose if required).</li>
	<li>Clip the fur and immobilize the mouse by 4-way restraints and prep the skin with povidone-iodine and drape in a sterile fashion.</li>
	<li>Make a 2 cm midline vertical abdominal incision and enter the abdominal cavity.</li>
	<li>Retract the bowel superiorly and externalized on to the chest. Keep wrapped in sterile moist gauze (sterile saline) throughout the case.</li>
	<li>Isolate the abdominal aorta and inferior vena cava (IVC) below the renal vessels and place 4-0 cotton ties around the aorta and IVC superior then inferior to the anastomosis site.</li>
	<li>Identify any lumbar vessels within the field and ligate with 10-0 nylon suture.</li>
	<li>Knot the cotton ties, first the inferior followed by the superior. In this way some blood is retained in the aorta making the aortotomy easier.</li>
	<li>Form the aortotomy with a 30 G needle to enter the lumen of the aorta. Extend the incision with fine micro scissors to a length of approximately 2 mm. This incision is made in a straight line along the longitudinal axis of the vessel.</li>
	<li>Make an end to side anastomosis of the donor aorta to the recipient aorta in the following fashion. Place a 10-0 nylon suture stay stitch in the donor aorta and to the inferior angle of the incision in the recipient aorta and tie. Place a second 10-0 nylon opposite the first in the donor aorta and the superior corner of the incision in the abdominal aorta and tie.</li>
	<li>Make a running suture line from superior to inferior in the lateral wall of the aorta and tie against the previously placed stay stitch. Be sure to bring the intimas (inner vessel surface) together as you stitch. Then suture the medial side in a running fashion and tie. The first and last stitch on each side should be placed as close to the stay stitches as possible. Then aim to have 3 evenly spaced stitches between these two making a total of 5 stitches.</li>
	<li>Make an end to side anastomosis of the donor pulmonary artery to the recipient IVC in the following fashion. Puncture the IVC with a 30 G needle and extend the incision for approx. 2 mm with fine micro scissors. This incision is made in a straight line along the longitudinal axis of the vessel.</li>
	<li>Tie the donor pulmonary artery to the inferior corner of the incision in the IVC with 10-0 nylon.&#160;Place a second 10-0 nylon opposite the first in the donor artery and the superior corner of the incision in the IVC and tie.</li>
	<li>Make a running suture line between the pulmonary artery and the IVC and tie. The first and last stitch on each side should be placed as close to the stay stitches as possible. Then aim to have 5 evenly spaced stitches between these two making a total of 7 stitches.</li>
	<li>Release the distal 4-0 cotton tie re-establishing venous flow.</li>
	<li>Once hemostasis of the venous anastomosis has been observed the proximal 4-0 cotton tie is gradually loosened and the arterial anastomosis observed for hemostasis. When both anastomoses are considered secure, remove the cotton ties from the mouse.</li>
	<li>Return the bowel to the abdomen. The abdominal wall is closed in two layers using 5-0 Silk suture in a running fashion.</li>
	<li>Administer a 1.0 ml bolus of sterile, warm normal saline into the abdomen as fluid resuscitation upon closing, and 0.8 ml of normal saline is injected subcutaneously post-operatively. No other supportive measures are required during the surgery. Recover the animal on a warming blanket. Total implant time is approx. 90-120 min for beginners, 45-60 min&#160;with experience.&#160;Administer buprenorphine analgesia, 0.05 mg/kg, SC, 0.1-0.2 ml at the beginning of the procedure and every 6-12 hr for 72 hr post-op.&#160;</li>
</ol>
3. Graft assessment
<ol>
	<li>Assess graft function daily by trans-abdominal palpation.</li>
	<li>Hold the mouse as though it were to be given an intraperitoneal injection.</li>
	<li>Gently press the tip of a forefinger against the abdominal wall and ascertain the beating strength and regularity of the graft.</li>
	<li>Give palpation quality a score from 4 (normal amplitude and frequency) to 0 (non-beating rejected graft).</li>
</ol>
IMPORTANT NOTES:
All instruments are sterilized, sterile gloves are worn throughout the procedure and a sterile field is maintained. The donor and recipient surgeries are performed with the use of an operating microscope. Ensure that the anastomoses are &#8220;clean&#8221;. That is, that the back walls are not caught when placing stitches. This will cause a significant constriction to flow that will more than likely result in a failed graft and in extreme cases to hind-limb paralysis. It is also vitally important that full thickness passes including the vascular adventitia and the intima of the suture needle are achieved. Evertion of the edges also ensures that there is intima-to-intima contact, which aids in sealing and healing of the anastomoses. Another vitally important factor is ensuring that the tension of the anastomotic suture lines is also optimal. Too loose and there will be irreversible leaking, too tight and stricture to flow will result. If on the arterial side this will result in poor perfusion of the graft, if on the venous side a congested heart will result.

<ol>
	<li>Corry, R. J., 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=Heart+transplantation+in+congenic+strains+of+mice.">Heart transplantation in congenic strains of mice.</a> Transplant Proc. 5 (1), 733 (1973).</li><li>Corry, R. J., 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=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343 (1973).</li><li>Niimi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+for+heterotopic+cardiac+transplantation+in+mice:+experience+of+3,000+operations+by+one+surgeon.">The technique for heterotopic cardiac transplantation in mice: experience of 3,000 operations by one surgeon.</a> J Heart Lung Transplant. 20 (10), 1123 (2001).</li><li>Plenter, R. J., Zamora, M. R., Grazia, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+decades+of+vascularized+heterotopic+cardiac+transplantation+in+the+mouse.">Four decades of vascularized heterotopic cardiac transplantation in the mouse.</a> J Invest Surg. 26 (4), 223 (2013).</li><li>Su, S., 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=Modified+suture+technique+in+a+mouse+heart+transplant+model.">Modified suture technique in a mouse heart transplant model.</a> Asian J Surg. 34 (2), 86 (2011).</li><li>Mao, 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=A+novel+and+knotless+technique+for+heterotopic+cardiac+transplantation+in+mice.">A novel and knotless technique for heterotopic cardiac transplantation in mice.</a> J Heart Lung Transplant. 28 (10), 1102 (2009).</li><li>Csencsits, K. L., Bishop, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+alloreactive+CD4++and+CD8++T+cells:+there's+more+to+it+than+MHC+restriction.">Contrasting alloreactive CD4+ and CD8+ T cells: there's more to it than MHC restriction.</a> Am J Transplant. 3 (2), 107 (2003).</li><li>Hasegawa, T., Visovatti, S. H., Hyman, M. C., Hayasaki, T., Pinsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+vascularized+murine+cardiac+transplantation+to+study+graft+arteriopathy.">Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy.</a> Nat Protoc. 2 (3), 471 (2007).</li><li>Linfert, D., Chowdhry, T., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytes+and+ischemia-reperfusion+injury.">Lymphocytes and ischemia-reperfusion injury.</a> Transplant Rev. 23 (1), (2009).</li><li>Grazia, T. 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=Acute+cardiac+allograft+rejection+by+directly+cytotoxic+CD4+T+cells:+parallel+requirements+for+Fas+and+perforin.">Acute cardiac allograft rejection by directly cytotoxic CD4 T cells: parallel requirements for Fas and perforin.</a> Transplantation. 89 (1), 33 (2010).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> J Vis Exp. (6), (2007).</li><li>Plenter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> European Society for Surgical Research. 45th Annual Congress. , (2010).</li></ol>

The authors have nothing to disclose.

This surgical technique is not easy to master, but once mastered is a powerful research tool. The researcher/surgeon is rewarded by consistency of technique and by attention to detail. Patience during the learning phase is key. As published by Niimi3, with the aid of a video based learning tool it takes an average of 11 attempts to achieve the first successful procedure and 78 attempts to achieve a 90% success rate. Videos have become an important teaching tool in surgery11,12.
Troubleshooting
Bleeding from the anastomoses may occur and this is likely due to either lack of correct tension in the sutures, or too few sutures. While a clotting inducing agent such as Gelfoam can be useful for reducing leaks, we recommend that the surgeon should rely on good technique. Congested non-beating heart is most commonly due to anastomoses that are too tight, particularly on the venous side. A non-beating, non-perfused graft is commonly caused by an air bubble that has traveled into one of the coronary arteries. It is important to maintain a damp-to-wet field to avoid the entry of bubbles into the vessels.

Limitations of the Technique
This technique is not suitable if a researcher wants to investigate effects on a fully functioning heart. That would require an orthotopic transplant technique, which to date has proved impossible to perform.
Significance with Respect to Existing Methods
If one wishes to study the effects on a fully vascularized, solid organ transplant in the mouse, then the heart model is probably the simplest to master. Mouse models of lung, kidney and liver transplantation do exist, but are much harder to learn and perfect.
Critical steps Within the Protocol
It is vitally important that full thickness passes including the vascular adventitia and the intima of the suture needle are achieved. Evertion of the edges also ensures that there is intima-to-intima contact, which aids in sealing and healing of the anastomoses. Another vitally important factor is ensuring that the tension of the anastomotic suture lines is also optimal. Too loose and there will be irreversible leaking, too tight and stricture to flow will result. If on the arterial side this will result in poor perfusion of the graft, if on the venous side a congested heart will result.
Above all, repetition, consistency of procedure and continual attention to detail will yield great results and fundable and publishable data.

In an era when it is possible to perform kidney, lung, liver and pancreas transplants in mice, the cornerstone of basic organ transplant and immunology research since 19731-4 remains the heterotopic heart transplant model in the mouse. In the intervening years several papers have been published detailing improvements/refinements5,6 to this procedure.
As a model of solid organ primarily vascularized transplantation this procedure is second to none. Once mastered this procedure lends itself to research into allogeneic rejection responses7, the development of chronic vasculopathies8 and the mechanisms of ischemia reperfusion injury9.
The keys to successfully learning this procedure are just like any other surgery, patience on the part of the instructor and the trainee and attention to detail. At the beginning of the process the new surgeon will find that they will spend many hours on each transplant. As experience is gained, surgical times, and therefore ischemia, will drastically reduce. Paying attention to the details of every step will sooner or later lead to success.
While the instructor can do their best to pass on, and to anticipate, all of the possible pit falls that may be encountered during these surgeries, the &#8220;creative&#8221; trainee will likely find some of their own!
The basics of the procedure are as follows. The donor ascending aortic arch is end-to-side anastomosed to the recipient abdominal aorta and the donor pulmonary artery is end-to-side anastomosed to the recipient abdominal inferior vena cava (IVC). Blood flows from the recipient aorta in retrograde fashion through the donor aorta to the coronary arteries. Once the blood has flowed through the coronary system it drains into the right atrium via the coronary sinus, is pumped into the right ventricle and then via the pulmonary artery into the recipient IVC. In this way the coronary system is supplied arterial blood and sinus rhythm returns to the graft within 1-2 min of reperfusion. Since the left chambers of the heart are essentially pressure under-loaded the left ventricular free wall will atrophy over time.

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		<tr height="17">
			<td height="17" style="height:17px;width:247px;">Instrument</td>
			<td style="width:109px;">Roboz #</td>
			<td style="width:147px;">Fine Science Tools #</td>
			<td style="width:105px;">Arosurgical #</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight micro-dissecting forcep #5</td>
			<td>RS-5015</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Curved micro-dissecting forcep #7</td>
			<td>RS-5047</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Curved serrated forcep</td>
			<td>RS-5137</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vannas micro-dissecting scissors, short</td>
			<td>RS-5610</td>
			<td></td>
			<td>09.140.08</td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Micro-dissecting scissors, straight, sharp, long</td>
			<td></td>
			<td>11.602.11</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro spring handle needle holder</td>
			<td></td>
			<td></td>
			<td>11.549.15</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight mosquito forcep</td>
			<td></td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-dissecting scissors, straight, blunt</td>
			<td>RS-5962</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-dissecting scissors, curved, blunt</td>
			<td>RS-5981</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro retractor</td>
			<td>RS-6540</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Instrument tray, 10&#8221; x 6 &#189;&#8221; x &#190;&#8221;</td>
			<td>RT-1350S</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Silk suture, 5/0, 22.5m spool</td>
			<td></td>
			<td>18020-50</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
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			<td height="17" style="height:17px;">Suture</td>
			<td></td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">10/0 nylon</td>
			<td></td>
			<td></td>
			<td>T4A10Q07</td>
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		<tr height="17">
			<td height="17" style="height:17px;">5/0 silk</td>
			<td></td>
			<td></td>
			<td>E19A05N</td>
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		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gloves</td>
			<td></td>
			<td>Drapes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biogel from Medex Supply</td>
			<td></td>
			<td>Precept, #64-9012-9</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Syringes</td>
			<td></td>
			<td>Cotton applicators</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-D 1cc insulin, #329424</td>
			<td></td>
			<td colspan="2">Fisher-brand, #23-400-100</td>
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		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Povidone-Iodine swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PDI, #B40600</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4/0 Cotton ties</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Domestic cotton autoclaved with instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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murine heterotopic heart transplant technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. 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 was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The utilization of this surgical technique opens the way for either simple graft survival/rejection studies, or quite complex experimental protocols. In the study briefly described in the figure below, we sought to define the involvement, if any, of Fas and/or perforin as mechanisms of CD4 T cell mediated cardiac rejection. This was made possible by the extraordinary array of mouse strains that are available today. Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell perforin expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51511/51511fig1highres.jpg" src="/files/ftp_upload/51511/51511fig1.jpg" /> 
Figure 1. Perforin and Fas Represent Obligate and Parallel Pathways of CD4 T cell-Mediated Cardiac Rejection B6, B6 PFPKO (perforin knock-out), and B6gld (Fas-ligand deficient) CD4 T cells were utilized to reconstitute B6rag-/- recipients of C3H wild type or Fas-deficient C3Hlpr cardiac allografts. Removal of Fas alone (&#9830;, p=NS vs. control Wt C3H + B6 CD4 T cells) from the donor hearts or removal or perforin alone from the CD4 T cells (&#9632;, p=NS vs. control Wt C3H + B6 CD4 T cells) did not abrogate rejection. Interestingly, the removal of FasL from effector CD4 T cells did delay rejection significantly (&#9679;, p &#60; 0.02 vs. Wt C3H + B6 CD4 T cells, p &#60; 0.01 vs. Wt C3H + B6 PFPKO CD4 T cells, and p&#60; 0.01 vs. C3Hlpr + B6 CD4 T cells). However, most allografts were still rejected (4 of 5). Significantly, the simultaneous removal of both donor Fas and CD4 T cell perforin completely abrogated rejection (&#9675;, p &#60; 0.002 vs. control Wt C3H + B6 CD4 T cells, Wt C3H + B6 PFPKO CD4 T cells, and C3Hlpr + B6 CD4 T cells). This abrogation was significantly more robust than the individual removal of CD4 T cell FasL (&#9675;, p &#60; 0.003 vs. control Wt C3H + B6gld CD4 T cells). From Grazia&#160;et al10. Reprinted with permission.&#160;

Watch this Scientific Journal Video about Murine Heterotopic Heart Transplant Technique at JoVE.com

Murine Heterotopic Heart Transplant Technique

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

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JoVE Journal

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16.7K Views.  University of Colorado Denver. The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Video: Murine Heterotopic Heart Transplant Technique

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

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 Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.