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.

<table><tbody><tr><td>&lt;strong&gt;Instrument&lt;/strong&gt;</td><td>&lt;strong&gt;Roboz #&lt;/strong&gt;</td><td>&lt;strong&gt;Fine Science Tools #&lt;/strong&gt;</td><td>&lt;strong&gt;Arosurgical #&lt;/strong&gt;</td></tr><tr><td>Straight micro-dissecting forcep #5</td><td>RS-5015</td><td>11295-51</td><td></td></tr><tr><td>Curved micro-dissecting forcep #7</td><td>RS-5047</td><td>11297-00</td><td></td></tr><tr><td>Curved serrated forcep</td><td>RS-5137</td><td>11052-10</td><td></td></tr><tr><td>Vannas micro-dissecting scissors, short</td><td>RS-5610</td><td></td><td>09.140.08</td></tr><tr><td>Micro-dissecting scissors, straight, sharp, long</td><td></td><td>11.602.11</td><td></td></tr><tr><td>Micro spring handle needle holder</td><td></td><td></td><td>11.549.15</td></tr><tr><td>Straight mosquito forcep</td><td></td><td>91308-12</td><td></td></tr><tr><td>Micro-dissecting scissors, straight, blunt</td><td>RS-5962</td><td>14078-10</td><td></td></tr><tr><td>Micro-dissecting scissors, curved, blunt</td><td>RS-5981</td><td>14079-10</td><td></td></tr><tr><td>Micro retractor</td><td>RS-6540</td><td></td><td></td></tr><tr><td>Instrument tray, 10&amp;rdquo; x 6 &amp;frac12;&amp;rdquo; x &amp;frac34;&amp;rdquo;</td><td>RT-1350S</td><td></td><td></td></tr><tr><td>Silk suture, 5/0, 22.5 m spool</td><td></td><td>18020-50</td><td></td></tr><tr><td>10/0 nylon</td><td></td><td></td><td>T4A10Q07</td></tr><tr><td>5/0 silk</td><td></td><td></td><td>E19A05N</td></tr><tr><td>Gloves</td><td></td><td>Biogel from Medex Supply</td><td></td></tr><tr><td>Drapes</td><td></td><td>Precept, #64-9012-9</td><td></td></tr><tr><td>Syringes</td><td></td><td>B-D 1 cc insulin, #329424</td><td></td></tr><tr><td>Cotton applicators</td><td></td><td>Fisher-brand, #23-400-100</td><td></td></tr><tr><td>Povidone-Iodine swabs</td><td></td><td>PDI, #B40600</td><td></td></tr><tr><td>4/0 Cotton ties</td><td></td><td></td><td>Domestic cotton autoclaved with instruments</td></tr></tbody></table>

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 ...

murine-heterotopic-heart-transplant-technique

Nanyang Technological University

Research

JoVE Journal

Medicine

16.8K 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

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

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.

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Biology

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 ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

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 ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Immunology and Infection

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...