This work was supported by the National Natural Science Foundation of China (81870304) to Jun Li.

All Animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals, Eight Edition, National Research Council (US) 2011. Procedures involving animal subjects have been approved by the Animal Care and Use Committee of the Chongqing University Cancer Hospital, Chongqing, China.&#160;Male BALB/c and C57BL/6 mice weighing 20-30 g, obtained from commercial sources (see Table of Materials), were used for allogeneic heart transplantation study. The C57BL/6 mice were used as donors and syngeneic recipients, while the BALB/c mice served as allogeneic recipients. A schematic of the protocol is shown in Figure 1. 
NOTE:&#160;All supplies used during surgery, including surgical instruments and solutions, are sterile. The surgical procedure adheres to the principle of&#160;aseptic operation technique.
1. Recipient procedure
<ol>
	<li>Induce general anaesthesia via inhalation of 5% isoflurane through a 15 x 10 x 10 cm induction chamber connected with a hood (see Table of Materials).</li>
	<li>Fix the recipient mouse on the operating table with a heating pad. Maintain anaesthesia with continuous inhalation of 2% isoflurane through a face mask over the nose and mouth. 
	&#8203;NOTE: Slow respiratory rate and rhythm, the disappearance of the corneal reflex, and the absence of the pedal reflex in the toes indicate the effectiveness of anaesthesia.</li>
	<li>After shaving the hair, disinfect the surgical area with three alternating rounds of povidone-iodine scrub followed by alcohol. Then,&#160;incise the skin&#160;1.5-2 cm&#160;parallel to the cervical midline from the right mandibular angle to the tail-end.</li>
	<li>Dissect ~1 cm of the right external jugular vein using an electro-coagulator and micro-forceps. Clip the vein at the proximal end with an atraumatic microvascular clamp and ligate it at the distal end.</li>
	<li>Pass the distal end of the vein through a 22 G polyurethane barbed cuff (see Table of Materials) with a bevel end and superficial grooves. Fix the vein with the handle of the cuff using a microvascular clamp.</li>
	<li>Remove the 8-0 ligation suture at the distal end, turn the lumen over the cuff hooked by the superficial barb inside out and fix with a 10-0 surgical suture in the grooves of the surface.</li>
	<li>Resect the right sublingual gland to form a fossa for implanting the cardiac graft, and reserve the right lobe of the submaxillary gland and the right sternocleidomastoid.</li>
	<li>Dissect the right common carotid artery for ~1 cm using micro-forceps, and clip the artery with an atraumatic microvascular clamp at the proximal end. At the distal end, ligate and cut off the artery.</li>
	<li>Pass the distal end of the artery through a 26 G polyurethane barbed cuff (see Table of Materials) with a bevel end and grooves on the surface. Fix the artery with the cuff&#39;s handle using a microvascular clamp.</li>
	<li>Remove the ligation suture at the distal end, turn the lumen inside out over the cuff, and fix with a superficial barb and grooves with a 10-0 surgical suture.</li>
	<li>After preparing the recipient&#39;s vessels, drop 100 IU/mL of heparin saline on the vessels to prevent thrombosis. Cover the cervical incision with sterile wet saline gauze for subsequent implantation.</li>
</ol>
2. Donor procedure
<ol>
	<li>Employ the same anaesthetic procedure (step 1.1) for the donor mouse.</li>
	<li>Shave the abdominal hair using an electric razor, and disinfect the surgical area with three alternating rounds of povidone-iodine scrub followed by alcohol.</li>
	<li>Incise the abdomen (2-3 cm) with a scissor along the midline from the symphysis pubis to the subxiphoid, and expand the incised area with a retractor.</li>
	<li>Dissect 1 cm of the abdominal aorta and inferior vena cava using an electro-coagulator and a micro-forceps, and perform heparinization by injecting 1 mL of physiological saline supplemented with 250 IU/mL of heparin through the inferior vena cava. After this, excise the abdominal aorta and inferior vena cava.</li>
	<li>Excise the thorax along the anterior axillary line on both sides using a surgical scissor to separate the chest wall. Ligate the superior vena cava with an 8-0 surgical suture.</li>
	<li>Insert a scalp needle at the suprahepatic inferior vena cava. Then, inject ice-cold physiological saline supplemented with 100 IU/mL of heparin through the scalp needle from suprahepatic inferior vena cava to perfuse the donor heart until the blood color fades.</li>
	<li>Re-perfuse the donor heart with 2-3 mL of ice-cold histidine-tryptophan-ketoglutarate (HTK) solution (see Table of Materials) using a scalp needle from the aortic arch to protect the donor myocardium. The mean warm ischaemia time is 5 min.</li>
	<li>Ligate the superior and inferior vena&#160;cava&#160;and the pulmonary vein with a 5-0 surgical suture. Dissect and cut off the donor aorta and pulmonary artery before their branching. After that, divide the superior and inferior vena&#160;cava&#160;and the pulmonary vein to remove the donor&#39;s heart.</li>
</ol>
3. Implantation
<ol>
	<li>Implant the donor heart into the cervical pocket of the recipient mouse in an inverted position.</li>
	<li>Pull the cuff with an everted recipient jugular vein into the lumen of the donor pulmonary artery to perform end-to-end anastomosis of the donor pulmonary artery to the recipient external jugular vein. Ligate the cuff using the grooves on the surface through a 10-0 surgical suture to fix the anastomosis.</li>
	<li>Employ a similar procedure for end-to-end anastomosis of the donor aorta to the recipient carotid artery.</li>
	<li>Release the atraumatic microvascular clamp of the jugular vein followed by the carotid artery to re-perfuse the donor&#39;s heart. The mean cold ischaemia time is 15 min.</li>
	<li>Fix the cardiac graft and suture it properly to prevent twisting of the graft.</li>
	<li>Close the cervical incision with continuous sutures using a 5-0 polyamide monofilament suture (see Table of Materials). 
	NOTE: Remove the suture after the wound is completely healed.&#160;</li>
	<li>Retain the recipient mouse inside a warm, dry, and clean cage until it recovers from anaesthesia. 
	NOTE: It takes 5-10 min to recover.</li>
	<li>Inject buprenorphine (0.05 mg/kg) subcutaneously into the recipient mouse every 6 h for 48 h for postoperative analgesia. 
	NOTE: The analgesia dosage was optimized for this study. However, the analgesia regimen can be extended/ modified if there is any sign&#160;of pain in accordance with the institutional animal use guidelines.&#160;</li>
</ol>

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+International+thoracic+organ+transplant+registry+of+the+international+society+for+heart+and+lung+transplantation:+Thirty-sixth+adult+heart+transplantation+report+-+2019;+focus+theme:+Donor+and+recipient+size+match.">The International thoracic organ transplant registry of the international society for heart and lung transplantation: Thirty-sixth adult heart transplantation report - 2019; focus theme: Donor and recipient size match.</a> The Journal of Heart and Lung Transplantation. 38 (10), 1056-1066 (2019).</li><li>Stehlik, 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=The+registry+of+the+international+society+for+heart+and+lung+transplantation:+29th+official+adult+heart+transplant+report--2012.">The registry of the international society for heart and lung transplantation: 29th official adult heart transplant report--2012.</a> The Journal of Heart and Lung Transplantation. 31 (10), 1052-1064 (2012).</li><li>Huang, H., 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=Combined+intrathymic+and+intravenous+injection+of+mesenchymal+stem+cells+can+prolong+the+survival+of+rat+cardiac+allograft+associated+with+decrease+in+miR-155+expression.">Combined intrathymic and intravenous injection of mesenchymal stem cells can prolong the survival of rat cardiac allograft associated with decrease in miR-155 expression.</a> Journal of Surgical Research. 185 (2), 896-903 (2013).</li><li>Eggenhofer, E., 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=Features+of+synergism+between+mesenchymal+stem+cells+and+immunosuppressive+drugs+in+a+murine+heart+transplantation+model.">Features of synergism between mesenchymal stem cells and immunosuppressive drugs in a murine heart transplantation model.</a> Transplant Immunology. 25 (2-3), 141-147 (2011).</li><li>Sula Karreci, E., 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=Brief+treatment+with+a+highly+selective+immunoproteasome+inhibitor+promotes+long-term+cardiac+allograft+acceptance+in+mice.">Brief treatment with a highly selective immunoproteasome inhibitor promotes long-term cardiac allograft acceptance in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (52), 8425-8432 (2016).</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> Journal of Visualized Experiments. 6, 238 (2007).</li><li>Lin, C. M., Gill, R. G., Mehrad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+killer+cell+activating+receptor,+NKG2D,+is+critical+to+antibody-dependent+chronic+rejection+in+heart+transplantation.">The natural killer cell activating receptor, NKG2D, is critical to antibody-dependent chronic rejection in heart transplantation.</a> American Journal of Transplantation. 21 (11), 3550-3560 (2021).</li><li>Ito, H., Hamano, K., Fukumoto, T., Wood, K. J., Esato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+blockade+of+CD4+and+major+histocompatibility+complex+class+II+molecules:+An+effective+immunosuppressive+treatment+in+the+mouse+heart+transplantation+model.">Bidirectional blockade of CD4 and major histocompatibility complex class II molecules: An effective immunosuppressive treatment in the mouse heart transplantation model.</a> The Journal of Heart and Lung Transplantation. 17 (5), 460-469 (1998).</li><li>Zhou, Y. X., 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+rejection+correlates+with+expression+of+major+histocompatibility+complex+class+I+antigens+on+peripheral+blood+CD3(+)CD8(+)+T-lymphocytes+following+skin+transplantation+in+mice.">Acute rejection correlates with expression of major histocompatibility complex class I antigens on peripheral blood CD3(+)CD8(+) T-lymphocytes following skin transplantation in mice.</a> Journal of International Medical Research. 39 (2), 480-487 (2011).</li><li>Oberhuber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> Journal of Visualized Experiments. (92), e50753 (2014).</li><li>Cui, D., Tan, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+technique+of+arterial+anastomosis+in+mouse+heart+transplantation.">An alternative technique of arterial anastomosis in mouse heart transplantation.</a> Clinical Transplantation. 32 (6), 13264 (2018).</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> Journal of Investigative Surgery. 26 (4), 223-228 (2013).</li><li>Fang, 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=A+simplified+two-stitch+sleeve+technique+for+arterial+anastomosis+of+cervical+heterotopic+cardiac+transplantation+in+mice.">A simplified two-stitch sleeve technique for arterial anastomosis of cervical heterotopic cardiac transplantation in mice.</a> American Journal of Translational Research. 5 (5), 521-529 (2013).</li><li>Matsuura, A., Abe, T., Yasuura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+mouse+cervical+heart+transplantation+using+a+cuff+technique.">Simplified mouse cervical heart transplantation using a cuff technique.</a> Transplantation. 51 (4), 896-898 (1991).</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), 343-350 (1973).</li><li>Fensterer, T. F., Miller, C. J., Perez-Abadia, G., Maldonado, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cuff+design+to+facilitate+anastomosis+of+small+vessels+during+cervical+heterotopic+heart+transplantation+in+rats.">Novel cuff design to facilitate anastomosis of small vessels during cervical heterotopic heart transplantation in rats.</a> Comparative Medicine. 64 (4), 293-299 (2014).</li></ol>

The authors have nothing to disclose.

The mouse heart transplantation model contributes to the investigation of rejection mechanisms after heart transplantation, contributing to the development of unique approaches to improve the long-term survival of cardiac allograft recipients. However, heart transplantation in mice is a complex and challenging task, requiring a high level of microsurgery techniques, especially in vascular anastomosis11,12,13. The mouse abdominal...

Heart transplantation has become the standard treatment for terminal heart failure. More than 5,500 heart transplantations per year are performed in the organizations registered under the International Society for Heart and Lung Transplantation. Among the allogeneic heart transplant recipients, the 1-year rejection rate is still &#62;10%, while the 3-year rejection rate increased to 36%1,2. However, effective prophylactic treatments for patients with cardiac allograft rejection are lacking. Therefore, animal model studies are warranted that elucidate the physiological mechanisms underlying the immunological and pathological rejection of cardiac allografts. Such studies would contribute to the investigation of novel targets required to develop efficacious drugs, which would help prevent cardiac allograft rejection and improve survival rates in those patient populations.
Some potential immunological and pathophysiological mechanisms of cardiac allograft rejection have been proposed recently in mouse model studies of heterotopic heart transplantation3,4,5. Consequently, mouse heterotopic heart transplantation became an ideal preclinical model to investigate the mechanisms of immune rejection and pathological injury occurring in cardiac allografts after heart transplantation because of their high homology with human genes. The prevalent concept is to perform heterotopic transplantation in a mouse model by an abdominal end-to-end anastomosis in the recipient aorta and inferior vena cava using stitches, similar to the normal human anatomy. However, this procedure may interfere with the recipient&#39;s blood supply and venous reflux of the lower body6. Therefore, a modified heterotopic heart transplantation procedure in a mouse model is proposed here.
The donor&#39;s heart is implanted with the recipient&#39;s carotid artery and jugular vein by an end-to-end cervical anastomosis using a modified Cuff technique. This modified procedure facilitated the operative feasibility and ensured the survival of the cardiac graft without interfering with the blood supply and venous reflux of the lower body.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;5-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>C3090954</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;8-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>C2090880</td>
			<td></td>
		</tr>
		<tr>
			<td>10-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>G0090781</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G polyurethane cuff</td>
			<td>B.Braun Medical Inc.</td>
			<td>4251628-02</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G polyurethane cuff</td>
			<td>Suzhou Linhua Medical Instrument Co., LTD</td>
			<td>REF383713</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia induction chamber</td>
			<td>RWD Life Science Co., LTD</td>
			<td>V100</td>
			<td></td>
		</tr>
		<tr>
			<td>Atraumatic microvascular clamp</td>
			<td>Beyotime</td>
			<td>FS500</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c and C57BL/6 mice (20&#8211;30 g)</td>
			<td>Centre of Experimental Animals (Army Medical University, Chongqing, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>US Biological life Sciences</td>
			<td>352004</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocoagulator</td>
			<td>Guangzhou Runman Medical Instrument Co., LTD</td>
			<td>ZJ1099</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Henan piaoan group Co., LTD</td>
			<td>10210402</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Guangzhou Dewei Biological Technology Co., LTD</td>
			<td>DK0032</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>North China Pharmaceutical Co., LTD</td>
			<td>2101131-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HTK solution</td>
			<td>Shenzhen Changyi Pharmaceutical Co., LTD</td>
			<td>YZB/Min8263-2013</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection syringe (10 mL)</td>
			<td>Shandong weigao group medical polymer Co., LTD</td>
			<td>20211001</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co., LTD</td>
			<td>21070201</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological saline</td>
			<td>Southwest pharmaceutical Co., LTD</td>
			<td>H50021610</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalp needle</td>
			<td>Hongyu Medical Group</td>
			<td>20183150210</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Beyotime</td>
			<td>FS600</td>
			<td></td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>RWD Life Science Co., LTD</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical operation microscope</td>
			<td>Tiannuoxiang Scientific Instrument Co. , Ltd, Beijing, China</td>
			<td>SZX-6745</td>
			<td></td>
		</tr>
		<tr>
			<td>Swab</td>
			<td>Yubei Medical Materials Co., LTD</td>
			<td>21080274</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 this mouse cervical heterotopic heart transplantation model, the survival rate of recipient mice was approximately 95.2% (20 out of 21 mice survived). The primary cause of death was postoperative bleeding. The fast heartbeat with a regular rhythm serves as an indicator of the survival of the implanted donor heart.
C57BL/6 and BALB/c mice were MHC (H-2b) and MHC (H-2d) types in this model, respectively7,8. These two strains differ by...

Watch this Scientific Journal Video about A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model at JoVE.com

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

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 ...

a-modified-cuff-technique-for-mouse-cervical-heterotopic-heart

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

3.0K Views.  Chongqing University Cancer Hospital. 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.

Video: A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

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 ...

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 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 ...

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 ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

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.

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 ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...