Name: a modified cuff technique for mouse cervical heterotopic heart transplantation model
Uploaded: 2022-02-07T15:00:00.000+00:00
Duration: 6 min 45 s
Description: Watch this Scientific Journal Video about A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model at JoVE.com

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>

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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 heterotopic heart transplantation model is performed using stitches by anastomosis of the donor aorta and pulmonary artery to the recipient aorta and inferior vena cava. The recipient&#39;s aorta and inferior vena cava need to be blocked in this operation. Therefore, ischaemia of the lower body and thrombosis of the inferior vena cava can increase the disability and death of recipient mice. To reduce the difficulties of vascular anastomosis during transplantation, Matsuura et al. first introduced a cervical heart transplantation model in mice using the cuff technique in 199114. In this model, the oversleeve-like everted anastomosis of vessels by ligation with cuff increased the anastomosis efficiency. In contrast to the anastomosis of vessels with sutures in an abdominal heart transplantation mouse model, it reduced the bleeding probability post-procedure. Therefore, the improvement of the anastomosis efficiency reduced the ischaemia time of cardiac muscle and increased the survival rate of cardiac grafts. Additionally, cervical implantation of the donor&#39;s heart does not interrupt the circulation of the recipient aorta and inferior vena cava compared with abdominal implantation15; therefore, the survival of recipient mice is increased.
A unique experimental mouse heterotopic heart transplantation model is described here, established by Rupert Oberhuber et al.10.&#160;The procedure involves an end-to-end cervical anastomosis of the donor aorta and pulmonary artery to the recipient carotid artery and jugular vein, following a modified Cuff technique. In this model, the systemic circulation of the recipient mice does not interfere with10, and the donor&#39;s heart was perfused from the inferior vena cava and aorta with heparin and HTK solutions for better myocardial protection. However, the critical component of this model differed from that of Oberhuber et al.10, which employed the modified barbed cuff with a bevel end and grooves similar to that of Finsterer et al.16. The bevel end facilitates an oversleeve-like evert of the vascular lumen. The grooves on the surface facilitate the fixation of everted vessel walls with a cuff using sutures, and the barb outside the cuff reduces the slippage of anastomosed vessel walls from the cuff (Figure 1). These modifications shorten the surgical time by 20% and improve cardiac grafts&#39; implantation efficiency and survival. Furthermore, the modified barbed cuff is produced from the most common polyurethane catheter used for scalp acupuncture, thus significantly reducing the cost of the procedure. A comparison of the current technique with that of Oberhuber et al.10 is shown in Table 1.
The unique features of this model need to be noted. First, the length and calibre of the cuff are essential for successful anastomosis. The suitable length of the cuff was ~3 mm with a handle (1 mm) (Figure 1). The suitable calibre of the cuff is 26 G and 22 G for artery and veins, respectively. The unsuitable length and calibre of the cuff would result in twisting or excessive tension of the anastomosed vessels. Second, the suitable lengths of the recipient&#39;s vessels are 1.5 to 2-folds of the cuff. Third, the donor&#39;s heart is not perfused with excessive pressure, potentially damaging the graft. Fourth, the cardiac graft is fixed, and the cuff is anastomosed in a suitable position by suturing after implantation to avoid movement or twisting of anastomosed vessels or grafts. Fifth, preservation of the submaxillary gland and sternocleidomastoid contributes to reducing twirling or twisting of the anastomosed vessels or graft when resecting the right sublingual gland to produce a fossa for cardiac graft. Sixth, to facilitate the oversleeve-like evert of the vascular lumenand reduce thrombosis after surgery, heparin solution (100 IU/mL) can be provided to the anastomosed vessels while performing anastomosis.
This cuff technique facilitates the anastomosis of donor and recipient vessels during implantation; however, the hardness of the cuff may, in turn, increase the risk of twisting the anastomosed vessels, resulting in an increase in thrombosis after transplantation. Optimization of the cuff material is warranted to reduce complications, increase graft survival, and increase the utilization rate of the models in subsequent experiments. Furthermore, the fibrous scar of the cut may limit the space for the cardiac graft and affect its long-term survival. In addition, ejection of the cardiac graft may interfere with recipient mice&#39;s normal haemodynamic blood flow. Finally, this model is non-functional and cannot be used to evaluate the cardiac function of grafts. Nevertheless, this study provides knowledge regarding heart transplantation&#39;s immunological and pathological functions.

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><tbody><tr><td>&amp;nbsp;5-0 Polyamide Monofilament suture</td><td>B.Braun Medical Inc.</td><td>C3090954</td><td></td></tr><tr><td>&amp;nbsp;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&amp;ndash;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 the H-2, which causes acute T-cell-mediated rejection9. Of all the cardiac allografts, 62.5% were lost within 7 days after transplantation, as assessed by palpating the heartbeat. All cardiac allografts were lost within 8 days after transplantation. In contrast, all the isogeneic heart transplants survived beyond 4 weeks (Figure 2). Mice that survived beyond 4 weeks were euthanized by CO2&#160;inhalation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63504/63504fig01.jpg" /> 
Figure 1: Schematic of the mouse cervical heterotopic heart transplantation model. (A) Protocol for preparing the recipient: after clipping the common carotid artery and external jugular vein at the proximal end, the vascular lumen of vessels is everted and fixed after passing through the barbed cuff with a bevel end and grooves on the surface. The dashed square shows the structure and usage of the cuff. (B)&#160;Donor heart resection: after the donor heart&#39;s perfusion with heparin and HTK solution from the inferior vena cava and aorta, the superior and inferior vena&#160;cava&#160;and pulmonary vein are ligated with sutures. The donor&#39;s heart is then resected by incising the vascular vessels. (C)&#160;Implantation of the donor&#39;s heart. The donor pulmonary artery and aorta is anastomosed to the recipient&#39;s external jugular vein, and carotid artery via the cuff with the recipient&#39;s vasculature turned inside out in an end-to-end pattern. <a href="https://www.jove.com/files/ftp_upload/63504/63504fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63504/63504fig02.jpg" /> 
Figure 2: Survival curve for cardiac grafts. The survival curve of cardiac grafts shows that allogeneic heart transplants are lost within 8 days after transplantation, which was assessed by palpation of the heartbeat. A total of 10 recipient mice underwent the modified cervical heterotopic heart transplantation in each group. All the isogeneic heart transplants survived more than 4 weeks. <a href="https://www.jove.com/files/ftp_upload/63504/63504fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Rupert Oberhuber et al.10</td>
			<td colspan="3">Xin Mao et al (present work)</td>
		</tr>
		<tr>
			<td>Anaesthesia</td>
			<td>xylazine and ketamine</td>
			<td colspan="3">isoflurane (Safe, continuous and stable)</td>
		</tr>
		<tr>
			<td>Right lobe of the submandibular gland</td>
			<td>removal</td>
			<td colspan="3">preservation (Reduce twirling of grafts)</td>
		</tr>
		<tr>
			<td>Right sternocleidomastoid</td>
			<td>removal</td>
			<td colspan="3">preservation (Reduce twirling of grafts)</td>
		</tr>
		<tr>
			<td>Cardiac perfusion</td>
			<td>Retrograde perfusion with 4 &#176;C HTK solution from the aortic arch</td>
			<td colspan="3">1. Anterograde perfusion with ice-cold physiological saline supplemented with 100 IU/mL heparin solution from the suprahepatic vena cava. 2. Retrograde reperfusion with ice-cold HTK solution from the aortic arch. (Reduce coagulation and increase myocardial protection)</td>
		</tr>
		<tr>
			<td>Cuff</td>
			<td>blunt end, with handle</td>
			<td colspan="3">bevel end, with handle, barb and grooves on the surface (Facilitate eversion and fixation)</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of heart transplantation techniques. The current mouse cervical heterotopic heart transplantation technique is modified from Oberhuber, R. et al.10 and possesses additional advantages for cardiac graft survival.

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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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

3.1K 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

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 ...

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 ...

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 ...

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. ...

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 ...

Strategies for Efficient Vascular Anastomosis in Mouse Liver Transplantation

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. However, the technical challenges pose a barrier to utilizing this valuable experimental model and passing on these skills to the next generation. The most challenging aspect of this procedure is vascular reconstruction, including the portal vein (PV), infrahepatic inferior vena cava (IHIVC), and suprahepatic inferior vena cava. The use of plastic cuffs, rather than sutures, allows for smoother PV and IHIVC reconstruction. Vessels are reconstructed by attaching a cuff made from an intravenous catheter to the tip of the graft vessel and interposing the cuff into the recipient vessel. The two most crucial aspects are properly visualizing the inner lumen of the vessel and avoiding the use of excessive force. Our aim is to provide a technical overview of vascular reconstructions using the cuff technique in recipient surgery. These technical tips for the cuff technique are expected to help microsurgeons facilitate vascular reconstruction and advance their research.

Author Spotlight: Exploring Cold Ischemia and Warm Reperfusion Injury in Fatty Liver Transplantation

This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...