Name: a modified cuff technique for mouse cervical heterotopic heart transplantation model
Uploaded: 2022-02-07T15:00:00
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 use...

<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 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 mouse heart transplantation model helps investigate cardiac allografts immunologic and the parasagittal rejection mechanisms. This understanding would help develop a unique approach to improving patient's long-term survival. This cervical transplantation surgery is performed without stitching using a cuff method.Hence, it decreases the complexity in vessel anastomosis and avoids the interference with blood supply and venous reflux of the lower body. Demonstrating the procedure will be Dr.Xin Mao, a surgeon from our laboratory. To begin, place the recipient mouse on a heating pad at the operating table and shave the hair over the surgical area.After disinfecting the surgical area, make a 1.5 to 2 centimeter incision parallel to the cervical midline from the right mandibular angle to the tail end. Dissect approximately one centimeter of the right external jugular vein using an electro-coagulator and micro-forceps. Clip the vein at the proximal end with an atruamatic microvascular clamp and ligate it at the distal end.Pass the distal end of the vein through a 22 gauge polyurethane barbed cuff with a bevel end and superficial grooves. Fix the vein with the handle of the cuff using a microvascular clamp. 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 it with a 10-0 surgical suture in the grooves of the surface.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. Dissect the right common carotid artery for approximately one centimeter using micro-forceps and clip the artery with an atraumatic microvascular clamp at the proximal end. Ligate and cut off the artery.Pass the distal end of the artery through a 26 gauge polyurethane barbed cuff with a bevel end and grooves on the surface. Fix the artery with the cuff's handle using a microvascular clamp. 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.After preparing the recipient's vessels, drop 100 international units per milliliter heparin saline on the vessels to prevent thrombosis. Cover the cervical incision with wet saline gauze for subsequent implantation. Shave the abdominal hair using an electric razor.After disinfecting the surgical area, incise the abdomen along the midline from the symphysis pubis to the subxiphoid and expand the incised area with a retractor. Dissect one centimeter of the abdominal aorta and inferior vena cava using an electro-coagulator and a micro-forceps and perform heparinization by injecting one milliliter of physiological saline supplemented with 250 international units per milliliter of heparin through the inferior vena cava. Excise the abdominal aorta and inferior vena cava.Excise the thorax along the anterior axillary line on both sides using a surgical scissor to separate the chest wall, then ligate the superior vena cava with an 8-0 surgical suture. Insert a scalp needle at the suprahepatic inferior vena cava. Then inject ice cold physiological saline supplemented with 100 units per milliliter heparin through the scalp needle from suprahepatic inferior vena cava to profuse the donor heart until the blood color fades.Reperfuse the donor heart with two to three milliliters of ice cold histidine-tryptophan-ketoglutarate solution from the aortic arch to protect the donor myocardium. Ligate the superior and inferior venae cavae and the pulmonary vein with a 5-0 surgical suture. Dissect and cut off the donor aorta and pulmonary artery before branching.Then divide the superior and inferior venae cavae and the pulmonary vein to remove the donor's heart. Implant the donor heart into the cervical pocket of the recipient mouse in an inverted position. Pull the cuff with an everted recipient jugular vein into the lumen of the donor pulmonary artery to perform end-to-end anastomosis.Ligate the cuff using the grooves on the surface through a 10-0 surgical suture to fix the anastomosis. Employ a similar procedure for end-to-end anastomosis of the donor aorta to the recipient carotid artery. Release the atraumatic microvascular clamp of the jugular vein followed by the carotid artery to reperfuse the donor's heart.Fix the cardiac graft and suture it properly to prevent twisting of the graft. Close the cervical incision with continuous sutures using a 5-0 polyamide monofilament suture. Using this mouse cervical heterotopic heart transplantation model, the survival rate of recipient mice was approximately 95.2%All cardiac allografts were lost within eight days after transplantation.In contrast, all the isogenic heart transplants survived beyond four weeks. grooves and cuff for better fixation of everted vessels, avoiding repeated operation of vessels. The modified cuff method for vessel anastomosis in this surgery is also applicable for another anastomosis in different organ transplantation animal models.

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