We would like to thank Editage (www.editage.com) for the English language editing.

All the animal procedures were conducted in accordance with &#34;An Outline of the Act on Welfare and Management of Animals&#34; and &#34;Standards Relating to the Care and Keeping and Reducing Pain of Laboratory Animals&#34; by the Ministry of the Environment, Government of Japan and the &#34;Guidelines for Proper Conduct of Animal Experiment&#34; by the Science Council of Japan16,17,18. The animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Tokyo (M-P19-065).
1. Heterotopic abdominal heart transplantation in rats
NOTE: Heterotopic abdominal heart transplantations were conducted in male Jcl:Wistar rats aged 7-9 weeks old. A microscope with 6.7x to 45x magnification was used to perform the procedure. The surgical instruments were autoclaved for sterilization.
<ol>
	<li>Donor heart harvest
	<ol>
		<li>Anesthetize the donor rat via an intraperitoneal injection of medetomidine (0.15 mg/kg), midazolam (2.0 mg/kg), and butorphanol (2.5 mg/kg).</li>
		<li>Place the rat in a supine position on a heating pad over the operative board, and fix the limbs using elastic bands. Remove as much fur as possible with an electric shaver and depilatory cream. Subsequently, clean the operating area with iodine and a 70% alcohol scrub three times for disinfection.</li>
		<li>Make a small incision of approximately 1 cm with scissors, followed by administering heparin to the abdomen (100 U).</li>
		<li>Extend the abdominal incision longitudinally from above the external urethral meatus to the subxiphoid process with scissors.</li>
		<li>Expose the abdominal aorta with sterilized cotton swabs, and transect with scissors to unload the donor&#39;s heart.</li>
		<li>Incise the diaphragm along the anterior chest wall, and open the thoracic wall with a V-shaped incision using scissors. Elevate the anterior chest wall beside the head, and fix it with pins.</li>
		<li>Initiate topical cooling of the heart using slushed ice.</li>
		<li>Tape the inferior vena cava with a 5-0 silk suture.</li>
		<li>Remove the thymus with scissors, and subsequently identify the ascending aorta and PA originating from the heart. The ascending aorta and PA are on the right and left, respectively.</li>
		<li>Transect the brachiocephalic artery originating from the aortic arch with Potts scissors for additional exsanguination.</li>
		<li>Insert a 23 G needle into the inferior vena cava, and administer 2-3 mL of cold-modified Krebs-Henseleit cardioplegic solution. After the administration, the heart gradually stops beating. Simultaneously, ensure that the blood within the heart is drained out as much as possible.</li>
		<li>Ligate the inferior vena cava with 5-0 silk, and divide it distally to the ligature using Potts scissors.</li>
		<li>After dissecting the connective tissue between the right lung and the esophagus with scissors, ligate the hilum of the right lung with 5-0 silk, and divide it distally to the ligature with scissors.</li>
		<li>Ligate the right superior vena cava with 5-0 silk, and divide it distally to the ligature with Potts scissors.</li>
		<li>Insert one blade of the Potts scissors into the transverse sinus and divide&#160;the ascending aorta and PA en bloc as distally as possible.</li>
		<li>Ligate the left superior vena cava with a 5-0 silk suture, and divide it distally to the ligature and proximally to the azygos vein with Potts scissors.</li>
		<li>After dissecting the connective tissue between the left lung and the esophagus with scissors, ligate the hilum of the left lung with a 5-0 silk suture, and divide it distally to the ligature with scissors.</li>
		<li>Ligate the base of the heart with a 5-0 silk suture, and divide it distally to the ligature with scissors. This silk is used to retract the donor&#39;s heart during the anastomosis of the donor&#39;s heart. At this time, remove the donor&#39;s heart from the pericardial cavity.</li>
		<li>After placing the donor&#39;s heart on the plate filled with slushed ice and cold normal saline, dissect the connective tissue between the ascending aorta and the PA using micro tweezers.</li>
		<li>Perfuse 2-3 mL of cold-modified Krebs-Henseleit cardioplegic solution from the ascending aorta into the coronary arteries by clamping the ostium of the ascending aorta with tweezers until the red color of the coronary arteries is diminished.</li>
		<li>Store the donor&#39;s heart in a cold-modified Krebs-Henseleit cardioplegic solution.</li>
	</ol>
	</li>
	<li>Recipient preparation 
	NOTE: Before harvesting the donor&#39;s heart, complete the recipient preparation.
	<ol>
		<li>Anesthetize the recipient rat via an intraperitoneal injection of medetomidine (0.15 mg/kg), midazolam (2.0 mg/kg), and butorphanol (2.5 mg/kg). After endotracheal intubation using a 16 G intravenous catheter, maintain the anesthesia via sevoflurane inhalation (induction at 5.0% and maintenance at 2.5% with an O2 flow rate of 0.3 L/min).</li>
		<li>Place the rat in a supine position on a heating pad laid over the operative board, and fix the limbs using elastic bands. Remove as much fur as possible with an electric shaver and depilatory cream. Additionally, clean the surgical site with iodine and a 70% alcohol scrub three times for disinfection.</li>
		<li>Using scissors, make a midline abdominal incision of approximately 6-7 cm above the external urethral meatus to the subxiphoid process.</li>
		<li>Retract the small intestine toward the upper-right side of the recipient with sterilized cotton swabs, and wrap it with gauze soaked with warm normal saline.</li>
		<li>Dissect the connective tissue between the small intestine and colon with scissors.</li>
		<li>Dissect the avascular area of the colon with scissors, and tape with two strip-like gauze pieces to retract the colon to the left.</li>
		<li>Expose the abdominal aorta and inferior vena cava with sterilized cotton swabs. Ligate the relatively large branches of both vessels with 9-0 nylon, and divide.</li>
	</ol>
	</li>
	<li>Heterotopic heart transplantation
	<ol>
		<li>Administer heparin to the recipient&#39;s abdomen (100 U) before initiating the donor heart transplantation.</li>
		<li>Clamp the abdominal aorta and inferior vena cava en bloc with side-biting clamp forceps.</li>
		<li>Puncture the abdominal aorta with a 90&#176; bent 23 G needle, and extend the hole longitudinally with Potts scissors to at least the diameter of the donor&#39;s ascending aorta. Subsequently, flush the lumen with heparinized normal saline (10 U/mL) to remove clots.</li>
		<li>Position the sterilized small plate on the right side of the recipient&#39;s abdominal aorta and inferior vena cava. Place the donor&#39;s heart on the plate filled with slushed ice and cold normal saline, which should be replenished every 5 min.</li>
		<li>Tie the donor&#39;s ascending aorta to the recipient&#39;s abdominal aorta with two 9-0 nylon stay sutures at the 12 o&#39;clock and 6 o&#39;clock positions.</li>
		<li>Rotate the operative board clockwise by 90&#176;.</li>
		<li>Anastomose the left side of the recipient&#39;s abdominal aorta and the donor&#39;s ascending aorta using a running 9-0 nylon suture from the caudal to the cranial direction, and tie with a 9-0 nylon stay suture at the 12 o&#39;clock position (total of seven to eight sutures).</li>
		<li>Rotate the operative board counterclockwise by 180&#176;. Translocate the donor&#39;s heart to the left of the recipient&#39;s abdominal aorta and inferior vena cava. It is easy to retract the donor&#39;s heart toward the left side with the 5-0 silk suture that was used when ligating the base of the donor&#39;s heart (step 1.1.18).</li>
		<li>Anastomose the right side of the recipient&#39;s abdominal aorta and the donor&#39;s ascending aorta using a running 9-0 nylon suture from the cranial to the caudal direction, and tie with a 9-0 nylon stay suture at the 6 o&#39;clock position (total of seven to eight sutures).</li>
		<li>Puncture the inferior vena cava using a 90&#176; bent 23 G needle distally to the anastomosis between the donor&#39;s ascending aorta and the recipient&#39;s abdominal aorta, and extend the hole longitudinally with Potts scissors to greater than the diameter of the donor&#39;s PA. Subsequently, flush the lumen with heparinized normal saline to eliminate clots.</li>
		<li>Tie the donor&#39;s PA to the recipient&#39;s inferior vena cava using a 9-0 nylon stay suture at the 6 o&#39;clock position.</li>
		<li>Anastomose the left side of the recipient&#39;s inferior vena cava and the donor&#39;s PA with a running 9-0 nylon suture from the caudal to the cranial direction (total of seven to eight sutures).</li>
		<li>Tie the donor&#39;s PA to the recipient&#39;s inferior vena cava with a 9-0 nylon stay suture at the 12 o&#39;clock position. Additionally, tie this stay suture to the 9-0 nylon on the left side of the anastomosis between the donor&#39;s PA and the recipient&#39;s inferior vena cava.</li>
		<li>Anastomose the right side of the recipient&#39;s inferior vena cava and the donor&#39;s PA using a running 9-0 nylon suture from the cranial to the caudal direction, and tie with the 6 o&#39;clock stay suture (total of 10-12 sutures).</li>
		<li>Apply hemostatic agents to both the anastomoses, and place gauze around them.</li>
		<li>After releasing the side-biting clamp forceps, compress the anastomoses gently with sterilized cotton swabs to facilitate hemostasis. Subsequently, apply warm normal saline to the donor&#39;s heart to provide warmth. The donor&#39;s heart begins to fibrillate within a few tens of seconds and recovers to sinus rhythm after a few minutes.</li>
		<li>Return the small intestine to the recipient&#39;s abdomen using sterilized cotton swabs. Care should be taken to prevent compressing the donor&#39;s heart or twisting the small intestine.</li>
		<li>Close the abdominal wall using a running 4-0 silk suture. After administering atipamezole (0.75 mg/kg) into the abdomen, close the skin with a running 4-0 silk suture.</li>
		<li>Discontinue the inhalation anesthesia, and inject 1 mL of 1% lidocaine under the incision. Additionally, inject 2 mL of warm normal saline subcutaneously to compensate for blood loss.</li>
		<li>Warm the recipient rat using a light-emitting diode lamp. The recipient rat regains consciousness and can be extubated at approximately 30-40 min after inhalation anesthesia is discontinued.</li>
	</ol>
	</li>
</ol>
2. Novel AR model using heterotopic abdominal heart transplantation in rats
NOTE: A novel AR model using heterotopic abdominal heart transplantation was generated using male Jcl:Wistar rats aged 7-9 weeks old. A microscope with 6.7x to 45x magnification was used to perform the procedure. The surgical instruments were autoclaved for sterilization.
<ol>
	<li>Donor heart harvest 
	NOTE: A modified Petri dish with a hole in the center, pliers, and a stiff guidewire are required to create the new AR model (Figure 1).
	<ol>
		<li>As described above, harvest the donor&#39;s heart using a similar procedure to the normal transplant in rats, except for the steps concerning the ascending aorta and PA transection (1.1.1-1.1.14, 1.1.16-1.1.17)</li>
		<li>Before transecting the ascending aorta and PA, remove the adipose tissue on the front of both vessels using Potts scissors. Subsequently, dissect the connective tissue between both vessels using tweezers.</li>
		<li>Insert one blade of the Potts scissors into the transverse sinus. Pull the Potts scissors toward the operator, and insert the blade between the ascending aorta and the PA. Subsequently, transect only the PA (Figure 2A), followed by transecting the aorta distally to the brachiocephalic artery using Potts scissors (Figure 2B).</li>
		<li>After ligating the base of the heart using a 5-0 silk suture and dividing it distally to the ligature with scissors, dissect the connective tissue between the ascending aorta and the PA, and perfuse cold-modified Krebs-Henseleit cardioplegic solution into the coronary arteries using a similar procedure to the normal transplant (steps 1.1.18-1.1.20).</li>
		<li>Fix the donor&#39;s heart with pliers (Figure 2C). Subsequently, cover the donor&#39;s heart with a modified Petri dish with a hole in the center, and immobilize the ascending aorta with a vascular clip.</li>
		<li>Insert a stiff guidewire into the brachiocephalic artery (Figure 2D). Push the guidewire further even after resistance is met, and pierce the aortic valve if a loss of resistance is confirmed. Perform multiple punctures if a severe AR model is to be created.</li>
		<li>Transect the aorta proximally to the brachiocephalic artery to remove the portion clamped with the vascular clip (Figure 2E). Subsequently, store the donor&#39;s heart in cold-modified Krebs-Henseleit cardioplegic solution using a procedure similar to the normal transplant (step 1.1.21).</li>
	</ol>
	</li>
	<li>Recipient preparation 
	NOTE: Before harvesting the donor&#39;s heart, complete the recipient preparation.
	<ol>
		<li>As described above, the recipient preparation procedure is similar to that of the normal transplant (steps 1.2.1-1.2.7).</li>
	</ol>
	</li>
	<li>Heterotopic heart transplantation
	<ol>
		<li>As described above, the donor&#39;s heart transplantation procedure is similar to the normal transplantation (steps 1.3.1-1.3.20).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Abbott, C. P., Lindsey, E. S., Creech, O., Dewitt, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+heart+transplantation+in+the+rat.">A technique for heart transplantation in the rat.</a> Archives of Surgery. 89, 645-652 (1964).</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>Niimi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+for+heterotopic+cardiac+transplantation+in+mice:+experience+of+3000+operations+by+one+surgeon.">The technique for heterotopic cardiac transplantation in mice: experience of 3000 operations by one surgeon.</a> The Journal of Heart and Lung Transplantation. 20 (10), 1123-1128 (2001).</li><li>Westhofen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterotopic+heart+transplantation+in+mice+as+a+small+animal+model+to+study+mechanical+unloading+-+Establishment+of+the+procedure,+perioperative+management+and+postoperative+scoring.">The heterotopic heart transplantation in mice as a small animal model to study mechanical unloading - Establishment of the procedure, perioperative management and postoperative scoring.</a> PLoS One. 14 (4), 0214513 (2019).</li><li>Hasegawa, T., 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=Heterotopic+vascularized+murine+cardiac+transplantation+to+study+graft+arteriopathy.">Heterotopic vascularized murine cardiac transplantation to study graft arteriopathy.</a> Nature Protocols. 2 (3), 471-480 (2007).</li><li>Wang, C., Wang, Z., Allen, R., Bishop, G. A., Sharland, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+method+for+heterotopic+mouse+heart+transplantation.">A modified method for heterotopic mouse heart transplantation.</a> Journal of Visualized Experiments. (88), e51423 (2014).</li><li>Weigle, C. A., 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=An+immunological+model+for+heterotopic+heart+and+cardiac+muscle+cell+transplantation+in+rats.">An immunological model for heterotopic heart and cardiac muscle cell transplantation in rats.</a> Journal of Visualized Experiments. (159), e60956 (2020).</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), e238 (2007).</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>Ono, K., Lindsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+of+heart+transplantation+in+rats.">Improved technique of heart transplantation in rats.</a> The Journal of Thoracic and Cardiovascular Surgery. 57 (2), 225-229 (1969).</li><li>Plenter, R. J., 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=Murine+heterotopic+heart+transplant+technique.">Murine heterotopic heart transplant technique.</a> Journal of Visualized Experiments. (89), e51511 (2014).</li><li>Munakata, 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=Aortic+conduit+valve+model+with+controlled+moderate+aortic+regurgitation+in+rats:+A+technical+modification+to+improve+short-+and+long-term+outcome+and+to+increase+the+functional+results.">Aortic conduit valve model with controlled moderate aortic regurgitation in rats: A technical modification to improve short- and long-term outcome and to increase the functional results.</a> Circulation Journal. 77 (9), 2295-2302 (2013).</li><li>Eskesen, 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=Sildenafil+treatment+attenuates+ventricular+remodeling+in+an+experimental+model+of+aortic+regurgitation.">Sildenafil treatment attenuates ventricular remodeling in an experimental model of aortic regurgitation.</a> Springerplus. 4, 592 (2015).</li><li>Plante, 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=Effectiveness+of+beta-blockade+in+experimental+chronic+aortic+regurgitation.">Effectiveness of beta-blockade in experimental chronic aortic regurgitation.</a> Circulation. 110 (11), 1477-1483 (2004).</li><li>Plante, 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=Left+ventricular+response+to+sustained+volume+overload+from+chronic+aortic+valve+regurgitation+in+rats.">Left ventricular response to sustained volume overload from chronic aortic valve regurgitation in rats.</a> Journal of Cardiac Failure. 9 (2), 128-140 (2003).</li><li>An Outline of the Act on Welfare and Management of Animals. Ministry of the Environment, Government of Japan Available from: <a target="_blank" href="https://www.care.nagoya-u.ac.jp/statute/public/02OutlineAct.pdf">https://www.care.nagoya-u.ac.jp/statute/public/02OutlineAct.pdf</a> (2007)</li><li>Standards Relating to the Care and Keeping and Reducing Pain of Laboratory Animals. Ministry of the Environment, Government of Japan Available from: <a target="_blank" href="https://www.env.go.jp/nature/dobutsu/aigo/2_data/laws/nt_h25_84_en.pdf">https://www.env.go.jp/nature/dobutsu/aigo/2_data/laws/nt_h25_84_en.pdf</a> (2013)</li><li>Guidelines for Proper Conduct of Animal Experiment. Science Council of Japan Available from: <a target="_blank" href="https://www.scj.go.jp/ja/info/kohyo/pdf/kohyo-20-k16-23.pdf">https://www.scj.go.jp/ja/info/kohyo/pdf/kohyo-20-k16-23.pdf</a> (2006)</li><li>Shimada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distention+of+the+immature+left+ventricle+triggers+development+of+endocardial+fibroelastosis:+An+animal+model+of+endocardial+fibroelastosis+introducing+morphopathological+features+of+evolving+fetal+hypoplastic+left+heart+syndrome.">Distention of the immature left ventricle triggers development of endocardial fibroelastosis: An animal model of endocardial fibroelastosis introducing morphopathological features of evolving fetal hypoplastic left heart syndrome.</a> Biomed Research International. 2015, 462469 (2015).</li><li>Shimada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+vascularized+heterotopic+neonatal+rat+heart+transplantation+model.">Development of a vascularized heterotopic neonatal rat heart transplantation model.</a> European Surgical Research. 57 (3-4), 240-251 (2016).</li></ol>

The authors declare no conflicts of interest.

Key steps were discovered to prevent the donor heart from stiffening during implantation. First, it is vital to transect the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart4,7. If the donor&#39;s surgical procedure is performed without endotracheal intubation, breathing ceases after the thoracotomy, which obstructs the donor&#39;s pulmonary circulation. Consequently, the donor&#39;s heart becomes overloaded, preventing good contracti...

Heterotopic abdominal heart transplantation in rats was first reported in 1964 by Abbott et al.1and has been used to study acute and chronic allograft rejection, cardiac allograft vasculopathy, ischemia-reperfusion injury, and cardiac remodeling2,3,4,5,6,7,8,9,10,11. Some modifications have been added to the procedure over the past 50 years. The fundamentals of the current procedure are as follows. The donor&#39;s ascending aorta and pulmonary artery (PA) are end-to-side anastomosed to the recipient&#39;s abdominal aorta and inferior vena cava, respectively. Although the donor&#39;s left atrium and ventricle do not receive any intracavitary flow, blood flows to the donor&#39;s coronary system; therefore, the donor&#39;s heart starts beating again after de-clamping.
Some experts with experience in hundreds or thousands of operations have reported a high success rate with short ischemia time for heterotopic abdominal heart transplantation2,3,4,5; however, it is difficult for beginners to achieve the short ischemia time from the outset. Sufficient cardioprotection is an important factor for obtaining good cardiac contraction of the donor&#39;s heart. Insufficient myocardial protection can stiffen the donor&#39;s heart. Therefore, we modified the transplantation procedure to strengthen the protection of the donor&#39;s heart. One of the aims of this study is to demonstrate a reproducible heterotopic abdominal heart transplantation procedure that beginners can easily perform since it prolongs the acceptable ischemia time.
Additionally, some researchers have reported an aortic regurgitation (AR) model in rats, which has been used to examine the effects of agents on left ventricular (LV) remodeling12,13,14,15. The conventional procedure includes the following: (1) a right lateral neck incision is made to expose the right carotid artery after anesthesia; (2) a catheter is cannulated from this vessel and advanced toward the aortic root; and (3) AR is induced by puncturing the native aortic valve under continuous echocardiographic guidance.
However, puncturing the aortic valve while holding the echocardiography probe and obtaining a good view of the ascending aorta, the aortic valve, and the catheter with an echocardiogram is challenging. Furthermore, cardiac failure following acute AR is another complication. Therefore, a novel AR model, which can be easily created and does not contribute to the recipient&#39;s circulation, has been established in this work to solve these challenges. The other aim of this study is to create an AR model by using heterotopic abdominal heart transplantation and damaging the donor&#39;s aortic valve using a guidewire after harvesting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antisedan (atipamezole)</td>
			<td>Nippon Zenyaku Kogyo Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Domitor (medetomidine)</td>
			<td>Nippon Zenyaku Kogyo Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dormicum (midazolam)</td>
			<td>Maruishi Pharmaceutical Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>heparin</td>
			<td>AY Pharmaceuticals Co.,Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jcl:Wistar rats</td>
			<td>CLEA Japan, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Orinpas Co., Ltd.</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>modified Krebs-Henseleit cardioplegic solution</td>
			<td>Merck KGaA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sevoflurane</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SURGICEL FIBRILLAR</td>
			<td>Johnson &#38; Johnson K.K.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vetorphale (butorphanol)</td>
			<td>Meiji Animal Health Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modified heterotopic abdominal heart transplantation and a novel aortic regurgitation model in rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

Regarding the normal model, good LV contraction was successfully established after de-clamping. The ischemia time of the transplanted heart and the recipient&#39;s anesthesia time were approximately 60 min and 130 min, respectively (Table 1).
Good LV contraction was also obtained after de-clamping in the new AR model. The ischemic time of the transplanted heart and the recipient&#39;s anesthesia time in the AR model were approximately 5 min and 10 min longer than the times of ...

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Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...

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1.8K Views.  The University of Tokyo. This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Video: Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

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 ...

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

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

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

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

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 ...

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. ...