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

<ol>
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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>

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 ...

Watch this Scientific Journal Video about Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats at JoVE.com

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 ...

Modified heterotopic abdominal heart transplantation and a novel aortic regurgitation model in rats.Introduction. Although some experts can perform heterotopic abdominal heart transplantation in a very short time, it is difficult for beginners to achieve a high success rate. We demonstrate a reproducible method which beginners can perform with ease.Additionally, we demonstrate the a AR model by using heterotropic abdominal heart transplantation and damaging the donor's aortic valve by a guidewire after harvesting the donor heart.Protocol. First, we explain heterotopic abdominal heart transplantation in rats. Donor heart harvest.Before surgery, remove as much fur as possible with an electric shaver and depilatory cream. In addition, clean the operating area with iodine and 70%alcohol scrub three times. After administering heparin and making a midline abdominal incision, transect the abdominal aorta in order to unload the donor's heart.Open the thoracic wall with a V-shaped incision. Initiate topical cooling with slushed ice. Tape the IVC with 5-0 silk.Remove the thymus with scissors. Transect the brachiocephalic artery due to additional exsanguination. Administer cold cardioplegic solution into the IVC.Ligate the IVC with 5-0 silk and divide it. Ligate the hilum of the right lung with 5-0 silk and divide it. Ligate the right SVC with 5-0 silk and divide it.Divide the ascending aorta and PA en bloc with Potts scissors. Ligate the left SVC with 5-0 silk and divide it. Ligate the hilum of the left lung with 5-0 silk and divide it.Ligate the base of the heart with 5-0 silk and divide it. Dissect the connective tissue between the ascending aorta and PA using micro tweezers. Perfuse cold cardioplegic solution into the coronary arteries.Recipient preparation. Retract the small intestine towards the upper right side. Dissect the connective tissue between the small intestine and the colon.Retract the colon to the left with two strip-like gauze pieces. Expose the abdominal aorta and IVC with cotton swabs. Heterotopic heart transplantation.Clamp the abdominal aorta and IVC en bloc with side-biting clamp forceps. Puncture the abdominal aorta with a bent 23-gauge needle and extend the hole longitudinally with Potts scissors. Place the donor heart on a small plate, filled with slushed ice and cold, normal saline.Tie the donor's ascending aorta to the recipient's abdominal aorta with two 9-0 nylon stay sutures at the six and 12 o'clock positions. Anastomose the left side of the recipient's abdominal aorta and the donor's ascending aorta with a running 9-0 nylon suture. Translocate the donor's heart to the left of the recipient's abdominal aorta and IVC.Anastomose the right side of the recipient's abdominal aorta and the donor's ascending aorta with a running 9-0 nylon suture. Puncture the IVC with a bent 23-gauge needle and extend the hole longitudinally with Potts scissors. Tie the donor's PA to the recipient's IVC with a 9-0 nylon stay suture at the six o'clock position.Anastomose the left side of the recipient's IVC and the donor's PA with a running 9-0 nylon suture. Tie the donor's PA to the recipient IVC with 9-0 nylon stay suture at the 12 o'clock position. Anastomose the right side of the recipient IVC and the donor's PA with a running 9-0 nylon suture.Apply hemostatic agent to both anastomoses. Release the side-biting clamp forceps. The donor's heart recovered to sinus rhythm with a few minutes.Second, we explain AR model using heterotopic abdominal heart transplantation in rats. Donor heart harvest. Harvest the donor's heart using a similar procedure as that of the normal transplant in rats, except for the ascending aorta and PA transection.Remove the adipose tissue on the front of the ascending aorta and PA with Potts scissors. Dissect the connective tissue between the ascending aorta and PA with tweezers. After inserting one blade of Potts scissors into the transverse sinus, transect only the PA.Transect the aorta distally to the brachiocephalic artery.Fix the donor's heart with pliers. Immobilize the ascending aorta with a vascular clip. Insert a stiff guidewire into the brachiocephalic artery and puncture the aortic valve.Transect the aorta proximally to the brachiocephalic artery with Potts scissors. Recipient preparation. The recipient preparation procedure is similar to that of the normal transplant.Heterotopic heart transplantation. The donor's heart transplantation procedure is similar to normal transplantation. Representative result.Table one shows operative records of the normal model. Also, our transplantation procedure showed approximately 60 minute of ischemia time. All cases had good airway contraction after the clamping, owing to the strengthened myocardial protection.Table two shows operative records of the AR model. The ischemia time in the AR model was only about five minutes longer than the normal model. Table three shows post-operative echocardiography data of the normal model and AR model.The new AR model developed a significant larger LV dimension and thinner LV wall than the normal model. This is the normal model. This is the AR model.LV dilatation was detected. Severe AR jet flow was detected. Figure four shows macroscopic findings of the AR model.Macroscopic examination showed LV dilatation and endocardial thickening. Figure five shows Masson's trichrome-stained microphotographs of the AR model. Masson's trichrome-stained samples demonstrated fibrotic changes in the myocardium and endocardium.Discussion.Key points of our modified transplantation procedure are as follows. First, transecting the donor's abdominal aorta before harvesting in order to unload the donor heart. Second, perfusing the donor's coronary arteries with cold cardioplegic solution.Third, topical cooling of the donor's heart during the anastomosis procedure. Additionally, this novel AR model has some benefits. It is possible to puncture the aortic valve easily and in a shorter time compared to the traditional AR model.Since this model does not contribute to the recipient circulation, the recipient can survive even when the donor heart showed severe AR.Conclusions. We believe that beginners can perform this study's procedure with ease and achieve a high success rate. Moreover, the novel AR model may contribute to studies on the pathomechanism of the myocardium and endocardium fibrosis and the evaluation of anti-fibrotic agents.Thank you for watching.

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