Name: rat model of normothermic ex-situ perfused heterotopic heart transplantation
Uploaded: 2023-04-21T13:00:00
Description: Watch this Scientific Journal Video about Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation at JoVE.com

这项工作得到了全南国立大学医院生物医学研究所的B2021-0991和韩国国家研究基金会的NRF-2020R1F1A1073921的支持

全南大学医院实验动物研究中心伦理委员会（批准文号）中国农业大学 IACUC - H - 2022-36） 批准了所有动物实验。本研究中使用的雄性Sprague-Dawley大鼠（350-450g）按照实验动物的护理和使用指南接受了护理。将大鼠饲养在温控室中，进行12小时的明暗循环，并提供标准食物和水。
1. 准备
注意：单个实验者可以进行所有实验程序。
<ol><li>手术前?...

<ol>
	<li>Langmuur, S. J. 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=Normothermic+ex-situ+heart+perfusion+with+the+organ+care+system+for+cardiac+transplantation:+A+meta-analysis.">Normothermic ex-situ heart perfusion with the organ care system for cardiac transplantation: A meta-analysis.</a> Transplantation. 106 (9), 1745-1753 (2022).</li><li>Ardehali, 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=Ex-vivo+perfusion+of+donor+hearts+for+human+heart+transplantation+(PROCEED+II):+a+prospective,+open-label,+multicentre,+randomized+non-inferiority+trial.">Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomized non-inferiority trial.</a> Lancet. 385 (9987), 2577-2584 (2015).</li><li>Dang Van, 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=Ex+vivo+perfusion+of+the+donor+heart:+Preliminary+experience+in+high-risk+transplantations.">Ex vivo perfusion of the donor heart: Preliminary experience in high-risk transplantations.</a> Archives of Cardiovascular Diseases. 114 (11), 715-726 (2021).</li><li>Zhou, P., 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=Donor+heart+preservation+with+hypoxic-conditioned+medium-derived+from+bone+marrow+mesenchymal+stem+cells+improves+cardiac+function+in+a+heart+transplantation+model.">Donor heart preservation with hypoxic-conditioned medium-derived from bone marrow mesenchymal stem cells improves cardiac function in a heart transplantation model.</a> Stem Cell Research and Therapy. 12 (1), 5f6 (2021).</li><li>Messer, S., Large, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resuscitating+heart+transplantation:+the+donation+after+circulatory+determined+death+donor.European.">Resuscitating heart transplantation: the donation after circulatory determined death donor.European.</a> Journal of Cardio-Thoracic Surgery. 49 (1), 1-4 (2016).</li><li>Trahanas, J. M., 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=Achieving+12+hour+normothermic+ex+situ+heart+perfusion:+an+experience+of+40+porcine+hearts.">Achieving 12 hour normothermic ex situ heart perfusion: an experience of 40 porcine hearts.</a> ASAIO Journal. 62 (4), 470-476 (2016).</li><li>Yang, Y., 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=Keeping+donor+hearts+in+completely+beating+status+with+normothermicblood+perfusion+for+transplants.">Keeping donor hearts in completely beating status with normothermicblood perfusion for transplants.</a> The Annals of Thoracic Surgery. 95 (6), 2028-2034 (2013).</li><li>Van Caenegem, O., 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=Hypothermic+continuous+machine+perfusion+enables+preservation+of+energy+charge+and+functional+recovery+of+heart+grafts+in+an+ex+vivo+model+of+donation+following+circulatory+death.">Hypothermic continuous machine perfusion enables preservation of energy charge and functional recovery of heart grafts in an ex vivo model of donation following circulatory death.</a> European Journal of Cardiothoracic Surgery. 49 (5), 1348-1353 (2016).</li><li>Lu, 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=Normothermic+ex+vivo+heart+perfusion+combined+with+melatonin+enhances+myocardial+protection+in+rat+donation+after+circulatory+death+hearts+via+inhibiting+NLRP3+inflammasome-mediated+pyroptosis.">Normothermic ex vivo heart perfusion combined with melatonin enhances myocardial protection in rat donation after circulatory death hearts via inhibiting NLRP3 inflammasome-mediated pyroptosis.</a> Frontiers in Cell and Developmental Biology. 9, 733183 (2021).</li><li>Pinnelas, R., Kobashigawa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+normothermic+perfusion+in+heart+transplantation:+a+review+of+the+TransMedics+Organ+Care+System.">Ex vivo normothermic perfusion in heart transplantation: a review of the TransMedics Organ Care System.</a> Future Cardiology. 18 (1), 5-15 (2022).</li><li>Fuchs, M., 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=Does+the+heart+transplant+have+a+future.">Does the heart transplant have a future.</a> European Journal of Cardiothoracic Surgery. 55, i38-i48 (2019).</li><li>Pahuja, M., Case, B. C., Molina, E. J., Waksman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+FDA's+circulatory+system+devices+panel+virtual+meeting+on+the+TransMedics+Organ+Care+System+(OCS)+Heart+-+portable+extracorporeal+heart+perfusion+and+monitoring+system.">Overview of the FDA's circulatory system devices panel virtual meeting on the TransMedics Organ Care System (OCS) Heart - portable extracorporeal heart perfusion and monitoring system.</a> American Heart Journal. 247, 90-99 (2022).</li><li>Jawitz, O. K., Devore, A. D., Patel, C. B., Bryner, B. S., Schroder, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+donor+pool:+quantifying+the+potential+impact+of+a+portable+organ-care+system+for+expanded+criteria+heart+donation.">Expanding the donor pool: quantifying the potential impact of a portable organ-care system for expanded criteria heart donation.</a> Journal of Cardiac Failure. 27 (12), 1462-1465 (2021).</li><li>van Suylen, V., 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=Ex+situ+perfusion+of+hearts+donated+after+euthanasia:+a+promising+contribution+to+heart+transplantation.">Ex situ perfusion of hearts donated after euthanasia: a promising contribution to heart transplantation.</a> Transplantation Direct. 7 (3), e676 (2021).</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), e0214513 (2019).</li><li>Qin, G., Jernryd, T., Sjoberg, S., Steen, S., Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+perfusion+for+human+heart+preservation:+A+systematic+review.">Machine perfusion for human heart preservation: A systematic review.</a> Transplant International. 35, 10258 (2022).</li><li>Dang Van, S., Brunet, D., Akamkam, A., Decante, B., Guihaire, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+the+donor+heart+during+ex+situ+perfusion:+insights+from+pressure-volume+loops+and+surface+echocardiography.">Functional assessment of the donor heart during ex situ perfusion: insights from pressure-volume loops and surface echocardiography.</a> Journal of Visual Experiments. (188), e63945 (2022).</li><li>Fu, X., Segiser, A., Carrel, T. P., Tevaearai Stahel, H. T., Most, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heterotopic+heart+transplantation+model+to+investigate+unloading-induced+myocardial+remodeling.">Rat heterotopic heart transplantation model to investigate unloading-induced myocardial remodeling.</a> Frontiers in Cardiovascular Medicine. 3, 34 (2016).</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>Qi, 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=The+evaluation+of+constant+coronary+artery+flow+versus+constant+coronary+perfusion+pressure+during+normothermic+ex-situ+heart+perfusion.">The evaluation of constant coronary artery flow versus constant coronary perfusion pressure during normothermic ex-situ heart perfusion.</a> The Journal of Heart and Lung Transplantation. 41 (12), 1738-1750 (2022).</li><li>Okahara, 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=A+novel+blood+viscosity+estimation+method+based+on+pressure-flow+characteristics+of+an+oxygenator+during+cardiopulmonary+bypass.">A novel blood viscosity estimation method based on pressure-flow characteristics of an oxygenator during cardiopulmonary bypass.</a> Artificial Organs. 41 (3), 262-266 (2017).</li><li>Quader, M., Torrado, J. F., Mangino, M. J., Toldo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+flow+rate+limit+the+optimal+ex-vivo+perfusion+of+the+heart+-+an+experimental+study.">Temperature and flow rate limit the optimal ex-vivo perfusion of the heart - an experimental study.</a> Journal of Cardiothoracic Surgery. 15 (1), 180 (2020).</li></ol>

我们建立这个模型的重点是复制正常的人类心脏移植。非射血模型是在 非原位 环境中保存供体心脏的常用首选技术16。虽然射血模型在 评估非原 位灌注期间的心脏功能方面具有许多优势17，但它们不适用于异位移植模型。在异位移植中，植入的供体心脏需要克服宿主心脏在受体循环系统中产生的收缩后负荷压力，导致供体心脏性能有限，并且在?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64954">Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation</a>. The Protocol section was updated.
Section 4 of the Protocol was updated from:
4. Implantation
<ol>
	<li>Preparation of recipient
	<ol>
		<li>Begin the recipient preparation 30 min before the cessation of ex situ perfusion.</li>
		<li>Anesthetize the recipient animal using the same method as mentioned in step 2.2.</li>
		<li>Place the rat in a supine position on the heating pad and insert the temperature probe into the rectum to maintain the body temperature at 37 &#176;C.</li>
		<li>Apply eye lubricant, shave the pubic to the epigastric area, and cleanse the area with an iodine-based scrub and 70% alcohol.</li>
	</ol>
	</li>
	<li>Medications
	<ol>
		<li>Inject 2 mL of warm saline subcutaneously to compensate for the fluid lost during the surgery. Inject 200 IU of heparin subcutaneously.</li>
		<li>Administer antibiotic prophylaxis by injecting 10 mg/kg cefazolin dissolved in 0.3 mL of saline subcutaneously or intramuscularly.</li>
		<li>Administer pain control by injecting 20 mg/kg of diclofenac subcutaneously.</li>
	</ol>
	</li>
	<li>Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure.</li>
	<li>Prevent dehydration by wrapping the abdominal organs with warm and wet gauze. Intermittingly spread warm saline with a 50 mL syringe during the surgery.</li>
	<li>Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs to expose the Abd. A. and IVC. Prepare the Abd. A and IVC for anastomosis and systematically implant the donor heart, in accordance with Figure 3 or previously documented methods15. 
	NOTE: Do not separate the Abd. A. and IVC.
	<ol>
		<li>Assuming vascular anastomosis to be placed infrarenal, prepare a sufficient portion of the aorta and IVC for clamping.</li>
		<li>Perform blunt preparation using cotton swabs or sharp-serrated forceps to remove the fats and fascia around the vessels.</li>
		<li>Place 5-0 silk ligatures to the mesenteric branches and both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures. Remember to spare the testicular arteries and veins and do not clamp them.</li>
		<li>Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Be sure to switch off the heating pad before placing the clamps, as excess heating can exacerbate limb ischemia.</li>
		<li>Puncture the aorta using a 27 G needle and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta (Asc. A), which is approximately 5 mm.</li>
		<li>Make a longitudinal incision on the IVC in the same way as the aortotomy, but make it 3 mm closer to the caudal side compared to the aorta incision.</li>
		<li>Starting the anastomoses, placed the donor heart on the right side of the recipient&#39;s abdomen and attach the donor Asc. A to the recipient&#39;s Abd. A with one simple interrupted stitch (9-0 polypropylene) at the cranial corner of the longitudinal incision.</li>
		<li>Move the heart to the left side of the recipient abdomen and perform anastomosis of the donor&#39;s Asc. A with the recipient&#39;s Abd. A using a running 9-0 polypropylene suture.</li>
		<li>Fixate the donor pulmonary artery to the IVC with two interrupted sutures (9-0 polypropylene) at the caudal and cranial corners of the longitudinal incision.</li>
		<li>Perform the first half of the venous anastomosis from the intraluminal side of the vessel and complete the second half from the extraluminal side of the vessel. Before tightening the knots, flush the field with saline to prevent air embolism.</li>
	</ol>
	</li>
	<li>De-airing and de-clamping
	<ol>
		<li>Remove the mesenteric vein clamp first after completing the anastomosis to allow the right side of the heart to fill with venous blood.</li>
		<li>Remove the air in the coronary circuit and Asc. A. by applying retrograde coronary perfusion for several seconds.</li>
		<li>Place a piece of gauze on both sides of the vessels and remove the caudal clamp and the cranial clamp.</li>
		<li>Apply gentle compression with cotton swabs for 1-2 min. After ensuring adequate hemostasis, remove the swabs and wash the anastomoses with warm saline. 
		NOTE: The heart should begin beating within the first minute of reperfusion. If the recipient rat&#39;s body temperature is below 35 &#176;C, the heart rhythm will normalize after the temperature reaches 36 &#176;C.</li>
	</ol>
	</li>
	<li>Replace the abdominal organs in a meander-like manner and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.</li>
</ol>
to:
4. Implantation
<ol>
	<li>Preparation of recipient
	<ol>
		<li>Begin the recipient preparation 30 min before the cessation of ex situ perfusion.</li>
		<li>Anesthetize the recipient animal using the same method as mentioned in step 2.2.</li>
		<li>Place the rat in a supine position on the heating pad and insert the temperature probe into the rectum to maintain the body temperature at 37 &#176;C.</li>
		<li>Apply eye lubricant, shave the pubic to the epigastric area, and cleanse the area with an iodine-based scrub and 70% alcohol.</li>
	</ol>
	</li>
	<li>Medications
	<ol>
		<li>Inject 2 mL of warm saline subcutaneously to compensate for the fluid lost during the surgery. Inject 200 IU of heparin subcutaneously.</li>
		<li>Administer antibiotic prophylaxis by injecting 10 mg/kg cefazolin dissolved in 0.3 mL of saline subcutaneously or intramuscularly.</li>
		<li>Administer pain control by injecting 20 mg/kg of diclofenac subcutaneously.</li>
	</ol>
	</li>
	<li>Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure.</li>
	<li>Prevent dehydration by wrapping the abdominal organs with warm and wet gauze. Intermittingly spread warm saline with a 50 mL syringe during the surgery.</li>
	<li>Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs to expose the Abd. A. and IVC. Prepare the Abd. A and IVC for anastomosis and systematically implant the donor heart, in accordance with Figure 3 or previously documented methods15. 
	NOTE: Do not separate the Abd. A. and IVC.
	<ol>
		<li>Assuming vascular anastomosis to be placed infrarenal, prepare a sufficient portion of the aorta and IVC for clamping.</li>
		<li>Perform blunt preparation using cotton swabs or sharp-serrated forceps to remove the fats and fascia around the vessels.</li>
		<li>Place 5-0 silk ligatures to the mesenteric branches and both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures. Remember to spare the testicular arteries and veins and do not clamp them.</li>
		<li>Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Switch off the heating pad before placing the clamps, as excess heating can exacerbate limb ischemia. Ensure to switch on the heating pad after de-clamping the vessels to avoid hypothermia.</li>
		<li>Puncture the aorta using a 27 G needle and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta (Asc. A), which is approximately 5 mm.</li>
		<li>Make a longitudinal incision on the IVC in the same way as the aortotomy, but make it 3 mm closer to the caudal side compared to the aorta incision.</li>
		<li>Starting the anastomoses, placed the donor heart on the right side of the recipient&#39;s abdomen and attach the donor Asc. A to the recipient&#39;s Abd. A with one simple interrupted stitch (9-0 polypropylene) at the cranial corner of the longitudinal incision.</li>
		<li>Move the heart to the left side of the recipient abdomen and perform anastomosis of the donor&#39;s Asc. A with the recipient&#39;s Abd. A using a running 9-0 polypropylene suture.</li>
		<li>Fixate the donor pulmonary artery to the IVC with two interrupted sutures (9-0 polypropylene) at the caudal and cranial corners of the longitudinal incision.</li>
		<li>Perform the first half of the venous anastomosis from the intraluminal side of the vessel and complete the second half from the extraluminal side of the vessel. Before tightening the knots, flush the field with saline to prevent air embolism.</li>
	</ol>
	</li>
	<li>De-airing and de-clamping
	<ol>
		<li>Remove the mesenteric vein clamp first after completing the anastomosis to allow the right side of the heart to fill with venous blood.</li>
		<li>Remove the air in the coronary circuit and Asc. A. by applying retrograde coronary perfusion for several seconds.</li>
		<li>Place a piece of gauze on both sides of the vessels and remove the caudal clamp and the cranial clamp.</li>
		<li>Apply gentle compression with cotton swabs for 1-2 min. After ensuring adequate hemostasis, remove the swabs and wash the anastomoses with warm saline. 
		NOTE: The heart should begin beating within the first minute of reperfusion. If the recipient rat&#39;s body temperature is below 35 &#176;C, the heart rhythm will normalize after the temperature reaches 36 &#176;C.</li>
	</ol>
	</li>
	<li>Replace the abdominal organs in a meander-like manner and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.</li>
	<li>After the surgery, place the anesthetized animal on a clean area over a heating pad until the body temperature reaches 37&#176;C.&#160; 
	NOTE: Do not initiate the postoperative examinations till the body temperature reaches 37&#176;C. Maintain anesthesia at 2-2.5% isoflurane until the end of the experiments.</li>
	<li>Monitor ECG of the transplanted donor heart for 3 h. Then, excise the heart under deep anesthesia for histological studies. 
	NOTE: Confirm anesthesia depth via lack of pedal reflex before excising the heart. The surgical procedure and the ECG monitoring take less than 6 h. Diclofenac, administered perioperatively (step 4.2.3.), enables pain management for the entire duration of this procedure. The analgesia regimen can be adjusted per the institutional animal use guidelines.</li>
</ol>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64954">Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation</a>. The Protocol section was updated.

Erratum: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

心脏移植仍然是难治性心力衰竭的唯一可行疗法1,2,3,4。尽管需要心脏移植的患者数量稳步增加，但尚未观察到供体器官可用性的比例增加5。为了解决这个问题，已经开发了保存捐赠者心脏的新方法，目的是改善挑战并增加捐赠者的可用性6,7,8,9。
使用器官护理系统（OCS）机器的常母异位心脏灌注（NESHP）已成为一种临床干预措施1,3。该技术被认为是传统静态冷藏（SCS）方法2,9的合适替代方案。NESHP有效缩短冷缺血的持续时间，减少代谢需求，并促进供体器官运输过程中的最佳营养供应和氧合10,11。尽管这种方法在改善供体器官保存方面具有明显的潜力，但其临床应用和进一步研究受到高成本的限制。因此，NESHP的临床前动物模型对于确定与该技术相关的关键技术挑战至关重要12,13。猪和大鼠由于其缺血耐受性而成为临床前研究的首选动物模型9。尽管猪模型是基础和转化研究的理想选择，但它受到其高成本以及护理和维护所需的密集劳动的限制。相比之下，大鼠模型更便宜且更容易处理14。
在本研究中，我们引入了NESHP的简化大鼠模型，然后是异位心脏移植，以评估保存技术对植入后移植条件的影响。该模型简单，具有成本效益，可以由单个实验者执行。 图 1 显示了该过程的原理图。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AES active evacuation system</td>
			<td>Smiths medical</td>
			<td>PC-6769-51A</td>
			<td>Utilize CO2 and excess isoflurane</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Smiths medical</td>
			<td>PC-8801-01A</td>
			<td>Mixes isoflurane and oxyegn and delivers to animal</td>
		</tr>
		<tr>
			<td>B20 patient monitor</td>
			<td>GE medical systems</td>
			<td>B20</td>
			<td>to observe mean aortic pressure and temperature</td>
		</tr>
		<tr>
			<td>Homeothermic Monitoring System</td>
			<td>Harvard apparatus</td>
			<td>55-7020</td>
			<td>To monitor and maintain animal&#39;s temperature</td>
		</tr>
		<tr>
			<td>Micro-1 Rat oxygenator</td>
			<td>Dongguan Kewei medical instruments</td>
			<td>Micro-MO</td>
			<td>For gas exchange in the langendorff circuit</td>
		</tr>
		<tr>
			<td>Micropuncture introducer Set</td>
			<td>COOK medical</td>
			<td>G48007</td>
			<td>for delivering cardioplegic solution to the arch through the abdominal aorta</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Amscope</td>
			<td>MU1403</td>
			<td>For zooming surgical field (Recipient)</td>
		</tr>
		<tr>
			<td>Surgical loupe</td>
			<td>SurgiTel</td>
			<td>L2S09</td>
			<td>For zooming surgical field (Donor)</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>AMP all</td>
			<td>SP-8800</td>
			<td>To deliver cardioplegic solution</td>
		</tr>
		<tr>
			<td>Transonic flow sensor</td>
			<td>Transonic</td>
			<td>ME3PXL-M5</td>
			<td>Perfusion circuit flow sensor</td>
		</tr>
		<tr>
			<td>Transonic tubing flow module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td>flow acquiring system</td>
		</tr>
		<tr>
			<td>Watson - Marlow pumps</td>
			<td>Harvard apparatus</td>
			<td>010.6131.DAO</td>
			<td>Peristaltic pump used for recirculate perfusate</td>
		</tr>
		<tr>
			<td>WBC-1510A</td>
			<td>JEIO TECH</td>
			<td>E03056D</td>
			<td>Heating bath</td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Samtako Bio Korea Co., Ltd., Osan City Korea</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medications</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioHAnce Gel Eye Drops</td>
			<td>SENTRIX Animal care</td>
			<td></td>
			<td>wet ointments for eye</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>JW pharmaceutical</td>
			<td></td>
			<td>For prophilaxis</td>
		</tr>
		<tr>
			<td>Custodiol</td>
			<td>DR, FRANZ KOHLER CHEMIE GMBH</td>
			<td></td>
			<td>For heart harvesting</td>
		</tr>
		<tr>
			<td>Diclofenac</td>
			<td>Myungmoon Pharm. Co. Ltd</td>
			<td></td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>JW pharmaceutical</td>
			<td></td>
			<td>Anticoagulant</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>JW pharmaceutical</td>
			<td></td>
			<td>hormon therapy</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>JW pharmaceutical</td>
			<td></td>
			<td>For hydration therapy</td>
		</tr>
	</tbody>
</table>

rat model of normothermic ex-situ perfused heterotopic heart transplantation

心脏移植是终末期心力衰竭最有效的治疗方法。尽管治疗方法和干预措施有所改善，但等待移植的心力衰竭患者人数仍在增加。常温 异地 保存技术已被确立为与传统静态冷藏技术相当的方法。该技术的主要优点是供体心脏可以在生理条件下保存长达12小时。此外，该技术允许在循环死亡后对供体心脏进行复苏，并应用所需的药物干预来改善植入后的供体功能。已经建立了许多动物模型来改进常温 异地 保存技术并消除与保存相关的并发症。虽然与小型动物模型相比，大型动物模型易于处理，但它既昂贵又具有挑战性。我们提出了一个正常变温异位供体心脏保存后异 位 腹部移植的大鼠模型。这种模型相对便宜，可以由单个实验者完成。

图1 显示了小动物模型中使用的实验设计。 图2 显示了改进的朗根道夫灌注装置，其中包括一个小动物氧合器。异位腹腔植入的吻合顺序如图 3所示。
图4 显示了用于评估 非原位 灌注期间心脏活力的参数，例如乳酸、钾和平均主动脉压。在这项研究中，使用常温 异位 保存?...

Watch this Scientific Journal Video about Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation at JoVE.com

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

在这里，我们提出了大鼠模型中正常变 位异地 保存后异位植入心脏的评估方案。

异位灌注异位心脏移植大鼠模型

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

我们开发了一个模型来减少与常温非原位灌注相关的并发症，并增加供体心脏的安全保存时间。与其他动物模型相比，该模型简单且具有成本效益，并且可以由单个实验者执行。该模型可用于测试药物和遗传干预对移植后供体心脏的长期有效性。外科手术需要显微外科技术。在这个模型中取得成功的关键因素是实践并专注于手稿中概述的关键步骤。首先，组装朗根道夫设备，包括氧合器、泵和灌注管路。用20毫升盐水溶液填充灌注回路，并循环直至用自体血灌注。在麻醉的供体大鼠上，做一个7厘米的中线腹部切口，然后从剑突到锁骨中部测量3厘米的双侧切口。从胸部区域取下毛皮。使用1毫升注射器通过下腔静脉注射1,000国际单位肝素溶解在0.3毫升等渗盐水中，用棉签轻轻压缩针孔以阻止针孔出血。将 5 法式股骨导管插入腹主动脉。确保导管尖端到达主动脉弓，并通过评估导管插入部分的大致长度来确认导管位置。通过插入腹主动脉的导管收集约10毫升血液。将心脏停搏溶液灌注管线连接到腹导管，并以每小时800毫升的速度用注射泵开始心脏停搏溶液给药。从横膈膜打开胸腔，切开靠近横膈膜的下腔静脉，以防止心室扩张。沿胸椎双侧切肋骨，直至胸廓入口。用蚊钳向上反射活动腹侧胸壁。给药所有心脏停搏液后，将主动脉弓与周围组织隔离，并小心地将其解剖在左锁骨下动脉下方。横断头臂动脉，左颈总动脉位于远处位置，留下主动脉弓较长的残端，以便在主动脉插管期间易于处理。然后将主肺动脉横断面尽可能靠近分叉处。用5-0丝线小心地结扎上腔静脉和下腔静脉，防止右心房和冠状窦阻塞。用湿纱布覆盖胸部左缘，将心脏放在其上，轻轻缩回上腔静脉和下腔静脉结扎，露出肺门。用5-0丝线将肺静脉和无齐静脉结扎在一起。切断结扎处的组织背侧并提取心脏。检查心脏是否有任何伤害后称量心脏。用血液灌注代替盐水灌注回路，并以每分钟两到三毫升的流速开始灌注，让灌注液从插管部位泄漏以去除任何气泡。放置永久性结扎后，确保心脏在几秒钟内开始收缩，并在 60 秒内达到正常节律。平均灌注压力为 55 至 65 毫米汞柱，37 摄氏度时冠状动脉流速为 3 至 4 毫升，表明灌注充分。从储液器中收集0.15毫升血液，并在灌注开始时和此后每20分钟检查一次血气分析。灌注120分钟后，以每小时250毫升的速度通过注射泵给予三毫升的Custodiol，以停止心脏。进行中线剖腹手术并插入牵开器以扩大腹腔。使用棉签将腹部器官移动到受体的左侧，为手术腾出空间。利用10倍放大倍率的手术显微镜，通过用棉签钝性解剖来调动十二指肠和近端空肠。暴露腹主动脉和下腔静脉。将5-0丝结扎放在主要血管的颅侧和尾侧的肠系膜分支上。抬高腹部血管，用 5-0 条丝线凝固或结扎腰支。切记要放过睾丸动脉和静脉，不要夹住它们。使用结扎器抬起血管并将微夹定位在大血管的肠系膜分支、尾部和颅侧，以阻止吻合部位的血流。使用27号针刺穿主动脉，并用微型剪刀将切口拉长至等于或略大于供体升主动脉开口的长度。以与主动脉切开术相同的方式对下腔静脉进行纵向切口，但与主动脉切口相比，使其更接近尾侧三毫米。开始吻合，将供体心脏放在受者腹部的右侧，并在纵向切口的颅角用一根简单的断针将供体升主动脉连接到受体的腹主动脉上。将心脏移动到受者腹部的左侧，并使用运行的9-0聚丙烯缝合线将供体的升主动脉与受体的腹主动脉进行吻合。将供体肺动脉固定在下腔静脉上，在纵向切口的尾角和颅角处进行两次间断缝合。吻合完成后，取下尾夹和颅夹。确保足够的止血，以蜿蜒的方式更换腹部器官，并使用连续的5-0聚丙烯缝合线关闭腹部切口的层。用于评估非原位灌注期间心脏活力的参数，如乳酸、钾和平均主动脉压，如图所示。在这里，使用常温离位保存将6例成功病例的总缺血时间减少到46.2正负4.7分钟，而总出体时间为166.2正负4.7分钟。从供体中提取心脏以及准备异位灌注和异位移植所需的时间为5.8正负1.3分钟。手术总成功率为70%，6例成功病例平均吻合时间为38.4正负3.4分钟。该图显示了供体心脏在获取之前和植入后的电生理性能。在所有实验中，植入后心率立即显着下降，但最终会随着时间的推移而恢复。这里显示了正常变位保存前、正常温原位保存后和异位植入两小时后供体心脏的宏观和微观外观。在进行此过程时，重要的是要取足够的血液进行灌注，小心地切开主动脉而不损坏升序部分，并在保存过程中密切观察生理参数。按照这一程序，我们可以测试新型保存溶液的有效性以及受体对新引入的保存方法的免疫反应。通过非原位灌注和异位移植的结合，我们能够通过延长保存时间来研究新技术，并观察对受体免疫反应的长期影响。

Research

JoVE Journal

Medicine

Murine Heterotopic Heart Transplant Technique

小鼠异位心脏移植技术

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 ...

科研

医学

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

肺动脉瓣使用异位心脏移植小鼠模型移植

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

大鼠异位心脏腹部/在卷装配置单肺移植

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

Ex Situ Normothermic Machine Perfusion of Donor Livers

Ex Situ Normothermic Machine Perfusion of Donor Livers

供肝易地常温灌注机

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Heterotopic Cervical Heart Transplantation in Mice

颈椎异位心脏移植小鼠

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

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

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

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

工作模式下的心功能和代谢评估

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

啮齿动物异位心脏瓣膜移植的简化模型

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

非原位负压通气肺灌注：肺功能和代谢的评估

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

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

一种猪异位心脏移植方案，用于向心脏同种异体移植物提供治疗药物

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