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

Dette arbejde blev støttet af et tilskud B2021-0991 fra Chonnam National University Hospital Biomedical Research Institute og NRF-2020R1F1A1073921 fra National Research Foundation of Korea

Det etiske udvalg for Laboratory Animal Research Center på Chonnam National University Hospital (godkendelsesnr. CNU IACUC - H - 2022-36) godkendte alle dyreforsøg. Hanrotter (350-450 g), der blev anvendt i denne undersøgelse, blev behandlet i overensstemmelse med retningslinjerne for pasning og brug af forsøgsdyrene. Rotterne blev anbragt i temperaturstyrede rum med en 12 timers lys-mørk cyklus med standard mad og vand til rådighed.
1. Forberedelse
<p class="jove_cont...

<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>

Vores fokus i etableringen af denne model var at replikere normoterm human hjertetransplantation. Ikke-udstødende modeller er den almindeligt foretrukne teknik til bevarelse af donorhjertet i et ex situ-miljø 16. Mens udstødningsmodeller giver mange fordele ved vurdering af hjertefunktion under ex situ perfusion17, er de ikke egnede til heterotopiske transplantationsmodeller. Ved heterotopisk transplantation skal det implanterede donorhjerte overvinde s...

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

Hjertetransplantation er fortsat den eneste levedygtige behandling for ildfast hjertesvigt 1,2,3,4. På trods af en støt stigning i antallet af patienter, der har behov for hjertetransplantation, er der ikke observeret en forholdsmæssig stigning i tilgængeligheden af donororganer5. For at løse dette problem er der udviklet nye tilgange til bevarelse af donorhjerter med det formål at forbedre udfordringerne og øge tilgængeligheden af donorer 6,7,8,9.
Normothermic ex situ hjerteperfusion (NESHP) ved hjælp af organplejesystem (OCS) maskiner er opstået som en klinisk intervention 1,3. Denne teknik er blevet anset for at være et egnet alternativ til den konventionelle metode 2,9 med statisk kold opbevaring (SCS). NESHP reducerer effektivt varigheden af kold iskæmi, mindsker metabolisk efterspørgsel og letter optimal ernæringsforsyning og iltning under transport af donororganer10,11. På trods af denne metodes klare potentiale til at forbedre donororganets bevaring har dens kliniske anvendelse og yderligere undersøgelse været begrænset af høje omkostninger. Derfor er prækliniske dyremodeller af NESHP afgørende for at identificere vigtige tekniske udfordringer forbundet med denne teknik12,13. Svin og rotter er de foretrukne dyremodeller til prækliniske undersøgelser på grund af deres iskæmiske tolerance9. Selvom svinemodellen er ideel til grundlæggende og translationel forskning, er den begrænset af dens høje omkostninger og det intensive arbejde, der kræves til pleje og vedligeholdelse. I modsætning hertil er rottemodeller billigere og lettere at håndtere14.
I denne undersøgelse introducerer vi en forenklet rottemodel af NESHP, efterfulgt af heterotopisk hjertetransplantation, for at evaluere virkningen af konserveringsteknikken på transplantatets tilstand efter implantation. Denne model er ligetil, omkostningseffektiv og kan udføres af en enkelt eksperimentator. Figur 1 viser skemaerne for proceduren.

<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

Hjertetransplantation er den mest effektive terapi til hjertesvigt i slutstadiet. På trods af forbedringerne i terapeutiske tilgange og interventioner stiger antallet af hjertesvigtpatienter, der venter på transplantation, stadig. Den normotermiske ex situ-konserveringsteknik er etableret som en metode, der kan sammenlignes med den konventionelle statiske kølelagringsteknik. Den største fordel ved denne teknik er, at donorhjerter kan bevares i op til 12 timer i en fysiologisk tilstand. Desuden tillader denne teknik genoplivning af donorhjerterne efter kredsløbsdød og anvender nødvendige farmakologiske indgreb for at forbedre donorfunktionen efter implantation. Talrige dyremodeller er blevet etableret for at forbedre normotermiske ex situ-konserveringsteknikker og eliminere bevaringsrelaterede komplikationer. Selvom store dyremodeller er nemme at håndtere sammenlignet med små dyremodeller, er det dyrt og udfordrende. Vi præsenterer en rottemodel af normoterm ex situ donorhjertekonservering efterfulgt af heterotopisk abdominal transplantation. Denne model er relativt billig og kan opnås af en enkelt eksperimentator.

Figur 1 illustrerer det eksperimentelle design, der anvendes i en lille dyremodel. Figur 2 viser det modificerede Langendorff-perfusionsapparat, som omfatter en oxygenator til små dyr. Rækkefølgen af anastomose for heterotopisk abdominal implantation er vist i figur 3.
Figur 4 viser de parametre, der anvendes til at vurdere hjertets levedygtighed under ex situ per...

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

Her præsenterer vi en vurderingsprotokol for et heterotopisk implanteret hjerte efter normoterm ex situ-konservering i rottemodellen.

Rottemodel af normamymisk ex-situ perfuseret heterotopisk hjertetransplantation

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

Vi har udviklet en model til at reducere komplikationer forbundet med normoterm ex-situ perfusion og øge den sikre konserveringstid for donorhjerter. Sammenlignet med de alternative dyremodeller er denne model enkel og omkostningseffektiv og kan udføres af en enkelt eksperimentator. Denne model kan være nyttig til at teste den langsigtede effektivitet af farmakologiske og genetiske indgreb på donorhjerterne efter transplantationer.Den kirurgiske procedure kræver mikrokirurgisk dygtighed. Nøglefaktoren for at opnå succes i denne model er at øve og fokusere på de kritiske trin, der er skitseret i manuskriptet. Til at begynde med skal du samle Langendorff-apparatet, herunder oxygenator-, pumpe- og perfusionsledningerne.Fyld perfusionskredsløbet med 20 ml saltopløsning, og cirkulere det, indtil det er primet med autologt blod. På den bedøvede donorrotte laves et 7 centimeter midterlinie abdominal snit og derefter bilaterale snit, der måler 3 centimeter fra xiphoidprocessen til midtkravebenet. Fjern skindet fra brystområdet.Injicer 1.000 international enhed heparin opløst i 0, 3 ml isotonisk saltvand gennem den ringere vena cava ved hjælp af en 1 ml sprøjte Stop enhver blødning fra nålehullet ved forsigtigt at komprimere det med en vatpind. Indsæt et 5-fransk lårbenskateter i abdominal aorta. Sørg for, at kateterspidsen når aortabuen, og bekræft kateterets placering ved at vurdere den omtrentlige længde af den indsatte del af kateteret.Saml omkring 10 ml blod via kateteret indsat i abdominal aorta. Tilslut perfusionslinjen til den kardioplegiske opløsningsledning til abdominkateteret, og start administrationen af den kardioplegiske opløsning med sprøjtepumpen med en hastighed på 800 ml i timen. Åbn brysthulen fra membranen, og skær den ringere vena cava tæt på membranen for at forhindre ventrikulær distension.Skær ribbenene bilateralt langs brystryggen op til brystindløbet. Reflekter den mobiliserede ventrale brystvæg overlegent med mygtang. Efter administration af al kardioplegisk opløsning isoleres aortabuen fra de omgivende væv og dissekeres forsigtigt lige under venstre subklaviske arterie.Transekter brachiocephalic og venstre fælles halspulsårer i en fjern position, efterlader de længere stubbe af aortabuen for nem håndtering under aorta kanylering. Derefter transekteres hovedlungearterien så tæt som muligt på bifurkationen. Juster forsigtigt den overlegne vena cava og ringere vena cava med 5-0 silkesuturer, hvilket forhindrer obstruktion af højre atrium og koronar sinus.Dæk venstre margen af thoraxen med vådt gasbind, læg hjertet på det, og træk forsigtigt de overlegne vena cava og ringere vena cava ligaturer tilbage for at udsætte hilum. Ligate lunge- og azygovenerne sammen med en 5-0 silkesutur. Afbryd vævet dorsalt til ligaturen og ekstraher hjertet.Vej hjertet efter at have undersøgt det for enhver skade. Udskift det saltholdige kredsløb med blodpriming, og start perfusionen med en strømningshastighed på to til tre milliliter i minuttet, så perfusatet lækker fra kanyleringsstedet for at fjerne eventuelle luftbobler. Efter placering af den permanente ligatur skal du sikre dig, at hjertet begynder at trække sig sammen inden for få sekunder og når en normal rytme på 60 sekunder.Et gennemsnitligt perfusionstryk på 55 til 65 millimeter kviksølv med en koronar strømningshastighed på tre til fire milliliter ved 37 grader Celsius indikerer tilstrækkelig perfusion. Saml 0,15 ml blod fra reservoiret, og kontroller blodgasanalysen i begyndelsen af perfusionen og hvert 20. minut derefter. Efter 120 minutters perfusion administreres tre ml Custodiol gennem sprøjtepumpen med en hastighed på 250 ml i timen for at arrestere hjertet.Udfør midterlinjen laparotomi og indsæt en retraktor for at udvide bukhulen. Mobiliser abdominale organer til venstre side af modtageren ved hjælp af vatpinde for at give plads til proceduren. Brug et kirurgisk mikroskop med en 10x forstørrelse, mobiliser tolvfingertarmen og proksimal jejunum ved stump dissektion med vatpinde.at udsætte abdominal aorta og ringere vena cava. Placer 5-0 silkeligaturer på de mesenteriske grene af både kraniale og kaudale sider af de store kar. Løft abdominalkarrene og koaguler eller liger lændehvirvelgrenene med 5-0 silkesuturer.Husk at skåne testikelarterierne og venerne, og klem dem ikke. Brug ligaturer til at løfte karrene og placere mikroklemmerne til mesenteriske grene, kaudale og kraniale sider af de store kar for at stoppe blodgennemstrømningen på anastomosestedet. Punktere aorta ved hjælp af en 27-gauge nål, og forlæng snittet med mikrosaks til en længde, der er lig med eller lidt større end åbningen af donorens stigende aorta.Lav et langsgående snit af den ringere vena cava på samme måde som aortotomien, men gør det tre millimeter tættere på den kaudale side sammenlignet med aorta-snittet. Start anastomoserne, placer donorhjertet på højre side af modtagerens mave og fastgør donorens stigende aorta til modtagerens abdominale aorta med en simpel afbrudt søm i kraniehjørnet af det langsgående snit. Flyt hjertet til venstre side af modtagerens mave, og udfør anastomose af donorens stigende aorta med modtagerens abdominale aorta ved hjælp af en løbende 9-0 polypropylensutur.Fastgør donorens lungearterie til den ringere vena cava med to afbrudte suturer i de kaudale og kraniale hjørner af det langsgående snit. Efter afslutning af anastomose skal du fjerne den kaudale klemme og kranieklemmen. Sikring af tilstrækkelig hæmostase, udskift abdominale organer på en meander-lignende måde og luk lagene af abdominal snit ved hjælp af kontinuerlige 5-0 polypropylen suturer.De parametre, der anvendes til at vurdere hjertets levedygtighed under ex-situ perfusion, såsom laktat, kalium og gennemsnitligt aortatryk, er vist i denne figur. Her reducerede brugen af normoterm ex-situ-konservering den samlede iskæmiske tid for seks vellykkede tilfælde til 46,2 plus-minus 4,7 minutter, mens den samlede ud-af-kroppen-tid var 166,2 plus-minus 4,7 minutter. Ekstraktionen af hjertet fra donoren og forberedelse til ex-situ perfusion og heterotopisk transplantation var 5,8 plus-minus 1,3 minutter.Den samlede succesrate for operationen var 70%, og den gennemsnitlige anastomosetid for de seks vellykkede tilfælde var 38,4 plus-minus 3,4 minutter. Donorhjertets elektrofysiologiske ydeevne før indkøb og efter implantation er præsenteret i denne figur. I alle eksperimenterne faldt hjertefrekvensen signifikant umiddelbart efter implantation, men den kom sig til sidst over tid.Det makroskopiske og mikroskopiske udseende af donorhjertet før normoterm ex-situ konservering, efter normoterm ex-situ konservering og efter to timers heterotopisk implantation er vist her. Når du udfører denne procedure, er det vigtigt at tage nok blod til perfusion, skære forsigtigt aorta uden at beskadige den stigende del og nøje overvåge de fysiologiske parametre under konservering. Efter denne procedure kan vi teste effektiviteten af nye konserveringsløsninger og modtagerens immunrespons på nyligt introducerede konserveringsmetoder.Med kombinationen af ex-situ perfusion og heterotopisk transplantation er vi i stand til at undersøge nye teknikker gennem forlænget konserveringstid og observere den langsigtede indvirkning på modtagerens immunrespons.

Research

JoVE Journal

Medicine

Murine Heterotopic Heart Transplant Technique

Murine Heterotop Heart Transplant Teknik

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

Transplantation af pulmonale ventilen med en musemodel af Heterotop Heart Transplantation

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

Rotte Heterotop Abdominal Heart / Single-lunge transplantation i en Volume-loaded konfiguration

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

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

Heterotopisk Livmoderhalskræft hjertetransplantation i mus

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

A Novel Microsurgical Model for Heterotop, En Bloc brystvæggen, Thymus, og hjertetransplantation i mus

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

Normothermic Ex Situ hjerte Perfusion i arbejde Mode: vurdering af hjertets funktion og stofskifte

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

En forenklet model til heterotopisk hjerteventiltransplantation hos gnavere

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

Normothermisk undertryksventilation ex situ lungeperfusion: evaluering af lungefunktion og metabolisme

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

En svine heterotopisk hjertetransplantationsprotokol til levering af terapi til en hjertealograft

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