Name: Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model (Video) | JoVE Journal
Uploaded: 2021-06-03T13:00:00.000+00:00
Duration: 8 min 37 s
Description: Learn a novel heterotopic abdominal heart transplantation technique in mice using modified blood circuit reconstruction. This method reduces warm ischemia time and improves graft success rates for alloimmune response studies.

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5 minutes

We thank Dr. Yun Xu for her help as voice actor, Dr. med. Jianhua Peng for her help in video editing and Dr. Annika Kuckhahn for her comments and support. This work was supported in part by the German Research Foundation (DFG) to promote international collaborations (HO2581/4-1 to AH), and the National Science Foundation of China (NSFC; #81760291 to FJ).

All animal experiments were conducted following the guidelines from the directive 2010/63/EU of the European Parliament on protection of animals used for scientific purposes (Ethic committee approved, #G1071/09).
NOTE: Preliminary preparation, anesthesia, post-operative care and monitoring work are the same as performed in traditional surgical methods1,2,4. BALB/c mice served as heart donors and C57BL/6J as transplant recipients. Mice were aged 8-12 weeks, weighed~30 g at transplantation and were housed under standard conditions.
1. Preparatory steps
<ol>
	<li>For anesthesia, give mice inhalative isoflurane (2%) until they fall asleep, followed by intraperitoneal injections of ketamine (100 mg/kg) + xylazine (10 mg/kg) + acepromazine (2 mg/kg). For postoperative analgesia, apply Metamizol (200 mg/kg) p.o. and Carprofen (5 mg/kg) s.c.. 
	NOTE: The application of antibiotics was abstained on purpose as these substances may influence immunological responses.</li>
	<li>For surgery, use a set of microscopic instruments including a micro-scissor, micro-forceps, a needle holder and micro hemostatic clamps. An electrosurgical pen is also necessary. Perform sutures using 7/0er, 10/0er and&#160;4/0er nylon types.</li>
	<li>Place the mouse in a box for isoflurane inhalation (2%) for 40-60 seconds. Determine the depth of anesthesia by squeezing the paw with tweezers. If there is a complete lack of response for this stimulus, go to the next step.</li>
	<li>Once the mouse has fallen asleep, weigh the mouse.</li>
	<li>Apply an intraperitoneal injection of ketamine (100 mg/kg) + xylazine (10 mg/kg) + acepromazine (2 mg/kg) to the anesthetized mouse.</li>
	<li>Clip the abdominal fur and place the mouse on the operation table. Perform disinfection using povidone iodide for 3 times, then properly drape the mouse using a fenestrated surgical towel.</li>
</ol>
2. Donor operation procedure
<ol>
	<li>Use scissors to cut the skin from the neck to the lower abdomen, and peel off the full layer of the skin to the midline of both axillae.</li>
	<li>Use scissors to cut the muscles of the abdominal wall and gently move the viscera to the left (from the operator&#39;s view). Wrap away the viscera with a saline imbibed gauze to safely expose the inferior vena cava.</li>
	<li>Use a 1 mL syringe to inject 0.4 mL of the heparin solution (contains 500 U heparin) slowly into the lower vena cava and wait for 1 minute before pulling out the needle.</li>
	<li>Pull out the needle and use a micro-scissor to cut both the inferior vena cava and the abdominal aorta&#160;to accelerate exsanguination.</li>
	<li>Use scissors to open the chest cavity by performing a u-shaped cut; completely expose the heart, lungs and all chest blood vessels.
	<ol>
		<li>Expose the thoracic aorta, cut 1/2 of the lumen, and then cut the pulmonary vein to facilitate irrigation and drainage.</li>
		<li>Insert an irrigation tube into the opening of the thoracic aorta, inject at least 2 mL of 4 &#176;C cold histidine-tryptophan-ketoglutarate cardioplegia solution (Custodiol HTK solution)9 until the pulmonary vein outflow is completely clear and the heart completely stops beating.</li>
	</ol>
	</li>
	<li>Pull out the irrigation tube and detach the sternum.</li>
	<li>Use micro scissors to remove the thymus and to slightly strip the fat around the aortic arch.</li>
	<li>Use straight and curved forceps to expose and ligate the trunk of the arteria pulmonalis (on the right side of the aortic arch) with a 10/0 suture.</li>
	<li>Use micro forceps to separate the fat and connective tissue attached to the IIVC, expose and ligate the superior vena cava (on the left side of the aortic arch) with a 7/0 suture and use micro scissors to cut it behind the ligation.</li>
	<li>Make a 7/0 suture around the base of the heart underneath the aortic arch, the IIVC, and both auricles. Then ligate the pulmonary artery branches and venous lung vessels.</li>
	<li>Use micro scissors to transect the aortic arch as distal as possible, the lung vessels underneath the ligature and the IIVC near the diaphragm. Remove the heart out of the chest.</li>
	<li>Place the explanted donor&#39;s heart into 4 &#176;C cold HTK cardioplegia solution and preserve temporarily.</li>
</ol>
3. Recipient operation procedure
NOTE: The initial operation steps are similar to those previously shown for the donor mouse, including anesthesia and disinfection.
<ol>
	<li>Perform the abdominal skin cut in a transverse manner, cover the abdominal organs with a wet gauze using saline solution.</li>
	<li>Use micro forceps to expose the inferior vena cava and abdominal aorta and free them from surrounding fat tissue.</li>
	<li>Use micro forceps to ligate or electrocauterize side branch vessels (lateral or under the vein/aorta) below the renal vessels.</li>
	<li>Use clip applicator forceps to position two micro hemostatic clamps at the abdominal part of vein/aorta coming from the right leaving more than 1 cm distance for both aorta/vein to ensure space for the construction of the anastomosis in between them.</li>
	<li>Use micro scissors to make an incision into the aorta a little nearer to the lower clamp than to the upper clamp. Alternatively, use a 30 G needle to make a small hole and open it up with micro scissors.</li>
	<li>Position the recipient mouse so that the aorta is facing the operator with the vena cava on the other side. Then place the heart into the abdominal cavity and cover it with a small wet gauze pad.</li>
	<li>Use a 10/0 suture to adapt and stitch the donor aorta to the recipient aorta starting caudally, make a knot and proceed with a running suture to the top of the incision (about 4-5 stitches). Next, flip the heart over to the right (from the subject&#39;s view), cover it again and continue the suture on the left side until reaching the caudal end and knot it.</li>
	<li>Use an irrigation tube to inject at least 0.5 mL of 4 &#176;C HTK cardioplegia solution to flush the donor&#39;s IIVC.</li>
	<li>Use micro-scissors to cut a round hole on the abdominal inferior vena cava of the recipient, which should have the same size of the donors IIVC lumen. The incision should be located above the aortic anastomotic opening. Make the vein incision larger than the aortic incision.</li>
	<li>Use a 10/0 suture to sew the donor IIVC to the recipient vena cava starting caudally. Tie a knot and perform a running suture until the top of the incision is reached. Use five stitches and continue the suture on the left. Finally, tie a knot in the tail corner, and carefully tighten (be careful not to pull too tight).</li>
	<li>Place the small parts of the hemostatic sponge around the vein and aortic anastomoses.</li>
	<li>Use clip applicator forceps to remove first the lower and then the upper micro-hemostatic clamps and rinse the abdominal cavity with 38.0&#176;C tempered 0.9% sodium chloride.</li>
	<li>Use micro forceps to take away the hemostatic sponge.</li>
	<li>Observe the heartbeat of the transplanted heart.</li>
	<li>Use forceps to put the intestines back into the abdominal cavity and two-layer sutures (abdominal muscles followed by skin) to close the abdominal wound with a 4/0 suture.</li>
	<li>Put the mice into an oxygen and temperature control workstation chamber (e.g., INVIVO2-400) to provide a warm and oxygen rich environment for the transplanted mice to recover, wait for the mice to wake up.</li>
	<li>For postoperative analgesia, directly give Metamizol 200 mg/kg per os after operation. Four and 16 hours after operation give Metamizol 200 mg/kg per os+ Carprofen (5mg/kg) s.c. In the further followup, give Carprofen (5 mg/kg) s.c to the transplanted mice every 24 hours for three consecutive days after operation.</li>
</ol>

<ol><li>Corry, R., Winn, H., Russell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primarily+vascularized+allografts+of+heart+in+mice%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Primarily vascularized allografts of heart in mice.</a> Transplantation. 16, 343-350 (1973).</li>
<li>Habertheuer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Donor+tissue-specific+exosome+profiling+enables+noninvasive+monitoring+of+acute+rejection+in+mouse+allogeneic+heart+transplantation%5BTitle%5D)+AND+%22Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Donor tissue-specific exosome profiling enables noninvasive monitoring of acute rejection in mouse allogeneic heart transplantation.</a> Journal of Thoracic and Cardiovascular Surgery. 155 (6), 2479-2489 (2018).</li>
<li>Plenter, R. J., Zamora, M. R., Grazia, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four+decades+of+vascularized+heterotopic+cardiac+transplantation+in+the+mouse%5BTitle%5D)+AND+%22Journal+of+Investigative+Surgery%22%5BJournal%5D)">Four decades of vascularized heterotopic cardiac transplantation in the mouse.</a> Journal of Investigative Surgery. 26, 223-228 (2013).</li>
<li>Wu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+technique+for+blood+circuit+reconstruction+in+mouse+heart+transplantation+model%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Novel technique for blood circuit reconstruction in mouse heart transplantation model.</a> Microsurgery. 26, 594-598 (2006).</li>
<li>Alamran, F. G., Shahkolahi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Total+arterial+anastomosis+heterotopic+heart+transplantation+model%5BTitle%5D)+AND+%22Transplantation+Proceedings%22%5BJournal%5D)">Total arterial anastomosis heterotopic heart transplantation model.</a> Transplantation Proceedings. 45, 625-629 (2013).</li>
<li>Melanie, L., et al. Potential of Heterotopic Cardiac Transplantation in Mice as a Model for Elucidating Mechanisms of Graft Rejection. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMelanie%2C+L+%22Cardiac+Transplantation%22">Cardiac Transplantation</a>. Moffatt-Bruce, S. D., et al. , IntechOpen (2012).</li>
<li>Westhofen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+heterotopic+heart+transplantation+in+mice+as+a+small+animal+model+to+study+mechanical+unloading+-+Establishment+of+the+procedure%2C+perioperative+management+and+postoperative+scoring%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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>Wang, C., Wang, Z., Allen, R., Bishop, G. A., Sharland, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Modified+Method+for+Heterotopic+Mouse+Heart+Transplantion%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A Modified Method for Heterotopic Mouse Heart Transplantion.</a> Journal of Visualized Experiments. (88), e51423(2014).</li>
<li>Mokbel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histidine-Tryptophan-Ketoglutarate+Solution+for+Donor+Heart+Preservation+Is+Safe+for+Transplantation%5BTitle%5D)+AND+%22Annals+of+Thoracic+Surgery%22%5BJournal%5D)">Histidine-Tryptophan-Ketoglutarate Solution for Donor Heart Preservation Is Safe for Transplantation.</a> Annals of Thoracic Surgery. 109 (3), 763-770 (2020).</li>
<li>Song, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modified+Suture+Technique+in+a+Mouse+Heart+Transplant+Model%5BTitle%5D)+AND+%22Asian+Journal+of+Surgery%22%5BJournal%5D)">Modified Suture Technique in a Mouse Heart Transplant Model.</a> Asian Journal of Surgery. 34 (2), 86-91 (2011).</li>
<li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+graft+function+in+rodent+models+of+heart+transplantation%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">Assessment of graft function in rodent models of heart transplantation.</a> Microsurgery. 28 (7), 565-570 (2008).</li>
<li>Wu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cold+storage+using+a+new+histidine-tryptophan-ketoglutarate-based+preservation+solution+in+isogeneic+cardiac+mouse+grafts%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">cold storage using a new histidine-tryptophan-ketoglutarate-based preservation solution in isogeneic cardiac mouse grafts.</a> European Heart Journal. 32 (4), 509-516 (2011).</li>
<li>Türk, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+of+chronic+graft+injury+with+a+new+HTK-based+preservation+solution+in+a+murine+heart+transplantation+model%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">Reduction of chronic graft injury with a new HTK-based preservation solution in a murine heart transplantation model.</a> Cryobiology. 64 (3), 273-278 (2012).</li>
</ol>

The surgical technique of heterotopic abdominal heart transplantation in mice is very challenging and this implicates a restriction to the widespread use of this model.
One of the disadvantages of the conventional technique is the limiting length of the donor&#39;s pulmonary artery (PA). It is usually of about 2 mm of length, whereas the length of the IIVC of the donor heart used in our model is generally about 1 cm (Figure 2). That means that in the modified mode...

The surgical technique of heterotopic abdominal heart transplantation in mice represents a standard model for research in transplantation immunology1,2,3. However, it is very challenging to perform and this implicates a restriction to the widespread use of this model4,5.
In traditional mouse heart transplantation (THTx), the donor aorta and the recipient abdominal aorta are anastomosed while the pulmonary artery is anastomosed to the recipient inferior vena cava6,7,8.
In this modified mouse heart transplantation technique, the donor aorta is anastomosed to the recipient abdominal aorta and the donor IIVC is anastomosed to the recipient inferior vena cava(3,4,6) (Figure 2 and Figure 3).

<table><tbody><tr><td>30G-needles</td><td>Braun</td><td>456300</td><td></td></tr><tr><td>acepromazine</td><td>CP Pharma</td><td>Tranquisol P</td><td></td></tr><tr><td>BALB/c AnNCrl mice</td><td>Charles River. Germany</td><td>no catalog number</td><td></td></tr><tr><td>Bepanthen eye ointment</td><td>Haus-Apotheke</td><td>PZN 01578675</td><td></td></tr><tr><td>Bonn Micro Forceps</td><td>FST</td><td>11083-07</td><td></td></tr><tr><td>Box for insulation and oxygen supply device</td><td>RUSKINN</td><td>INVIV</td><td></td></tr><tr><td>C57BL/6J&amp;nbsp; mice</td><td>Charles River. Germany</td><td>no catalog number</td><td></td></tr><tr><td>Carprofen</td><td>Zoetis</td><td>Rimadyl 50 mg/ml</td><td></td></tr><tr><td>CATHETER-FEP 26G</td><td>TERUMO</td><td>Surflo-W</td><td></td></tr><tr><td>Clip Applicator Forceps Style</td><td>FST</td><td>18057-14</td><td></td></tr><tr><td>Curved forceps</td><td>WPI</td><td>14114-G</td><td></td></tr><tr><td>custodiol/HTK</td><td>Dr. Franz K&amp;ouml;hler Chemie</td><td>no catalog numer</td><td></td></tr><tr><td>Cutasept skin disinfection</td><td>VWR</td><td>BODL980365</td><td></td></tr><tr><td>electrosurgical pen</td><td>Bovie</td><td>CHANGE-A-TIP</td><td></td></tr><tr><td>gauze pads, cotton swabs</td><td>Lohmann-Rauscher</td><td>13353</td><td></td></tr><tr><td>Heating mat</td><td>THERMO MAT PRO 30W</td><td>HTP-30</td><td></td></tr><tr><td>Hemostatic sponge</td><td>CuraSpon</td><td>J1276A</td><td></td></tr><tr><td>heparine-solution</td><td>Haus-Apotheke</td><td>PZN 03029820</td><td></td></tr><tr><td>Ice box</td><td>PETZ</td><td>No Catalog Number available</td><td></td></tr><tr><td>Inhalation anesthesia device</td><td>GROPPLER</td><td>BKGM 0616</td><td></td></tr><tr><td>insulation and oxygen supply device</td><td>RUSKINN</td><td>INVIV</td><td></td></tr><tr><td>isoflurane</td><td>CP Pharma</td><td>Isofluran CP 1 ml/ml</td><td></td></tr><tr><td>ketamine</td><td>Zoetis</td><td>no catalog numer</td><td></td></tr><tr><td>metamizole</td><td>WDT</td><td>no catalog numer</td><td></td></tr><tr><td>Micro scissors</td><td>FST</td><td>15000-00,15000-10</td><td></td></tr><tr><td>Micro Serrefine ( Clamp ) Angled / 16 mm</td><td>FST</td><td>18055-06</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>LEICAMZ6</td><td></td></tr><tr><td>Microscope light</td><td>SCHOTT</td><td>KL2500LED</td><td></td></tr><tr><td>Saline solution (NaCl 0.9%)</td><td>Haus-Apotheke</td><td>PZN 06178437</td><td></td></tr><tr><td>Scissors</td><td>Peha Instruments</td><td>991083/4</td><td></td></tr><tr><td>small Petri dish</td><td>Sarstedt</td><td>833900</td><td></td></tr><tr><td>Straight forceps</td><td>WPI</td><td>14113-G</td><td></td></tr><tr><td>surgical tape</td><td>BSN</td><td>4120</td><td></td></tr><tr><td>Suture Tying Forceps - 10 cm</td><td>FST</td><td>18025-10</td><td></td></tr><tr><td>Sutures(10-0)</td><td>Medtronic</td><td>N2540</td><td></td></tr><tr><td>Sutures(4-0)</td><td>ETHILON</td><td>V4940H</td><td></td></tr><tr><td>Sutures(7-0)</td><td>ETHILON</td><td>1647H</td><td></td></tr><tr><td>Syringe (0.3 mL)</td><td>BD</td><td>324826</td><td></td></tr><tr><td>Syringe (1 mL)</td><td>BD</td><td>320801</td><td></td></tr><tr><td>xylazine</td><td>Bayer</td><td>Rompun</td><td></td></tr></tbody></table>

blood circuit reconstruction in an abdominal mouse heart transplantation model

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

Here, a modified technique of heterotopic abdominal heart transplantation in mice that has been previously developed in our laboratory and has proven useful for the last 16 years is presented. Previously, it was reported that in overall 40 cases of vena cava to vena cava (V-V group) compared to 40 cases of the traditional pulmonary artery to vena cava (P-V group) anastomosis procedure4 (Table 1) the vessel anastomosis took 20.8±1.3 min in the V-V group, which was significantly sho...

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

blood-circuit-reconstruction-an-abdominal-mouse-heart-transplantation

Research

JoVE Journal

Medicine

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response.  However, the techniques involved are still challenging.  By modifying standard techniques we have had success with more than 1000 transplants.

Heterotopic Heart Transplantation in Mice

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach. Herein, we provide additional details of this technique to supplement the video.

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

A Modified Method for Heterotopic Mouse Heart Transplantion

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Murine Heterotopic Heart Transplant Technique

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotopic Cervical Heart Transplantation in Mice

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

This protocol presents a simplified, end-to-side suture anastomosis technique for murine cervical heart transplantation, providing a robust and reproducible model for studying transplant immunology.

Cervical Heterotopic Mouse Heart Transplantation using End-to-side Anastomosis Technique to Study Transplant Immunology

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

Orthotopic Liver Transplantation in Rats

Learn a novel heterotopic abdominal heart transplantation technique in mice using modified blood circuit reconstruction. This method reduces warm ischemia time and improves graft success rates for alloimmune response studies.

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

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