This work was supported by the National Health and Medical Research Council (NHMRC) of Australia Project Grant 1029205, by the Microsearch Foundation of Australia, and by the Myee Codrington Medical Research Foundation.

Prior to commencement of experiments, obtain approval from the relevant institution&#8217;s Animal Care Ethics Committee for the planned experiments. Maintain mice in the accordance with the requirements of your institution. The following protocol has been approved by Sydney University and Royal Prince Alfred Hospital committees.
Prior to commencing surgery, all instruments should be cleaned thoroughly and sterilized by soaking in 80% ethanol. Some institutions stipulate autoclaving however this can damage fine microsurgical instruments over the long term. &#160;Where possible use sterile disposable equipment.
1. Anesthesia
<ol>
	<li>Anesthetize mouse with isoflurane in a sealed container then place it supine on an operating board, quickly connecting anesthetic nose cone. Test adequacy of anesthesia by pinching the hind foot to ensure there is no reflex.</li>
	<li>Shave the skin with a surgical blade and sterilize with 80% ethanol. In addition, 2% chlorhexidine in 70% isopropyl alcohol can be used for skin sterilization.&#160;During induction of anesthesia the isoflurane concentration is 3% but reduce this to 1-1.5% for maintenance. Make minor adjustments to this concentration to maintain regular breathing and heart rate. Maintain temperature of the animal on a heating pad.</li>
</ol>
2. Donor Operation
<ol>
	<li>Make a thoracotomy to expose the heart and vessels by cutting the chest through both sides of the rib cage from the rib edge up to the axilla followed by a transverse cut at the level of the xiphisternum to form a chest wall flap. Lift this flap up beside the head and pin it to the operating board. Tear off pericardium to expose the heart and vessels.</li>
	<li>Lift the inferior vena cava with forceps in one hand and with the other inject 1 ml cold heparinized saline proximal to heart through the IVC, then put a small artery clamp on the IVC to prevent flow of perfusion solution back through the needle hole.</li>
	<li>Using gauze and a cotton bud, retract the heart downward to expose the ascending aorta and pulmonary artery. Pass one blade of a pair of microscissors through the channel (transverse sinus) posterior to the bundle of aorta and pulmonary artery and cut the aorta and pulmonary artery together as far distally as possible to ensure sufficient length for anastomosis.</li>
	<li>Tie and divide the IVC, right superior vena cava (SVC), left SVC and pulmonary veins using 6-0 silk thread. Tie the IVC and right SVC separately then place a single thread posterior to the heart to encircle the left SVC together with pulmonary veins and tie it. Harvest the heart from the donor site by cutting the vessels distal to the ties, then store it in cold sterile saline at 4 &#176;C until transplantation. This results in death of the donor by exsanguination.</li>
</ol>
3. Recipient Operation
<ol>
	<li>Anesthetize the recipient as above (section 1). Carefully shave abdomen to avoid irritation,&#160;then make a laparotomy by midline incision from pubis to xiphisternum and retract using paper clips bent to form retractors. Wrap the bowel in warm sterile saline soaked gauze and retract to the upper right of the abdomen.</li>
	<li>To expose the infra-renal aorta and IVC, free the bundled segments of aorta and IVC from the left renal artery and vein to the iliac bifurcation by dividing them from the lumbar vessels using a cautery device. Take care that the cautery is at the correct temperature and used for sufficient time to divide and seal the vessel ends.</li>
	<li>Apply small atraumatic vessel clamps to the aorta and IVC segments distally and proximally. Make an incision in the front wall of the aorta by first puncturing a hole with a 30 G&#160;needle; then cut an incision vertically with microscissors to match the size of the donor aorta. Flush the aorta lumen with heparinized saline to remove any blood clots.</li>
	<li>Bring the donor heart to the recipient site covered with gauze soaked in cold saline and place it to the right side of the abdomen. Make sure the donor aorta is positioned next to the incision on the recipient aorta and the donor pulmonary artery positioned next to the recipient IVC.</li>
	<li>Anastomose the donor aorta end-to-side to the recipient aorta using running 10-0 nylon sutures starting from the proximal corner and suturing along the left side first until reaching the distal corner, at which stage the animal is rotated through 180&#176;. Gently move the donor heart to the left side of the abdomen to expose the right side of the aorta and continue suturing through the right side of the aortic wall from the distal end to the proximal end. Before closing the aortic anastomosis, gently flush the lumen with heparinized saline to remove any clot and air.</li>
	<li>Anastomose the donor pulmonary artery (PA) end-to-side with the recipient IVC. Make an incision vertically with microscissors in the front wall of the IVC at a site in accordance with the artery anastomosis. Anastomose the donor PA to recipient IVC with running 10-0 nylon sutures starting from the distal end of the left wall within the lumen of the IVC. After reaching the proximal end, continue sutures along the front right side wall up to the distal end to complete the anastomosis. Before closing the anastomosis, gently flush the lumen to remove any clots and air.</li>
	<li>Before releasing the vessel clamps, place pieces of Gelfoam around the anastomosis sites, and apply gentle pressure with a cotton applicator until hemostasis is achieved. At the time of revascularization, release the distal clamp first, followed by the proximal clamp.</li>
	<li>After revascularization, apply warm saline at 37 &#176;C to the graft externally to assist its recovery. The graft usually starts fibrillation immediately and reverts spontaneously to sinus rhythm within a few minutes. Inject 0.6 ml warm saline subcutaneously to maintain hydration of the recipient. Inject buprenorphine subcutaneously for analgesia prior to completion of surgery</li>
	<li>Close the abdominal wound with one 5-0 absorbable running suture for&#160;both the layers. Start by completing the inner layer and continue along the skin.</li>
</ol>
4. Recovery and Graft Monitoring
<ol>
	<li>Inject ampicillin for prophylaxis of infection and place the recipient on a heating pad at 37 &#176;C for recovery. Most animals recover rapidly and are usually drinking and often eating within 3 hr. If the mouse shows signs of distress, examine closely to determine cause. If no obvious cause, treat with buprenorphine and monitor closely. Consult a veterinarian if symptoms are severe or persist more than 8 hours and consider euthanasia. If necessary, give continuing 12&#160;hourly injections of buprenorphine until symptoms resolve.&#160;If the mouse shows signs of distress after 8 hr, give continuing 12 hourly injections of buprenorphine until they resolve. Consult the veterinarian if symptoms continue for more than 48 hr and euthanize if necessary.</li>
	<li>Monitor graft heart beat by direct abdominal palpation and record the strength of beat as ++++ for a healthy graft to + for weak beat due to advanced rejection and &#8211; as non beating due to complete rejection of the graft. Monitor mice daily for the first 10 days, then 3x per week for the duration of the experiment.</li>
</ol>

<ol>
	<li>Aramaki, 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=Interleukin-10+but+not+transforming+growth+factor-beta+is+essential+for+generation+and+suppressor+function+of+regulatory+cells+induced+by+intratracheal+delivery+of+alloantigen.">Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen.</a> Transplantation. 79, 568-576 (2005).</li><li>Chen, R. H., Bushell, A., Fuggle, S. V., Wood, K. J., Morris, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+granzyme+A+and+perforin+in+mouse+heart+transplants+immunosuppressed+with+donor-specific+transfusion+and+anti-CD4+monoclonal+antibody.">Expression of granzyme A and perforin in mouse heart transplants immunosuppressed with donor-specific transfusion and anti-CD4 monoclonal antibody.</a> Transplantation. 61, 625-629 (1996).</li><li>Poulin, L. F., 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=Interleukin-9+promotes+eosinophilic+rejection+of+mouse+heart+allografts.">Interleukin-9 promotes eosinophilic rejection of mouse heart allografts.</a> Transplantation. 76, 572-577 (2003).</li><li>Ono, K., Lindsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+of+heart+transplantation+in+rats.">Improved technique of heart transplantation in rats.</a> J. Thorac. Cardiovasc. Surg. 57, 225-229 (1969).</li><li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+heart+in+mice.">Primarily vascularized allografts of heart in mice.</a> Transplantation. 16, 343-350 (1973).</li><li>Larsen, C. 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=CD40-gp39+interactions+play+a+critical+role+during+allograft+rejection.+Suppression+of+allograft+rejection+by+blockade+of+the+CD40-gp39+pathway.">CD40-gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway.</a> Transplantation. 61, 4-9 (1996).</li><li>Saitovitch, D., Bushell, A., Mabbs, D. W., Morris, P. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+induction+of+transplantation+tolerance+with+a+nondepleting+anti-Cd4+monoclonal+antibody+and+donor-specific+transfusion+before+transplantation.+A+critical+period+of+time+is+required+for+development+of+immunological+unresponsiveness.">Kinetics of induction of transplantation tolerance with a nondepleting anti-Cd4 monoclonal antibody and donor-specific transfusion before transplantation. A critical period of time is required for development of immunological unresponsiveness.</a> Transplantation. 61, 1642-1647 (1996).</li><li>Callaghan, C. 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=Regulation+of+allograft+survival+by+inhibitory+FcgammaRIIb+signaling.">Regulation of allograft survival by inhibitory FcgammaRIIb signaling.</a> J. Immunol. 189, 5694-5702 (2012).</li><li>Qian, 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=Impact+of+donor+MHC+Class+I+or+Class+II+antigen+deficiency+on+first-+and+second-set+rejection+of+mouse+heart+or+liver+allografts.">Impact of donor MHC Class I or Class II antigen deficiency on first- and second-set rejection of mouse heart or liver allografts.</a> Immunology. 88, 124-129 (1996).</li><li>Wang, C., 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=Spontaneous+acceptance+of+mouse+kidney+allografts+is+associated+with+increased+Foxp3+expression+and+differences+in+the+B+and+T+cell+compartments.">Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments.</a> Transpl. Immunol. 24, 149-156 (2011).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Heart+Transplantation+in+Mice.">Heterotopic Heart Transplantation in Mice.</a> J. Vis. Exp. , (2007).</li><li>Mottram, P. L., 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=Electrocardiographic+monitoring+of+cardiac+transplants+in+mice.">Electrocardiographic monitoring of cardiac transplants in mice.</a> Cardiovasc. Res. 22, 315-321 (1988).</li></ol>

The authors have nothing to disclose.

Mouse heart transplantation is a challenging microsurgical method that requires considerable surgical skill to master. The most challenging aspect is the small diameter of the vessels. In addition, it is necessary to limit the recipient operation time and bleeding. The technique for mouse heart transplantation was first described by Corry et al. in 1973 and subsequently by Mottram et al12. Our modifications include the following points. Firstly, immediate perfusion of the donor heart with col...

Animal models of organ transplantation can provide valuable information for improving treatment of clinical transplant patients. Mouse models are particularly useful for studying the immune mechanism of organ transplant rejection or acceptance due to the wide range of genetically modified mice and reagents specific for mice that are not available for other animal models1-3. A challenge with mouse models of transplantation is the small size of the donors and recipients which requires considerable technical skill to achieve satisfactory outcomes.
A technique was first described for heterotopic transplantation of hearts in rats4 which was subsequently adapted for mouse heart transplantation by Corry et al5. This technique involved preparing the recipient prior to the donor operation and no perfusion of the donor heart, both of which are likely to compromise survival of the transplanted heart or the recipient. The procedure has been widely used as originally described to examine the mechanisms of transplant rejection and tolerance6-8. Others have adapted the original rat heart transplant procedure of Ono and Lindsey for heart transplants in mice9,10. More recently, a technique was published for mouse heart transplantation that overcame some of the problems associated with the method of Corry et al11. The protocol described here incorporates our modifications, based on the method of Mottram et al12, which include: in situ perfusion with cold heparinized saline immediately after thoracotomy and performing the donor operation prior to the recipient operation to minimize recipient operation time. In addition, we use small atraumatic vessel clamps instead of 6-0 silk ties. Although vessel clamps have the disadvantage of taking more space they are easier to control than ties, which should not be too tight or loose and are less straightforward to remove than clamps. Our method uses a single running suture for vessel anastomosis, although in the initial learning stages an alternative is to use stay sutures at the proximal and distal corners to ensure evenness of sutures and thus patency of the anastomosis.

<table border="0" cellpadding="0" cellspacing="0" width="831">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Operating microscope&#160;</td>
			<td style="width:247px;">Leica, Heerbrugg, Switzerland</td>
			<td style="width:124px;">M651</td>
			<td style="width:167px;">10x - 25x magnification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthetic machine&#160;</td>
			<td>Vet Quip Pty Ltd, Sydney, Australia</td>
			<td style="width:124px;">Vett3</td>
			<td>Capable of delivering a mixture of isoflurane and oxygen in air&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Operating board&#160;</td>
			<td style="width:247px;">Hardware store or office supplier</td>
			<td style="width:124px;"></td>
			<td>Dense cork or synthetic capable of taking pins</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:293px;">Heparinised saline (5 U/ml, 4oC)</td>
			<td>Pfizer, USA</td>
			<td style="width:124px;">FW25</td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:293px;">Normal saline (37oC)</td>
			<td style="width:247px;">AstraZeneca Pty Ltd, Australia</td>
			<td style="width:124px;">4538</td>
			<td>In 1ml syringe with 23 gauge needle on warm pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Sutures 10- nylon, 5-0 Vicryl</td>
			<td>Ethicon, Inc. NJ, USA</td>
			<td style="width:124px;">2870G/J421H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine (0.05mg/kg in 0.1ml saline)</td>
			<td>Reckitt Benckiser, Sydney, Australia</td>
			<td style="width:124px;"></td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampcillin (5mg/kg in 0.1 ml saline)&#160;</td>
			<td>Aspen, Sydney, Australia</td>
			<td style="width:124px;"></td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Foam&#160;</td>
			<td>Pharmacia &#38; Upjohn Co. USA</td>
			<td style="width:124px;">801289304</td>
			<td>Cut into small pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High temperature cautery device&#160;</td>
			<td>Medtronic, USA</td>
			<td style="width:124px;">8444000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microsurgery instruments:</td>
			<td colspan="2">Shanghai Medical Instruments Co. Ltd.,&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-needleholders</td>
			<td style="width:247px;">Shanghai, China</td>
			<td style="width:124px;">WT2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro scissors</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WT1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-forceps (straight tip)</td>
			<td style="width:247px;">&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WA3010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-forceps (curved tip)</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WA3020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-mosquito clamps (1 pair)</td>
			<td style="width:247px;">&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">W40350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-vessel atraumatic clamps (1 pair)</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">W40130/W40150</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Heating Pad/Right Temp</td>
			<td style="width:247px;">Kent Scientific Corporation, Turrington, CT 06790</td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified method for heterotopic mouse heart transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

After an initial training period, 227 cases of mouse heterotopic heart transplantation in our group were analyzed. The success rate within the first 24 hr was 90.3% and at 48 hr it was 86.8%. Of the 30 (13.2%) failures within 48 hr, 5 (2.2%) experienced hind limb paralysis and had to be euthanized, 10 (4.4%) had a non-beating heart due to graft ischemia injury and/or thrombosis, while 15 (6.6%) died within 48 hr. Some experimental graft survivals are shown in Table 1 with a variety of strain combinations...

Watch this Scientific Journal Video about A Modified Method for Heterotopic Mouse Heart Transplantion at JoVE.com

A Modified Method for Heterotopic Mouse Heart Transplantion

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

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

a-modified-method-for-heterotopic-mouse-heart-transplantion

Research

JoVE Journal

Medicine

12.0K 조회수.  University of Sydney. 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.

비디오: A Modified Method for Heterotopic Mouse Heart Transplantion

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

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

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

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

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

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

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

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

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

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

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

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

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

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

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

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

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

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

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

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 transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

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 ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

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

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

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