M.-A. Deutsch is supported by Dr. Rusche Forschungsprojekt (2014) Deutsche Stiftung f&#252;r Herzforschung and Deutsche Gesellschaft f&#252;r Thorax- Herz- und Gef&#228;&#223;chirurgie. M. Krane is supported by Deutsche Stiftung f&#252;r Herzforschung (F/37/11), Deutsches Zentrum f&#252;r Herz Kreislauf Forschung (DZHK B 13-050A; DZHK B 14-013SE) and Deutsche Forschungsgemeinschaft (KR3770/7-1; KR3770/9-1)

All experiments were approved by the German Heart Center Munich and the Government of Bavaria, Germany (Gz. 55.2-1-54-2532.3-17-13). Animals were housed in a specific pathogen free facility and received human care in compliance with the &#8221;Principles of Laboratory Animal Care&#8221; formulated by the National Society for Medical Research and the &#8221;Guide for the Care and Use of Laboratory Animals&#8221; provided by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication no. 86-23, revised 1985). 
NOTE: A complete set of sterile microsurgical instruments, including two curved forceps and vessel dilatators, is necessary for the procedure (Figure 2a). Perioperative antibiotic prophylaxis is not routinely necessary but may be indicated in immunocompromised animals. Animals were sacrificed using terminal isoflurane inhalation.
Donor Preparation and Graft Harvest
<ol>
	<li>Anesthetize the animal by intraperitoneal (i.p.) injection of midazolam (5.0 mg/kg), medetomidine (0.5 mg/kg) and fentanyl (0.05 mg/kg body weight). Place the mouse in a supine position on the operative field and confirm surgical tolerance by the pedal withdrawal reflex.</li>
	<li>Disinfect the skin three times with chlorhexidine and perform a midline abdominal incision. Retract the intestines with wet gauze to the left to expose the inferior vena cava.</li>
	<li>Inject 50 IU of heparin diluted in 0.4 ml saline through the inferior vena cava for anticoagulation. Incise the vessel 1 min after heparinization.</li>
	<li>Open the diaphragm and perform a bilateral thoracotomy to expose the heart. The anterior chest wall is reflected superiorly and held in place with a clamp.</li>
	<li>Remove the thymus and bluntly dissect the connective tissue between aorta and pulmonary trunk. Incise the right superior vena cava.</li>
	<li>Puncture the mobilized ascending aorta at the level of the brachiocephalic trunk with a 30 G needle mounted on a 5 ml syringe and slowly perfuse the heart retrogradually without excessive pressure with 3 ml of 4 &#176;C cold cardioplegic solution. Avoid the entry of air into the coronary circulation. Drop ice-cold saline solution onto the graft to further enhance cardiac arrest.</li>
	<li>Transect the aorta proximally to the origin of the brachiocephalic trunk.</li>
	<li>Ligate the inferior and both superior venae cavae close to the heart with 8-0 silk and transect them distally to the ligatures.</li>
	<li>Dissect free the pulmonary trunk as distally as possible. Ligate the pulmonary arteries close to the bifurcation with 8-0 silk and transect them distally.</li>
	<li>Ligate the pulmonary veins all together with a single 8-0 silk ligature and transect them.</li>
	<li>Remove the heart from the donor and store it in ice-cold cardioplegic solution until implantation.</li>
</ol>
2. Recipient Preparation
<ol>
	<li>Prepare the cuffs for anastomoses by cutting the polyimide tubing with a No. 11 scalpel&#160;or a scissor&#160;under the microscope. The cuff consists of a cylindrical body and an extension (1/3 of the circumference) for the placement of the vascular clamp, each of a length of 1 mm (Figure 2b).</li>
	<li>Anesthetize the animal and confirm surgical tolerance as described above. Inject 0.25 ml warm saline solution subcutaneously (s.c.) for fluid replacement.</li>
	<li>Remove the hair of the right cervical region using an electric shaver and place the animal in a supine position on a warm pad. Secure the legs with strands of tape while avoiding overstretching the front limbs as this can compromise respiration. Cover the eyes using ophthalmic ointment to prevent dryness. Disinfect the surgical site three times with chlorhexidine.</li>
	<li>Make a transverse skin incision from the right mandibular angle to the jugular notch.</li>
	<li>Bluntly mobilize the external jugular vein. Cauterize side branches with a bipolar forceps and transect them.</li>
	<li>Divide the external jugular vein between ligatures at the level of the confluence with the vein from the submandibular gland.</li>
	<li>Pull the external jugular vein through the cuff and occlude the vessel proximally by placing a vascular clamp on the extension of the cuff. Remove the ligature and irrigate the vessel lumen with 1:10 heparinized saline.</li>
	<li>Evert the vessel wall over the cuff and fix it with a preset circumferential 8-0 silk ligature.</li>
	<li>Remove the superficial part of the sternocleidomastoid muscle.</li>
	<li>Mobilize the right common carotid artery and transect the vessel between ligatures below the carotid bifurcation.</li>
	<li>Pull the carotid artery through the cuff and occlude the vessel proximally by placing a Yasargil clamp on the extension of the cuff. Remove the ligature and irrigate the vessel lumen with 1:10 heparinized saline.</li>
	<li>Gently dilate the arterial lumen with a vessel dilatator, evert the vessel wall over the cuff and fix it with a preset circumferential 8-0 silk ligature.</li>
	<li>To gain enough space for graft implantation, remove the right lobe of the submandibular gland.</li>
	<li>Keep the operative field moist until implantation.</li>
</ol>
3. Graft Implantation
<ol>
	<li>Place the graft in the recipient cervical region upside down with the aorta orientated medially and the pulmonary artery laterally.</li>
	<li>Pull the aorta of the graft over the cuff with the carotid artery and secure the anastomosis with a circumferential 8-0 silk ligature.</li>
	<li>Incise the pulmonary trunk on its anterior aspect and pull it over the cuff with the external jugular vein. Again secure the anastomosis with a circumferential 8-0 silk ligature.</li>
	<li>Remove the clamp on the external jugular vein, following by unclamping of the carotid artery. The hearts fills immediately with blood. To enhance reperfusion drop 37 &#176;C warm saline solution onto the graft. Normally the heart develops sinus rhythm within one minute. Remove the cuff extension at the venous site.</li>
	<li>Ensure correct graft position, anastomoses must be tension-free and vessels without torsion. Do not cauterized vessels of the skin since this may impair wound healing. Bleeding usually stops spontaneously. Close the skin with a single 8-0 continuous suture.</li>
</ol>
4. Postoperative Care
<ol>
	<li>Inject 0.25 ml warm saline solution s.c. for fluid replacement.</li>
	<li>To antagonize narcotics inject naloxone (1.2 mg/kg), flumazenil (0.5 mg/kg) and atipamezol (2.5 mg/kg body weight) s.c. Left the animal on the warm pad until it has regained sufficient consciousness to maintain sternal recumbency. Observe the animal for at least one additional hour to register any abnormal behavior. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>Provide preemptive analgesia with buprenorphine (0.05 mg/kg s.c. three times per day) or an alternative analgesia according to a local institutional protocol for a minimum duration of 72 hr after the procedure.</li>
	<li>The surgeon and a veterinarian should see animals regularly. In case of weight loss &#62;15%, apathy, swelling or surgical side infection animals are euthanized using terminal isoflurane inhalation.</li>
</ol>

<ol>
	<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=Heart+transplantation+in+congenic+strains+of+mice.">Heart transplantation in congenic strains of mice.</a> Transplant Proc. 5, 733-735 (1973).</li><li>Chen, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+of+cervical+heterotopic+heart+transplantation+in+mice.">A technique of cervical heterotopic heart transplantation in mice.</a> Transplantation. 52, 1099-1101 (1991).</li><li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+graft+function+in+rodent+models+of+heart+transplantation.">Assessment of graft function in rodent models of heart transplantation.</a> Microsurgery. 28, 565-570 (2008).</li><li>Schramm, R., 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+subepicardial+microcirculation+in+heterotopically+transplanted+mouse+hearts:+an+intravital+multifluorescence+microscopy+study.">The subepicardial microcirculation in heterotopically transplanted mouse hearts: an intravital multifluorescence microscopy study.</a> J Thorac Cardiovasc Surg. 134, 210-217 (2007).</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 : JoVE. 238, (2007).</li><li>Gong, W., 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=Introduction+of+modified+cervical+cardiac+transplant+model+in+mice.">Introduction of modified cervical cardiac transplant model in mice.</a> Exp Clin Transplant. 10, 158-162 (2012).</li><li>Matsuura, A., Abe, T., Yasuura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+mouse+cervical+heart+transplantation+using+a+cuff+technique.">Simplified mouse cervical heart transplantation using a cuff technique.</a> Transplantation. 51, 896-898 (1991).</li><li>Chen, H., Zhang, Y., Zheng, D., Praseedom, R. K., Dong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+kidney+transplantation+in+mice:+technique+using+cuff+for+renal+vein+anastomosis.">Orthotopic kidney transplantation in mice: technique using cuff for renal vein anastomosis.</a> PloS one. 8, e77278 (2013).</li><li>Maglione, 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=A+novel+technique+for+heterotopic+vascularized+pancreas+transplantation+in+mice+to+assess+ischemia+reperfusion+injury+and+graft+pancreatitis.">A novel technique for heterotopic vascularized pancreas transplantation in mice to assess ischemia reperfusion injury and graft pancreatitis.</a> Surgery. 141, 682-689 (2007).</li><li>Sucher, R., 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> J Vis Exp : JoVE. , (2010).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47-50 (1979).</li><li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+rats.">Orthotopic liver transplantation in rats.</a> J Vis Exp : JoVE. , (2012).</li><li>Okazaki, 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=A+mouse+model+of+orthotopic+vascularized+aerated+lung+transplantation.">A mouse model of orthotopic vascularized aerated lung transplantation.</a> Am J Transplant. 7, 1672-1679 (2007).</li><li>Suzuki, H., Fan, L., Wilkes, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+obliterative+bronchiolitis+in+a+murine+model+of+orthotopic+lung+transplantation.">Development of obliterative bronchiolitis in a murine model of orthotopic lung transplantation.</a> J Vis Exp : JoVE. , (2012).</li><li>Zou, 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=Mouse+model+of+venous+bypass+graft+arteriosclerosis.">Mouse model of venous bypass graft arteriosclerosis.</a> Am J Pathol. 153, 1301-1310 (1998).</li><li>Zhou, Y., Gu, X., Xiang, J., Qian, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+suture+versus+cuff+anastomosis+in+mouse+cervical+cardiac+transplant.">A comparative study on suture versus cuff anastomosis in mouse cervical cardiac transplant.</a> Exp Clin Transplant. 8, 245-249 (2010).</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> J Heart Lung Transplant. 20, 1123-1128 (2001).</li><li>Arras, M., Autenried, P., Rettich, A., Spaeni, D., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intraperitoneal+injection+anesthesia+in+mice:+drugs,+dosages,+adverse+effects,+and+anesthesia+depth.">Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.</a> Comp Med. 51, 443-456 (2001).</li></ol>

The authors have nothing to disclose.

We have completed over 200 cardiac transplants with a success rate over 90% in various experimental settings. With the technique described here, the complete procedure can be done within 60 min by an experienced surgeon. By preparing the cervical vessels before organ procurement, total graft ischemia time can be limited to 20 min. Time for implantation (warm ischemia time) is significantly shorter using the cuff technique compared to the suture technique16.
Instead of simply excisin...

The heart transplantation model is frequently used to investigate I/R-injury, allograft rejection, post-transplant vasculopathy and the efficiency of immunomodulating agents. Mouse models combine many advantages like the known immunological and genetic backgrounds, the availability of many inbred and transgenic strains and relatively low experimental costs.
In 1973, a technique of abdominal heart transplantation in mice was first described by Corry et al1. In this model grafts are revascularized by end-to-side anastomoses of the ascending aorta to the recipient&#8217;s abdominal aorta and of the main pulmonary artery to the inferior vena cava. In 1991, a suture technique for cervical heart transplantation in mice was described by Chen et al2. In this model a retrograde coronary artery perfusion is established by anastomosis of the recipient&#8217;s carotid artery to the ascending aorta of the graft. The venous blood drains via the coronary sinus into the right atrium, the right ventricle and is ejected through the pulmonary artery into the recipient external jugular vein (Figure 1). In comparison to the abdominal graft implantation the cervical model is less invasive and the superficial location of the graft enables a very easy accessibility for additional follow-up examinations (e.g. palpation, echocardiography, intravital fluorescence microscopy)3,4.
Despite technical improvements5,6, a more widespread use of this model has been limited due to technical difficulties to perform sutured anastomoses of small vessels with a high incidence of anastomosis leakage and thrombosis. In 1991, Matsuura et al. introduced the cuff technique in which the vessel wall is everted over a synthetic cylinder, which greatly facilitates the model of cervical heart transplantation7. The technique was further adopted for various experimental transplant models including renal8, pancreatic9, limb10, hepatic11,12, lung13,14 and vascular transplantation15. In comparison to the suture technique the required time for anastomoses and, thus, the warm ischemia time is shorter and there are significantly less vascular complications using the cuff technique16. Moreover, it enables surgeons with little training in microsurgery to perform the operation with a high success rate. A disadvantage of the cuff technique is the prerequisite ligation of the common carotid artery, which may alter cerebral perfusion.
In this video paper we present an improved and simplified cuff technique for cervical heart transplantation in mice.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps JFC-7.2</td>
			<td>S&#38;T</td>
			<td>P-00036</td>
			<td>Curved tip 0.20 mm</td>
		</tr>
		<tr>
			<td>Vessel dilatator D-5a</td>
			<td>S&#38;T</td>
			<td>S-00124</td>
			<td></td>
		</tr>
		<tr>
			<td>Adventita scissor</td>
			<td>S&#38;T</td>
			<td>S-00102</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamp B-1V</td>
			<td>S&#38;T</td>
			<td>S-00396</td>
			<td></td>
		</tr>
		<tr>
			<td>Yasargil Clip</td>
			<td>Peter Lazic</td>
			<td>65,097</td>
			<td>3.5 mm lenght</td>
		</tr>
		<tr>
			<td>Bipolar forceps</td>
			<td>Micromed</td>
			<td>148-100-011</td>
			<td>tip 0.25 mm</td>
		</tr>
		<tr>
			<td>Polyimide tubes</td>
			<td>River Tech Medical</td>
			<td></td>
			<td>19-24 gauge</td>
		</tr>
		<tr>
			<td>8.0 silk ligatures</td>
			<td>Catgut</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol HTK solution</td>
			<td>Essential Pharmaceuticals</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Our team could successfully perform the described technique after approximately 20 training surgeries. The most challenging part is to evert the vessel wall of the carotid artery over the cuff, because it easily flips back and multiple attempts may lead to tearing of the wall. After the training period, the mean total operative time was 60 &#177; 8 min. Around 20 min are required for donor harvest, 25 min for preparation of the cervical vessels and 15 minutes for anastomoses and skin closure.
...

Watch this Scientific Journal Video about Heterotopic Cervical Heart Transplantation in Mice at JoVE.com

Heterotopic Cervical Heart Transplantation in Mice

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 ...

heterotopic-cervical-heart-transplantation-in-mice

Research

JoVE Journal

Medicine

11.1K 조회수.  German Heart Center Munich, Technische Universität München. 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.

비디오: Heterotopic Cervical Heart Transplantation in Mice

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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 ...

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.

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 ...

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 ...

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 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 ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

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.

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...