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Ann Thorac Surg 1999;68:265-268
© 1999 The Society of Thoracic Surgeons

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Technique for heterotopic pig heart xenotransplantation in primates

^a Primate Xenotransplantation Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

Address reprint requests to Dr Adams, Division of Cardiac Surgery, Brigham & Women’s Hospital, 75 Francis St, Boston, MA 02115;
e-mail: dhadams{at}bics.bwh.harvard.edu

	Abstract

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The primate is a commonly utilized model for the human immune response after heart transplantation. This report describes our experience with heterotopic abdominal transplantation of porcine hearts into primate recipients. Abdominal graft placement was surprisingly well tolerated, and we found this approach to be particularly useful in the setting of significant donor-recipient size mismatch. Continuous monitoring with an implantable monitoring system facilitated assessment of graft viability in awake recipients; progressive graft bradycardia and decreasing QRS amplitude were predictive of ensuing graft failure.

	Introduction

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Heart transplantation has emerged as the treatment of choice for patients with irreversible heart failure. Due to the shortage of human donors, much interest is now directed toward pigs as a possible source. Pigs grow to human size rapidly and are easy to raise in large numbers. Importantly, genetic manipulation of pig donors is possible, and organs from transgenic pigs expressing human complement regulatory proteins have been shown to be protected against hyperacute rejection in primates [1]. Continued studies with these organs are necessary in order to understand the immunological barriers to be overcome before clinical application.

Heterotopic heart transplantation offers advantages over the orthotopic technique in the investigation of graft failure as well as in the primary development of immunologic strategies [2–4]. Heterotopic engraftment is easier to perform, and serial biopsies and graft explantation with animal survival is possible. Previous reports detailing heterotopic heart transplantation in primates have described graft preparation with partial resection of the mitral valve, creation of an atrial septal defect, and isolated closure of each cava and left atrial free wall [5], with graft implantation in either the neck [5] or the iliac fossa [6]. In this report, we now summarize modifications employed in a pig to primate model. To simplify donor preparation, we applied the technique of inflow exclusion with a simple tie originally described by Ono and Lindsey in the rat model [3], avoiding intragraft manipulation. To accommodate donor-recipient size mismatch, we utilized abdominal positioning. An implantable telemetric monitoring system was employed to follow graft function in awake primates. We have been satisfied with this approach, and have not encountered serious complications attributable to the abdominal placement of grafts.

	Material and methods

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Animals
Olive baboons (Papio anubis) (10 to 30 kg) served as transplant recipients. Donors (5 to 15 kg) were either wild-type or transgenic pigs constitutively expressing human complement regulatory proteins, including CD59, DAF, and/or MCP (Nextran, Princeton, NJ). All animals received humane care in accordance with the guidelines of the Harvard University Animal Care Committee, and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources as published by the National Institutes of Health (NIH publications No. 86-23, revised 1985).

Anesthesia and intraoperative monitoring
Baboons and pigs were sedated with ketamine hydrochloride (10 mg/kg im) and telazol (5 mg/kg im), respectively. Anesthesia was induced with inhalational isoflurane (1.3%–2.0%), and each animal was supported on a veterinary ADS 1000 pressure-controlled ventilator (Engler Engineering, Hialeah, FL). A central line was placed in the right internal jugular vein, and blood pressure was monitored using an extremity pressure cuff.

Recipient preparation
A low midline abdominal incision was made in the baboon, and the small bowel and colon were packed and retracted under the superior border of the incision. The aorta and inferior vena cava (IVC) were identified. Vessel loops were placed around the dissected aorta above the bifurcation. Posterior lumbar arteries were identified and clipped. The corresponding IVC segment was similarly dissected and looped. Posterior venous branches were typically present and controlled with a third vessel loop.

Cardiac harvest
Median sternotomy was performed in the pig, and the pericardial sac was incised to expose the heart. After heparin bolus (100 IU/kg, iv), the heart was retracted inferiorly, and the superior vena cava (SVC) and the aorta at the level of the inominate artery were divided. The heart was then retracted superiorly-anteriorly, and the IVC, hemiazygos vein, pulmonary artery (PA), and pulmonary veins were divided blindly at the level of pericardial reflection. The heart was transferred to a 4°C normal saline slush bath. The aorta and the pulmonary artery were quickly separated. Cold cardioplegia (80 mL/kg) (dextrose 2.5%, sodium chloride 0.45%, potassium 30 meq/L, bicarbonate 5 meq/L) was infused with an 18-gauge catheter after aortic clamping. The aorta and the PA were then trimmed to approximately 1 cm above the valve commissures. The pulmonary veins, SVC, and IVC were identified, and a 2-0 silk suture was used to doubly circumscribe and ligate both atria such that all inlet vessels were excluded (Fig 1A). Occasionally, the SVC was ligated with a separate simple silk suture. The heart was left in 4°C normal saline bath.

View larger version (22K): [in this window] [in a new window]   Fig 1. (A) The SVC, IVC, hemiazygous and pulmonary veins were identified by forceps, and a silk ligature was used to ligate vascular inlets. (B) The pulmonary artery was sutured end-to-side to the IVC, while the ascending aorta was sutured in a similar fashion to the abdominal aorta.

Closure and graft monitoring
We developed a practice of carefully placing the abdominal contents around the heart in order to provide support for the graft and to minimize bowel kinking. The abdominal fascia was closed with running #2 Ethilon sutures, and the skin was closed with subcuticular 4-0 Vicryl sutures.

Recipients were originally sedated daily to assess graft viability by palpation. More recently, we have employed an implantable telemetric system (Data Science International, St. Paul, MN) to continuously follow graft cardiac rhythm as an indicator of viability in awake recipients. Monitoring epicardial leads were sutured on the left ventricle with Prolene 7-0 sutures, and the indwelling telemetric console was sutured inside the peritoneal wall with 3-0 silk sutures. Grafts were biopsied through the previous abdominal incision routinely every third or fourth day or when there was evidence suggesting deterioration of graft function.

	Results

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We have performed 22 pig-to-baboon heterotopic heart transplants in the abdominal cavity. Graft preparation time varied between 5 and 10 minutes and implantation time varied between 20 and 30 minutes. Grafts resumed sinus rhythm with vigorous contraction after reperfusion. Wild-type pig hearts all hyperacutely rejected after 20 to 80 minutes and were explanted intraoperatively. Transgenic pig heart survival ranged from 60 minutes to 16 days, with an average survival of 5.25 days. Early grafts were monitored by palpation, and the last 13 transplants were monitored by continuous telemetry. Progressive bradycardia and decreasing QRS amplitude were predictive of imminent graft failure (Figs 2A, 2B, and 2C). Rejected grafts underwent explantation and selected recipients were survived for posttransplant studies.

View larger version (14K): [in this window] [in a new window]   Fig 2. Continuous telemetric monitoring of abdominal heart xenotransplants: (A) immediately after implantation, the heart was in a tachycardiac, narrow complexed rhythm; (B) on postoperative day 6, the heart rate was slower and demonstrated broadening and decreasing amplitudes of the QRS complexes; and (C) on postoperative day 7, bradycardia with low QRS amplitudes correlated with rejection of the graft.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
Heterotopic heart transplantation is commonly used to assess immunologic modulation and response. Iliac graft positioning has been described for use in small primates less than 2 kg [6]. The technique of cervical implantation in primate-to-primate transplantation described by Michler and associates [5] is ideal for appropriately sized adult primate recipients (> 12 kg) and pig donors (< 6 kg). We have used this technique successfully and would recommend it in appropriate size-match combinations. It is technically easier to perform than abdominal implantation, subsequent visual assessment of graft function is possible, and graft accessibility for biopsies is simplified.

Unfortunately, donor pigs grow rapidly at young ages, and we frequently encountered considerable pig donor-primate recipient size disparity. Our concern for possible tracheal compression and inability to close cervical wounds led us to abdominal cardiac graft positioning. Even though postoperative ileus and bowel obstructions were potential concerns, we did not encounter these problems in our recipients. Baboons resumed feeding almost immediately after surgery. Occasionally, animals exhibited temporary anorexia. We never encountered prolonged ileus, and all recipients maintained intake and excretory function. Our only technical complication occurred early in our experience and involved a torn aortic anastomosis when the baboon recipient assumed a vertical position, probably due to the downward displacement of abdominal contents onto the graft. We have not had any further problem since we began carefully distributing the bowels around the graft before closure.

In an attempt to simplify graft preparation, we discontinued the practice of intragraft shunt creation [5], and have not been disappointed with graft performance. Previously, we used running Prolene sutures to close each separate right and left atrial inflow site. We have found tying off the majority of atrial tissue, including inflow sites, with simple silk sutures to be quick and hemostatic in our pig hearts.

One major disadvantage associated with abdominal implantation was difficulty with graft monitoring. To palpate the graft, we had to repeatedly sedate recipients. Recently, with the use of implantable telemetry, we were able to continuously monitor the graft in awake baboons. This advance has made the abdominal approach a much more practical option for primate xenotransplantation. We observed that graft failure was invariably preceded by progressive bradycardia and decreasing amplitude of the QRS complex. Consequently, the monitoring system was helpful in guiding us regarding the time of open exploration and biopsy.

	Acknowledgments

 
Dr Chen is an American College of Surgeons Research Scholar 1998–2000 and recipient of National Institutes of Health Individual National Research Service Award (NRSA) 1F32HL0996601. This work was supported by a Nextran, Inc,-sponsored research grant. Primate quarantine work was supported by the New England Primate Research Center grant P51RR00168-37.

We would like to thank Matthew Carty and Dean Santerre for technical support and John Logan, PhD and Lisa Diamond, PhD for their continued collaboration.

	References

Top Abstract Introduction Material and methods Results Comment References

 

1. Chen R.H., Naficy S., Logan J.S., Diamond L.E., Adams D.H. Hearts from transgenic pigs constructed with CD59/DAF genomic clones demonstrate improved survival in primates. Xenotransplantation 1999;6:122-132.
2. Mann F.C., Priestly J.T., Markowitz J., Yater W.M. Transplantation of the intact mammalian heart. Arch Aurg 1933;26:219-224.
3. Ono K., Lindsey E. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969;57:225-229.[Medline]
4. Jamieson S.W., Burton N.A., Shumway N.E. A new method for heterotopic cardiac transplantation in the dog. J Surg Res 1982;32:150-153.[Medline]
5. Michler R., McManus R., Smith C., Sadeghi A., Rose E. Technique for primate heterotopic cardiac xenotransplantation. J Med Primatol 1985;14:357-362.[Medline]
6. Minanov O., Kwiatkowski P., Popilskis S., Michler R. Modified technique for heterotopic heart transplantation in small primates. Ann Thorac Surg 1997;63:258-260.[Abstract/Free Full Text]

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