Total Arterial Anastomosis Heterotopic Heart Transplantation Model

Total Arterial Anastomosis Heterotopic Heart Transplantation Model F.G. Al-Amran and M.M. Shahkolahi ABSTRACT Background. Mouse transplant models offer a valuable platform for studying the biology of a spectrum of diseases, particularly those of the immune system. We have developed a modified abdominal heterotopic heart transplantation (AHHT) model with a total arterial anastomosis and compared the results with the cervical heterotopic heart transplantation (CHHT) and the non-modified AHHT models. Methods. Mice were randomly assigned to four groups: sham, AHHT, CHHT, and modified AHHT groups. Each group (except for the sham) included donor and recipient animals. Postoperative outcome, operative mortality, operative time, and tissue damage were assessed by measuring plasma levels of tumor necrosis factor ␣. Result. The modified AHHT group had significantly lower values. However, hind limb paralysis was observed equally and only in AHHT and modified AHHT models. The modified AHHT group had the highest success rate of functioning hearts. ORONARY heart disease, which leads to ischemic heart disease and the subsequent development of heart failure, is the single largest cause of cardiovascular disease.1 Heart transplantation is an effective treatment for intractable failure. The global ischemia/reperfusion event may cause myocardial injury in addition to rejection.2 Animal transplantation models have been used to develop novel treatments to reduce myocardial injury.3 Among rodents used extensively for those studies, the mouse is preferred because, of the more than 1300 strains of genetically modified animals available for studying transplantation immunology.4 – 6 Furthermore, the high degree of similarity between the mouse H-2 major histocompatibility complex complex and the human leukocyte antigen system (HLA) provides clinically relevant mechanistic insights. The first model of mouse heart transplantation was the abdominal heterotopic heart transplant (AHHT), which was first reported in 1973.7 A novel technique for blood circuit reconstruction in AHHT anastomoses the donor ascending aorta and pulmonary artery to the recipient abdominal aorta and inferior vena cava (IVC), respectively.8 A subsequent model heterotopically transplants the heart in the cervical area (cervical heterotopic heart transplantation; CHHT): The common carotid artery and external jugular vein of the recipients are cuffed to reconstruct blood flow.4,9 We compared the results of a modified AHHT procedure with a total arterial anastomosis, analyzing anas-

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tomosis time, postsurgical recovery, technical errors, and operative mortality with the three other models. METHODS Experimental Protocols We used 105 C57/B6 male mice aged 10 to 12 weeks with protocols reviewed and approved by The Institutional Animal Care and Use Committee of Howard University. After a week of acclimatization, the mice were randomly assigned to groups: sham, AHHT, CHHT, and modified AHHT groups. The sham group included 15 hearts; the other cohorts, 30 animals, including 15 donors and 15 heart transplant recipients.

Anesthesia Intraperitoneal injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) rendered both unconscious within 5 to 10 minutes.10

Surgical Procedure A CHHT was performed using the cuff method as previously described with some modifications (Fig 1).11 A transverse abdomFrom the Surgical Department, (F.G.A.) Medical College, Kufa University, Najaf, Iraq and Howard University College of Medicine (M.M.S.), Washington, DC. Address reprint requests to Fadhil G. Al-Amran, MD, FRCS, FACS, P.O. Box 18, Surgical Department, Medical College, Kufa University, Najaf, Iraq. E-mail: [email protected]; [email protected] 0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.10.039 625

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AL-AMRAN AND SHAHKOLAHI

Fig 1. Image of cervical heterotopic heart transplant.

inal incision was used to slightly retract the viscera to the left side before 100 U of heparin was injected through the infrahepatic IVC. Approximately 3 minutes later, the thorax was opened via left and right dorsolateral incisions with a pair of rigid scissors. The diaphragm was separated from the anterior chest wall at the same time. The right superior vena cava (SVC) was ligated and transected at the distal side leaving a segment of suture. The first branch of the aorta was ligated with a 6-0 silk suture, with a portion retained at the first branch. After transecting the aorta at a site distal to its first branch, the heart was gently retracted upward by the silk sutures, which were attached to the right SVC and the first branch of the aorta. Retraction was maintained with a forceps that was fixed to the corkboard. The left SVC, pulmonary arteries, pulmonary veins, and azygos vein were then ligated together with a

Fig 3. Image of modified abdominal heterotopic heart transplant. 4-0 silk suture. Then, the harvested heart was stored in cold lactated Ringer’s solution.

Recipient Operation Via a right anterolateral incision parallel to the neck, the jugular vein was isolated from incoming branches. An 18-gauge cuff was sleeved, everted, and secured with a circumferential 6-0 silk suture using the conventional method. An 18-gauge cuff was always attached in this series. The common carotid artery was ligated as far from the subclavian artery as possible. Gentle manipulation prevented injury to the endothelium with thromby formation. The cuffing techniques for the artery were the same as those with the jugular vein. After setting a stay suture in the posterior wall of the right SVC of the heart graft, the transplant was gently slipped over the cuff that had been fixed to the jugular vein of the recipient to be secured with a circumferential 6-0 silk suture. The anastomosis between the donor aorta and recipient common carotid artery was the same as that used for the vein. In the final step, the bulldog clamps were removed, and the beating graft evaluated to ensure that it was free of congestion.

AHHT An AHHT was performed as previously described, with some modifications (Fig 2).12

Donor Heart Retrieval

Fig 2. Image of conventional abdominal heterotopic heart transplant.

Animals anesthetized with ketamine (100 mg/kg, intraperitoneally) and xylazine (10 mg/kg, intraperitoneally) had the chest opened from both sides in the supine position proceeding from lateral to create a flap to expose the heart and its major vessels. The aorta and pulmonary artery were cleaned from the surrounding tissue, especially the thymus. After 1 mL of heparin (250 U/mL) was injected into the IVC using a 29-G needle, silk ligatures (6-0) were applied to both vena cavae and the pulmonary vein. After transection preserving maximal length, the heart was immediately transferred to and rinsed in cold normal saline (4 °C).

HETEROTOPIC HEART TRANSPLANTATION

627 with a continuous suture. After the anastomosis, the distal ligature was released to check bleeding; if there was little to none we removed the proximal ligature. The donor heart filled with blood immediately; its color became red, and it began to beat. The intestines were returned to the abdomen, which was closed with 6-0 continuous sutures. The recipient mouse was placed in a warm area for 1 hour recovery. During surgery, anesthetized animals were warmed with a heating blanket. The loss of body fluid was compensated by intraperitoneal injection of normal saline (0.5 mL 2 to 3 times). The core body temperature was monitored with a digital rectal thermometer.

The Novel Modified AHHT Model

Fig 4. Total operative time and the separate recipient vessel anastomosis in the three studied groups.

Recipient Mouse Preparation Animals anesthetized with ketamine (100 mg/kg, intraperitoneally) and xylazine (10 mg/kg, intraperitoneally) had the abdominal area shaved and sterilized, with the mouse placed in a supine position on the operative field. After a long midline abdominal incision, the abdominal contents were retracted outside the carity with gauze to expose the aorta and IVC. The vascular branches were ligated with 10-0 sutures with placement of proximal and distal ligatures using microscissors. Venotomy using a 30-gauge needle was extended to equal the donor’s pulmonary aorta. Afterward, an aortotomy was performed with a 30-gauge needle, and lumina were irrigated with saline solution. The donor heart, placed on the left side of the recipient abdomen, was covered with gauze. Afterward, stay sutures were placed at the proximal and distal apices of the abdominal aorta and donor ascending aorta, and the anastomosis of the right side was completed with continuous sutures. Two stay sutures placed at the proximal apex and distal apex of the IVC and donor pulmonary artery guided the second anastomosis. The left wall of the IVC and donor’s pulmonary artery was closed with continuous sutures inside the IVC. After knotting the proximal apex stay suture, the right wall of the IVC and the donor pulmonary artery were closed with continuous sutures outside the IVC. The donor heart was relocated to the right side of the abdomen. The right wall of the abdominal aorta and the ascending aorta was anastomosed

The donor heart retrieval was exactly the same as above; the recipient procedure included the following modifications (Fig 3): Only the aorta was dissected; proximal and distal ligatures were placed around it. The aorta was then transected in the middle; the donor ascending aorta was anastomosed to the proximal end, and the donor pulmonary artery to the distal end. Therefore, coronary artery inflow emerged from the proximal abdominal aorta, while venous outflow drained into the distal abdominal aorta. The anastomosis was performed with 10-0 prolene sutures, using four interrupted sutures for the proximal and distal anastomoses. The subsequent steps were completed as in the conventional AHHT.

Study Metrics Operative time was studied for the three surgical groups to compare efficiency to evaluate anastomosis time specifically. Intraoperative mortality was assessed in all groups, including that due to technical errors, such as injury to major vessels, disruption of anastomosis, or failure to accomplish the anastomosis. The postoperative outcome included the morbidity and mortality during the 24-hour after surgery. Morbidity included delayed recovery or no recovery due to cerebrovascular insult or brain death and limb paralysis. At 24 hours after transplantation, the animals were sacrificed to determine the function of the transplanted heart and to assess the magnitude of tissue damage through measurement of plasma tumor necrosis factor ␣ (TNF␣) compared with the sham group.

RESULTS

The modified AHHT model showed the lowest operative time. Figure 4 reveals the times for the three models, including donor and recipient operations, and anastomosis time. The modified AHHT model was completed at an average of 74 minutes compared with 118 and 101 minutes

Table 1. Operative Mortality in All Groups Causes Groups

Bleeding

Cardiac Arrest

Failure of Completion of Anastomosis

Apnea

Overall Operative Mortality

CHHT AHHT Modified AHHT Sham Causes in All Groups

4 (26.66%) 5 (33.33%) 1 (6.66%) 00 (0%) 10 (16.66%) N ⫽ 60

0 (0%) 1 (6.66%) 1 (6.66%) 1 (6.66%) 3 (5%)

1 (6.66%) 1 (6.66%) 0 (0%) 0 (0%) 2 (2.33)

1 (6.66%) 0 1 (6.66%) 1 (6.66%) 3 (5%)

5 (33.33%) 7 (46.66%) 3 (20%) 2 (13.33%) 12 (30%)

Abbreviations: CHHT, cervical heterotopic heart transplantation; AHHT, abdominal heterotopic heart transplantation. *Versus sham group. † Versus AHHT, CHHT groups.

P Value

⬍ .05* ⬍ .05* ⬍ .05†

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AL-AMRAN AND SHAHKOLAHI Table 2. Postoperative Outcome in All Groups

Outcome Groups

Nonrecovery

Hind Limb Paralysis

Four-Limb Paralysis or Cerebrovascular Accident

Postoperative Mortality

CHHT AHHT Modified AHHT Sham All groups

1 (6.66%) 0 (0%) 0 (0%) 1 (6.66%) 2 (3.33%)

0 (0%) 1 (6.66%) 1 (6.66%) 0 (0%) 2 (3.33%)

2 (13.33%) 0 (0%) 0 (0%) 0 (0%) 2 (3.33%)

1 (6.66%) 1 (6.66%) 0 (0%) 0 (0%) 2 (3.33%)

P Value

⬍.05* ⬍ .05* ⬍ .05†

Abbreviations: CHHT, cervical heterotopic heart transplantation; AHHT, abdominal heterotopic heart transplantation. *Versus sham group. † Versus AHHT, CHHT groups.

for AHHT and CHHT procedures, respectively. Recipient vessel anastomosis was the lowest in the modified AHHT model (10 minutes) compared with the AHHT and CHHT models (19 and 13 minutes, respectively). As shown in Table 1, the most common cause of operative mortality was bleeding during major vessel dissection, which was most commonly observed among AHHT procedures. Table 2, shows nonrecovery and four-limb paralysis events that were reported mostly in CHHT (3, 20%). Hind limb paralysis was observed equally and only in AHHT and modified AHHT groups (1, 6.66%). No postoperative mortality was observed in the modified AHHT group compared with the other two groups (1 [6.66%] postoperative mortality in each group). After 24 hours, the modified AHHT model apparently engendered less tissue damage than the conventional AHHT. The plasma TNF␣ levels were increased in the AHHT group (17.9 pg/mL) compared with both the CHHT and modified AHHT groups. The modified AHHT group (11.5 pg/mL) showed the lowest TNF␣ plasma levels except for the sham group (4.4 pg/mL; Fig 5). As shown in Fig 6, the modified AHHT group displayed the highest success rate of beating transplanted hearts among surviving animals (100%) compared with CHHT (66.66%) and AHHT groups (85.7%).

We have developed a novel, efficient surgical procedure for heart transplantation. It can be accomplished in significantly less time than other methods, which results in less tissue damage and inflammation, with a higher rate of beating hearts. The introduction of the CHHT model requires less experience.4 However, it did not show much advantage over the AHHT model. The abdominal approach to heterotopic heart transplantation is demanding and time consuming, although it was the first murine model for the procedure.12 The modified AHHT model has the lowest operative time, which was mainly due to the decreased anastomosis and blood vessel dissection times because dissection of the IVC in the conventional AHHT model and the external jugular vein in the CHHT model usually extends the time. These dissections are omitted in the modified AHHT model while providing an easily accessible field for total arterial anastomosis. The merits of the new model allow for not just a shorter procedure but they also render the surgical technique, steps, and exposure easier. The modified AHHT model showed a decreased operative mortality. Overall operative mortality was commonly due to bleeding, which occurred mainly during major vessel

Fig 5. Plasma levels of tumor necrosis factor ␣ in all groups 24 hours after transplantation.

Fig 6. Function of transplanted heart in surviving animals after 24 hours.

DISCUSSION

HETEROTOPIC HEART TRANSPLANTATION

dissection or from anastomotic disruption. The introduction of the new model decreases mortality, mainly due to modifications in the dissection of major blood vessels and the anastomosis technique. The interruption of brain circulation may be blamed for death in the CHHT model, which was not observed in either the modified or conventional AHHT model, although hind limb paralysis, a particular postoperative complication of the abdominal approach, was observed equally in the AHHT and the modified model. However, this outcome did not affect the animal being a candidate to study the transplanted heart. The large degree of tissue destruction observed in CHHT and AHHT models, as assessed by increased plasma inflammatory markers, might interfere with investigations. A model with less tissue destruction may be considered to be useful to improve the accuracy of results regarding inflammation. Our experience showed the new, rapid method to be reproducible, representing an advance in studies of ischemia reperfusion injury. In conclusion, the novel HHT model appeared to be superior to other models in terms of lower operative time, tissue damage, as well as operative mortality and success rate at 24 hours. REFERENCES 1. Banner NR, Khaghani A, Fitzgerald M, Mitchell AG, Radley Smith R, Yacoub MH. The expanding role of cardiac transplantation. In: Unger R, ed. Assisted Circulation III. Berlin, Heidelberg: Springer-Verlag; 1989:448 – 467.

629 2. Kirklin JK, McGiffin DC, Pinderski LJ, Tallaj J. Selection of patients and techniques of heart transplantation. Surg Clin North Am. 2004;84:257–287. 3. Kadner A, Chen RH, Adams DH. Heterotopic heart transplantation: experimental development and clinical experience. Eur J Cardiothorac Surg. 2000;17:474 – 481. 4. Gong W, Thornley T, Whitcher GH, Ge F, Yuan S, Liu DJ, et al. Introduction of modified cervical cardiac transplant model in mice. Exp Clin Transplant. 2012;10(2):158 –162. 5. Erickson RP. Mouse models of human genetic disease: which mouse is more like a man? Bioessays. 1996;18(12):993–998. 6. Ge F, Gong W. Strategies for successfully establishing a kidney transplant in a mouse model. Exp Clin Transplant. 2011; 9(5):287–294. 7. Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation. 1973;16(4):343–350. 8. Wu K, Zhang J, Fu J, Wu S, Philipp T, Uwe H, et al. Novel technique for blood circuit reconstruction in mouse heart transplantation model. Microsurgery. 2006;26(8):594 –598. 9. Gong W, Klpfel M, Reutzel-Selke A, et al. High weight differences between donor and recipient affect early kidney graft function—a role for enhanced IL-6 signaling. Am J Transplant. 2009;9(8):1742–1751. 10. Nian M, Lee P, Khaper N, et al. Inflammatory cytokines and postmyocardial infarction remodeling. Circ Res. 2004;94(12):1543– 1553. 11. Cha J, Wang Z, Ao L, Zou N, Dinarello CA, Banerjee A, et al. Cytokines link toll-like receptor 4 signaling to cardiac dysfunction after global myocardial ischemia. Ann Thorac Surg. 2008;85: 1678 –1685. 12. Liu F, Kang SM. Heterotopic heart transplantation in mice. Vis Exp. 2007;6:238.