Objectives: Our objective was to investigate the techniques of vessel suturing in the abdominal mouse cardiac transplant model and animal outcomes.

Materials and Methods: Our mouse group included 92 female inbred mice, in which 46 abdominal mouse heart transplants were performed. During transplant, the openings for the aorta and the inferior vena cava were not parallel. With everted anastomosis, the ascending aortae and the pulmonary arteries of donors were connected to the abdominal aortae and interior vena cavae of recipients, respectively, with 11-0 Prolene suture. We recorded complications and animal survival.

Results: The first 2 mice that underwent the procedure died of blood loss on day 1. However, the remaining 44 mouse recipients were alive for greater than 14 days, resulting in 14-day survival rate of 95.65%. Range of time to complete the whole procedure, including learning curve, was 47 to 72 minutes. At histology, implanted hearts from recipients with long-term survival appeared normal.

Conclusions: The procedure for everted suturing in the abdominal mouse heart transplant model was easy to perform and had a high success rate.

Key words : Animal model, Cardiac transplant, Inferior vena cava

Introduction

The abdominal mouse heart transplant model has been widely used for decades in transplant immunology studies because the abdominal cavity is relatively spacious on its advent.¹ Although it is a well-established animal model, the abdominal mouse heart transplant procedure is technically demanding. Having a model with simplified techniques and higher success rates can save thousands of mice and benefit related studies. In this study, we constructed a model that everted suturing for inferior vena cava (IVC).

Materials and Methods

Donors and recipients were female inbred C57BL/C mice weighing 18 to 28 g and age 8 to 10 weeks old. All mice had free access to food and water. All procedures were in accordance with the Animal Care and Welfare Act.

Microsurgical instruments included Leica microscope M651 (Leica, Solms, Germany), micro­vascular clamps, microsurgery scissors, tweezers, needle holder, 6-0 and 8-0 silk sutures, and 11-0 Prolene suture.

Surgical methods
Donor mice were anesthetized by intraperitoneal injection of ketamine (200 mg/kg) and xylazine (10 mg/kg). The abdomen was shaved, and the limbs were fixed with tape on the surgical movable pad. After satisfactory anesthesia was achieved (verified by no movement observed after tail and toe pinch), the surgical area was disinfected with 75% alcohol, the abdominal cavity was entered through the midline incision, and the gut was pulled out to the left side to expose the IVC. We injected 2 mL of normal saline containing 100 U of heparin sodium into the IVC to heparinize the donor. About 1 minute later, the abdominal aorta was transected to let blood out, and the diaphragm was quickly pierced. The anterior chest wall was removed along the anterior axillary line on both sides of the ribs; ice was placed in the chest cavity to cool down the heart. The pericardium and the thymus were removed, and the superior vena cava and IVC were ligated respectively close to the atrium with 6-0 silk suture.

The heart was retrograde flushed again with 1 mL of normal saline through the ascending aorta, which was pierced with a 25-gauge needle until the heart was palish. During the procurement of the heart, the area was irrigated several times with 0°C saline to keep the donor’s heart cool. Using micro-spring scissors, we transected the ascending aorta below the brachiocephalic artery. The main pulmonary artery was transected as distally as possible, and the pulmonary veins and the left superior vena cava were ligated proximally en bloc with 6-0 silk suture. The donor heart was retrieved and placed in physiological saline solution at 0°C. In general, procurement time was 10 to 13 minute, warm ischemia time was about 1 minute, and cold ischemia time was less than 60 minutes.

The anesthesia, shaving, fixation, and disinfection procedures for recipients were similar to those for the donors. The abdominal cavity was entered midline from the xiphoid to the pubic symphysis, with care taken not to damage the bladder. The abdominal wall was retracted using a speculum retractor (Fine Science Tools, North Vancouver, BC, Canada). The mesosigmoid was divided, and the gut was pulled out of the cavity on the left side to fully expose the IVC and the abdominal aorta. The abdominal aorta and IVC were dissected free from the surrounding tissues in the retroperitoneum, exposing the lumber arteries and veins, which branch off posteriorly to the great vessels in 2 or 3 groups between the left renal vessels and the iliac bifurcations. Two or 3 groups of vessels from the dorsal lumbar regions were ligated individually with 8-0 silk sutures. Dissection was made from the left renal vein down to the iliac bifurcation. Two microclamps (Titanium Micro Serrefines; Fine Science Tools) were positioned, with one just below the left renal vein and one just above the bifurcation of the iliac vessel, interrupting the flow in both the aorta and IVC. The right ureter was covered and protected with gauze. A 30.5-gauge needle was used to pierce the aorta and IVC to make aortotomy and venotomy, which were extended with the microscissor. The opening in the aorta was equivalent to the ascending aorta, the opening in the IVC was slightly larger than that in the pulmonary artery, and the aorta and IVC openings were not in juxtaposed positions (Figure 1).

The donor heart was placed on the left side of the mouse abdominal cavity and treated several times with 0°C saline shower. With the 2-point technique, the first suturing was conducted using a 11-0 Prolene suture from the recipient’s aorta to the donor’s ascending aorta at the distal opening, and continuous everted suturing (right side) was made to the right proximal side. This generally involved 3 or 4 stitches on the right side. The anastomosis of the donor pulmonary artery to the recipient IVC was made in a manner to evert suturing using 4 or 5 stitches (Figure 2). The surgical pad was then moved, with suturing of the IVC and the aorta on the left side made in the same manner (Figure 3). Suturing was everted on both sides of the aorta and IVC. The proximal and distal vascular clamps were released sequentially. The transplanted heart then began to beat, which we observed for 5 minutes (Figure 4). After observation, the abdominal cavity was closed using 2 layers.

Postoperative management and observation
The mice were kept warm under an infrared lamp and allowed to wake after 5 to 10 minutes. Mice had free access to water and food. An animal handler used visual observation to check palpation of heartbeat. A mouse that survived for 7 days was considered to have a successful transplant procedure.

Results

Duration for the donor operation was about 10 to 13 minutes. Because of blood loss, the first 2 mice that underwent the procedure died. The remaining 44 mice survived and were alive for greater than 14 days (14-day survival rate of 95.65%). We had no graft failures due to thrombi secondary to transplant and no hind limb paralysis. Duration for the recipient surgical procedure was about 47 to 72 minutes, which included our learning curve (Figure 5). Histologic analyses of transplanted heart after 120-day survival showed normal results (data not shown).

Discussion

Everted suturing is a basic principle in vascular surgery. For the mouse heart transplant model developed by Corry and associates,¹ the connection of the recipient’s IVC to the donor’s pulmonary artery in the left side was performed in a standard manner. That is, suturing of the recipient’s IVC to the donor’s pulmonary artery is intraluminal because the openings of the IVC and aorta are parallel. However, this is a difficult technique, and it is easy to damage the vascular intima, especially when the sigmoid and the rectus are filled with feces and especially for inexperienced surgeons. The first stitch is more difficult, and the anastomosis of IVC and the pulmonary artery of the left side is made only on the right side through intraluminal suturing.² Blood loss and thrombosis can easily occur, with these complications being the main reasons for failed procedures.

For the procedures reported here, the openings were not parallel. Although the procedure for anastomoses of donor aorta to recipient aorta was identical to that described by Corry with everted suturing, the suturing of the donor’s pulmonary artery to the recipient’s IVC was everted on both sides of the IVC, in which the surgical pad was moved. This was different from the standard manner mentioned above. It is noted that the donor’s pulmonary artery was long enough to have unparalleled openings. We had no problem with pulmonary artery length for the donor. Su and associates selected the donor’s thoracic IVC for their procedure.³

For everted suturing, the anastomosis margins and spacing must be easy to grasp to avoid damaging the vascular intima; this also allows no difficulty with the first stitch. A factor that may have contributed to our good outcomes was the experience of our microsurgeon, who has performed mouse liver transplants for several years. Although the first 2 mouse deaths were due to learning the new procedure, after familiarization, the procedures were stable and consistent. In conclusion, everted suturing in the vascular anastomosis can be easily performed.

References:

1. Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of heart in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation. 1973;16(4):343-350.
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2. Liu F, Kang SM. Heterotopic heart transplantation in mice. J Vis Exp. 2007;(6):238.
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3. Su S, Türk TR, Wu S, et al. Modified suture technique in a mouse heart transplant model. Asian J Surg. 2011;34(2):86-91.
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