Objectives: Heterotopic heart transplant in rats has been accepted as the most-commonly used animal model to investigate the mechanisms of transplant immunology. Many ingenious approaches to this model have been reported. We sought to improve this model and compare survival rates and histologic features of acute rejection in cervical and abdominal heart transplants.
Materials and Methods: Rats were divided into cervical and abdominal groups. Microsurgical techniques were introduced for vascular anastomoses. In the abdominal heart transplant group, the donor’s thoracic aorta was anastomosed end-to-side to the recipient’s infrarenal abdominal aorta, and the donor’s pulmonary artery was anastomosed to the recipient’s inferior vena cava. In the cervical heart transplant group, the donor’s thoracic aorta was anastomosed to the recipient’s common carotid artery, and the donor’s pulmonary artery was anastomosed to the recipient’s external jugular vein. Survival time of the 2 models was followed and pathology was examined. Histologic features of allogeneic rejection also were compared in the cervical and abdominal heart transplant groups.
Results: The mean time to recover the donor’s hearts was 7.4 ± 2.2 minutes in the cervical group and 7.2 ± 1.8 minutes in the abdominal group. In the cervical and abdominal heart transplant models, the mean recipient’s operative time was 23.2 ± 2.6 minutes and 21.6 ± 2.8 minutes. Graft survival was 98% and 100% in the cervical and abdominal heart transplant groups. There was no significant difference in graft survival between the 2 methods. Heart allografts rejected at 5.7 and 6.2 days in the cervical and abdominal transplant groups. There was no difference in the histologic features of acute allogenic rejection in cervical and abdominal heart transplant.
Conclusions: Both cervical and abdominal heart transplants can achieve a high rate of success. The histologic features of acute allogeneic rejection in the models are comparable.
Key words : Heart transplant, Rat, Microsurgical technique, Rejection, Graft survival
Heterotopic heart transplant model in rats has been frequently used to investigate transplant immunology because of the shorter operative time and ease in monitoring graft function.1, 2 Abbott and associates developed the first experimental accessory heart transplant rat model in 1964,3 which was modified by Ono and Lindsey.4 In their modified model, the heart thoracic aorta was anastomosed end-to-side to the recipient’s abdominal aorta, and the pulmonary artery was anastomosed end-to-side to the recipient’s inferior vena cava. Unfortunately, this method is associated with a high rate of graft loss because it is technically demanding.5, 6 Cervical heart transplant by using a sutureless “cuff technique” was introduced by Heron and associates in 1971,7 which shortened operative time and made the model easier to perform. Rao and Lisitza developed a femoral heart accessory transplant model in 1985 for retransplant.8
Although many modifications have been adopted to make the anastomosis simpler and improve the survival rate,9-12 each has its own strengths and weakness; abdominal heart transplant is still the classic model. However, some authors suggest that cervical heart transplant in rats may be easier and have a higher survival rate.13 This study sought to compare the survival rates and histologic features of cervical and abdominal heart transplants.
Materials and Methods
Male Lewis and DA rats, weighing from 200 to 300 g (purchased from the Medical Experimental Animal Center, Sun Yat-sen University) were used as donors and recipients. They were housed in the animal facility under a 12-hour light/dark cycle and allowed free access to food and water. Procedures were performed in accordance with the guidelines in the Animal Welfare Act using protocols approved by the Institutional Animal Care and Use Committee of the University of Sun Yat-sen University.
Surgical techniques (Figure 1)
Animals were divided into cervical and abdominal groups. There were 56 cervical and 56 abdominal heart transplants performed. There were 28 cervical syngeneic grafts and 28 abdominal syngeneic grafts. All operations were performed with the animals under anesthesia by isoflurane inhalation using a commercial system (Surgivet, Waukesha, WI, USA). All steps were conducted under sterile conditions using a surgical microscope. Cervical and abdominal heart transplants were done using a modified technique described previously.3, 4 Time to recover the donor heart, vascular reconstruction time, scores of the transplanted heartbeat strength, survival time, and histology were recorded.
Donor heart recovery
After the donor was anesthetized, it was placed in a supine position. A long midline abdominal incision was made to expose the abdominal aorta and inferior vena cava. One mL of heparin (100 µ/mL) was injected into the recipient’s inferior vena cava. The ribs were divided bilaterally, and the anterior chest wall was reflected upward and fixed with a needle to provide exposure. The inferior vena cava was clamped before 10 mL of Ringer’s solution at 4°C was perfused into the inferior vena cava. Then, the inferior vena cava and the superior vena cava were ligated. The thoracic aorta and the pulmonary artery were freed from the surrounding tissue and transected. A silk suture was placed around the right atrium to ligate the vessels, except the thoracic aorta and the pulmonary artery. The heart was then removed and stored in ice-cold Ringer’s solution.
Abdominal heart transplant (Figure 2A)
A midline incision was made to enter the abdominal cavity. The branches of the abdominal aorta and inferior vena cava were ligated. An aortotomy was made in the anterior wall of the abdominal aorta with a 30-gauge needle. The opening was extended with microscissors to a long longitudinal incision. A venotomy was made inferior to the aortotomy in the same way, but somewhat dextrally to the midline of the inferior vena cava. The recipient’s abdominal aorta was anastomosed end-to-side to the donor’s thoracic aorta. (Air bubbles should be avoided inside the vessel when the anastomosis is finished.) The donor’s pulmonary artery was anastomosed to the recipient’s inferior vena cava. The distal clamp was removed first, followed by the proximal clamp to restore the blood supply to the graft. A dry cotton swab was placed around the anastomosis to stop the small amount of bleeding. The abdominal incision was closed in 2 layers with 5-0 silk sutures.
Cervical heart transplant (Figure 2B)
A longitudinal incision 3 cm long was made from the submaxilla to the xiphoid. The right common carotid artery and external jugular vein were dissected from the surrounding tissues. The donor’s pulmonary artery and the recipient’s external jugular vein were anastomosed end-to-end. The distal portion of recipient’s common carotid artery was ligated, and the proximal portion of common carotid artery was trimmed. The opening of the donor’s thoracic aorta was closed 50% to 75% with 8-0 suture to match the size of the proximal opening of the recipient’s common carotid artery. The donor’s thoracic aorta was obliquely anastomosed end-to-end to the recipient’s common carotid artery with 10-0 suture. The clamp on the jugular vein was removed first after the anastomosis, followed by the clamp on the carotid artery. The donor’s heart became red and began to beat right after restoration of blood flow. A space was created subcutaneously to accommodate the heart graft. The cervical incision was closed with a continuous suture in 2 layers.
Ringer’s solution (2 mL) was administered directly to the abdominal cavity for fluid loss. A dose of carprofen (Pfizer, New York, NY, USA) was immediately administered. Rats were monitored in a single cage. Pain was assessed hourly by observing respirations, vocal stress, and locomotion. Any pain was managed with buprenorphine (0.1 mg/kg, sc, Sigma-Aldrich, St. Louis, MO, USA). If pain and discomfort persisted longer than 3 hours, the animals were killed.
Accessory heart palpation
Technically, a successful transplant was defined as a functional graft of at least 48 hours. Grafts were evaluated daily by monitoring the heartbeat. The donor’s heartbeat strength was scored as 1 to 4, as described by Gordon and associates.14
Survival time of syngeneic and allogeneic rats in both the cervical and abdominal heart transplant groups was followed.
Transplanted hearts were recovered from syngeneic rats at 100 days and from allogeneic rats after acute rejection. Tissue specimens were fixed and stained with hematoxylin and eosin. All sections were reviewed by 2 certified histo-cytopathologists independently.
Values are expressed as means ± SD. Differences of continuous parameters between the groups were compared using the t test. Values for P < .05 were considered significant.
Fifty-six cervical and 56 abdominal heterotopic heart transplants were performed.
The mean time to recover the donors’ hearts was 7.4 ± 2.2 minutes in cervical group and 7.2 ± 1.8 minutes in abdominal group. There was no significant difference in the time to recover the donor organ between the 2 groups (P > .05).
The time of vascular reconstruction was 23.2 ± 2.6 minutes and 21.6 ± 2.8 minutes for cervical and abdominal heart transplants. The time to recover the donor heart and vascular anastomosed time in the cervical model were slightly longer than those in the abdominal model, but there was no significant difference in the vascular reconstruction time between the 2 groups (P > .05).
Scores of the transplanted heartbeat strength are shown in Table 1. With palpation, the transplanted hearts showed no changes in beat intensity or size from right after the transplant until the third or fourth day after transplant, and a strong beat was found. From postoperative days 4 to 6, the beat became weak. There were further decreases in beat intensity, and the beat became weak. From postoperative day 7 forward, no heartbeat was found in all allogeneic grafts.
The survival rate of the syngeneic grafts was 98% at 3 months after the operation in the cervical heart transplant model. One graft loss was caused by a vascular thrombosis. The survival rate of the syngeneic grafts was 100% at 3 months after the operation in the abdominal heart transplant model. There was no statistical difference in graft survival between the 2 models (P > .05). Allogeneic grafts were rejected at 5.7 days after the operation in the cervical heart transplant group. Allogeneic grafts were rejected at 6.2 days after the operation in the abdominal heart transplant group. There was no statistical difference in graft rejection time after the operation between the 2 models (P > .05).
In the abdominal heart transplant group, the syngeneic transplant heart group showed no inflammatory cell infiltration and no cardiac muscle cell necrosis. In the DA→Lewis allogeneic group, pathological changes of immunity injury included a large number of lymphocytes, mononuclear cell infiltration, and cardiac muscle cell necrosis (Figure 3). In the cervical heart transplant group, the syngeneic heart transplant group showed no inflammatory cell infiltration or cardiac muscle cell necrosis on postoperative day 6. In the allogeneic group, severe injuries, with a large number of lymphocytes, mononuclear cells infiltration, and cardiac muscle cell necrosis were observed (Figure 4).
Heterotopic heart transplant models in rats have been widely used in transplant immunology, transplant pathology, and organ protection research.15, 16 Currently, there are 3 models of rat heterotopic heart transplant used: abdominal, cervical, and femoral heart transplant. Abdominal heart transplants in rats are used more often, and heterotopic cervical and/or femoral heart transplants make heart retransplants and even a third heart transplant possible.
Rat heart transplants into the femoral or cervical regions have several advantages compared with transplants into the abdominal cavity: These include easy vascular dissection, shorted operative time, reduced blood loss, decreased cold ischemia time of the donor heart, and easy graft monitoring. However, there is a major discrepancy in the size of the donor and recipient vessels. The donor’s aorta is much larger than the recipient’s femoral or common carotid artery, and this could be a contraindication to the operation. To solve this problem, some investigators have modified the models by using cuff or sleeve techniques in vascular reconstruction, instead of traditional suture technique. In this study, we anastomosed the donor’s aorta with the recipient’s common carotid artery in an end-to-end fashion, whereas the donor’s pulmonary artery was anastomosed by a conventional technique. The excessive wall of the donor’s vessel was closed. Small vessels reduce the number of stitches, and therefore, shorten operative time. However, the vessels are more prone to twist or develop a thrombosis with cervical heart transplant, and venous anastomoses are even more difficult to do because of the thin vessels. Therefore, the rate of thrombosis in cervical heart transplants is higher. Abdominal heart transplants can be done easily with a high rate of success and without severe trauma to the recipients. In this study, only 1 graft was lost by thrombosis in the allograft group. There was no statistical difference in graft survival between the 2 models. In a skilled microsurgeon’s hand, our results of cervical and abdominal transplant compare favorably to the techniques described previously. According to our research and the literature, the 2 different models are not associated with any difference in graft rejection intensity, so it should not be problematic to decide on the site of the heart transplant.
Palpation of the recipient’s abdomen is a simple and reliable method of determining the time of rejection. Rejection was defined as the first noticeable drop in the intensity of pulsation by palpation.17 The rejection day was the time point when no graft palpation was found.18 However, correct diagnosis requires skill and experience. It is important to apply the right pressure on the abdomen. If the pressure is too high or too low, the contractility will be underestimated. Because abdominal palpation is a subjective classification system for detecting heart rejection, it is necessary to develop an objective score to assess the contractility of the heart graft. There are many alternative diagnostic methods of rejection (eg, electrocardiogram and echocardiogram) to develop objective and quantitative indices of rejection, but the validity of these methods remains controversial.19-20 The electrocardiogram is so sensitive in recording electrical activity that it usually shows longer graft survival compared with palpation.21 Echocardiograms require technical expertise and special equipment. Owing to limited spatial resolution, several echocardiographic parameters were found to be insensitive for early detection of acute rejection after heart transplant. Recently, murine echocardiography using tissue Doppler imaging was considered as an objective method for noninvasive detection of chronic rejection in heterotopic rat heart transplant model. Tissue Doppler imaging indices proved to be a strong predictor of histologic evidence of rejection.22 In rodent models, histologic biopsy is the criterion standard for diagnosing acute rejection after heart transplant.23, 24 However, biopsy is done only when the animal is killed and inflammatory cellular infiltration typical of rejection can be found early even while the graft is still beating.
The skill of the microsurgeon, knowledge of animal vascular anatomy, and gentle manipulation during the operation are important factors contributing to successful heart transplant in rats. Before this study, microsurgeons in our team already had more than 10 years’ experience with rat liver, mouse heart, and mouse liver transplant.
In conclusion, both cervical and abdominal heart transplants can achieve a high survival rate. The histologic features of acute allogeneic rejection in these models are comparable.
Volume : 9
Issue : 2
Pages : 128 - 133
From the Organ Transplantation Center, the First Affiliated Hospital, Sun
Yat-Sen University, Guangzhou 510080, China
Acknowledgements: Dr. Guodong Wang and Dr. Yi Ma contributed equally to the work and should be considered coauthors. Dr. Guodong Wang’s e-mail is firstname.lastname@example.org.
This study was sponsored by the Guangdong Natural Science Foundation (No. 9151008901000052 and No. 10151008901000226) and the Science and Technology Planning Project of Guangdong Province (No. 2010B031600216 and No. 2009B030801012).
The authors declare there were no conflicts of interest in this study.
Address reprint requests to: Yi Ma, MD, Organ Transplantation Center, the First Affiliated Hospital, Sun Yat-Sen University, No. 58 Zhongshan 2nd St, Guangzhou 510080, China
Figure 1. A: Trimmed donor heart; B: Transplanted heart with blood supply recovery in an abdominal heart transplant; C: Arterial anastomosis in a cervical heart transplant; D: A transplanted heart with blood supply recovery in a cervical heart transplant.
Figure 2. A: Vascular anastomoses of an abdominal heart transplant. The donor thoracic aorta was anastomosed end-to-side to the recipient’s infrarenal abdominal aorta, and the donor pulmonary artery was anastomosed to the recipient’s inferior vena cava. B: Vascular anastomoses of a cervical heart transplant. The donor thoracic aorta was narrowed by 50% to 75% and anastomosed end-to-end to the recipient’s common carotid artery; the donor’s pulmonary artery was anastomosed to the recipient’s external jugular vein.
Figure 3. Abdominal heart transplant: A: Inflammatory cell infiltration and cardiac muscle cell necrosis were not detected in the isogeneic transplant heart group; B: In the DA→Lewis group, pathological changes of immunity injury showed a large number of lymphocytes, mononuclear cells infiltration, and cardiac muscle cell necrosis.
Table 1. Palpable donor heartbeat strength score.
Figure 4. Cervical heart transplant: A: Inflammatory cell infiltration and cardiac muscle cell necrosis were not detected in an isogeneic heart transplant group on postoperative day 6; B: In the DA→Lewis group, pathological changes of immunity injury showed a large number of lymphocytes, mononuclear cells infiltration, and cardiac muscle cell necrosis.