Objectives: We sought to develop a better approach in establishing a heterotopic aortic transplant model.

Materials and Methods: We used “sleeve” opera-tion and set up 2 heterotopic aortic transplant experimental groups (groups 1 and 2). In group 1, donors (BALB/c (H-2^d) mice; weight, 25-30 g) had the same weight as the recipients (C57BL/6 (H-2^d) mice; weight, 25-30 g); in group 2, donors had lower weights (BALB/c (H-2d) mice, weight, 15-20 g). Grafts were examined macroscopically and histologically 60 days after the transplant.

Results: The thrombosis incidence was 10% in group 1 (5 of 50) and 2% in group 2 (1 of 50) (P < .05). Intestinal obstructions led to a high mortality rate in both groups: 8% in group 1 and 10% in group 2 (P < .05).

Conclusions: The issue of size discrepancy between donor and recipient aortas is not a problem in a heterotopic aortic transplant model with the use of a sleeve technique, while the high incidence of intestinal obstructions must be considered.

Key words : Heterotopic aortic transplant, Mouse model, Sleeve technique, Thrombosis, Size discrepancy

Introduction

Chronic vascular rejection or graft vascular disease remains a major obstacle to long-term survival of solid-organ transplant. Mouse aortic transplant models are used to investigate chronic vascular rejection or graft vascular disease. The first aortic transplant mouse model was developed by Koulack and associates who used a thoracic aorta as the donor artery for a heterotopic transplant using the suture method.¹ Considering the size discrepancy between thoracic and abdominal aortas, a new mouse model was developed that used the infrarenal aorta as the donor artery using a sleeve technique to enable orthotopic aortic transplant.^2-4 However, few studies have used the sleeve technique to establish a heterotopic transplant model to compare the success rates of orthotopic and heterotopic transplant.

When we tried to establish an orthotopic transplant model as Calise and associates reported,² we found that the separation of the donor infrarenal aorta from the inferior vena cava was difficult and time consuming, and aortic grafts shortened, and the lumen diameter narrowed, after removal. When we performed a heterotopic aortic transplant, we found it easier to obtain donor aortas, and we observed no noticeable deformation of the grafts.

We thought that the size discrepancy between thoracic and abdominal aortas (as Calise and associates reported) may be caused by the incorrect donor selection. Thus, we used unmatched thoracic aortas and matched thoracic aortas as the donor aortas for transplant, and compared the success rate of establishing this model and the incidence of thrombosis. Our results show that a heterotopic aortic transplant model has more advantages than an orthotopic aortic transplant model using the sleeve technique.

Materials and Methods

Animals and experimental design
Specific pathogen-free female C57BL/6 mice (25-30 g) were purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China) as recipients, and female BALB/c mice (15-20 g or 25-30 g) were used as donors. The experiment protocols were approved by Animal Studies Committee of Xiamen University, and all studies adhered to the National Institutes of Health publication, Principles of Laboratory Animal Care.

Two experimental groups of heterotopic aortic transplant were used to compare the success rates and thrombosis incidence. In group 1, the donors’ thoracic aortas had a great size discrepancy compared with recipients; while in group 2, the donors’ thoracic aortas matched the abdominal aortas of recipients. To choose the 2 kinds of donors, we measured the external diameter of the thoracic aortas. It was 1.05 to 1.1 mm in 25 to 30 g BALB/c donor mice, and 0.7 to 0.8 mm in 15 to 20 g BALB/c donor mice. The diameter of the infrarenal aortas in C57BL/6 recipient mice was 0.75 to 0.8 mm, measured by a sliding caliper as the aortas were engorged in vivo.

Preparation of sleeves
Sleeves used for vascular anastomosis were made of Teflon and matched with the infrarenal aorta of recipients to guarantee normal blood flow in the mice. The sleeves’ specifications were external diameter 0.75 mm, internal diameter 0.7 mm, and length 1.0 to 1.5 mm.

Operative procedures
All procedures were performed with an operating microscope while mice were under intraperitoneal anesthesia. Anesthesia was performed as previously described.1 Briefly, a stock anesthetic solution was prepared by mixing 2 mL diazepam (5 mg/mL), 1 mL sodium pentobarbital (65 mg/mL), and 7 mL normal saline. Mice were given an intraperitoneal injection of 0.5 to 0.75 mL/100 g of the mixture. The induction time was approximately 3 minutes, and the anesthesia lasted 60 to 90 minutes. Normal saline (about 0.2 mL) was given subcutaneously after the induction. No antibiotics were used.

In donor mice, a transverse incision at the sternal level and 2 longitudinal incisions were made along both axillary midlines of the thorax. The thorax was opened to expose the heart, vessels, and lungs. The heart and lungs were moved aside to expose the thoracic aorta. Connective and fatty tissues covering the thoracic aorta were stripped off, and electrocoagulation was applied to the lateral branch vessels from the aortic root to the diaphragmatic hiatus. The removed aortic section was flushed with 3 mL heparinized saline (100 U/mL) and placed in normal saline at 4°C.

Recipient mice were anesthetized with an intraperitoneal injection of pentobarbital and placed on a rectangular piece of plastic with an electric heating pad underneath. A midline abdominal incision was made with a scissors. The intestines were exteriorized and wrapped with a moist gauze on the left. Under ×25 magnification, the infrarenal aorta was separated from the inferior vena cava. Anastomosis of the donor artery was performed with the sleeve technique (Figure 1).^2,5

Postoperative care
After surgery, mice were laid on electrically heated pads until they awoke; they were then placed in autoclaved cages and raised under gnotobiotic conditions. No antibiotics or immunosuppressive drugs were used. Mice that appeared unwell or paralyzed were killed by cervical dislocation after anesthesia with 5% isoflurane and were autopsied.

Histopathologic examination
Transplant grafts were examined macroscopically and histologically 60 days after the transplant. The grafted aortas were removed, cut into paraffin sections, and stained with hematoxylin and eosin and Van Gieson’s stain.

Statistical analyses
Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 12.0, IBM Corporation, Armonk, NY, USA). Comparison between groups was assessed by the t test. Values for P < .05 were accepted as statistically significant.

Results

The success rate of transplants was calculated within a day; thrombosed grafts were declared within 12 hours by paraplegia or death. The incidence of thrombosis was 10% in group 1 (5 of 50), and 2% in group 2 (1 of 50) (P < .05). Unexpectedly, postoperative intestinal obstructions were observed 3 to 5 days after the transplant. Recipient mice could not defecate, and their abdomen distended severely. The incidence of intestinal obstruction was 8% in group 1, and 10% in group 2 (P < .05, Table 1).

We recorded the survival of recipients on 1, 7, 15, 30, 60, and 90 days after transplant. We found that the mortality rate was high the first week after transplant; afterwards, the survival rate stabilized (Table 2). We defined the success of transplant as survival without paralysis and intestinal obstruction until the time of planned death. The survival rates of the recipients showed similar trends in the 2 groups, with no significant difference at each time point (P < .05, Figure 2).

Grafts were assessed 60 days after transplant, and pathological changes were evaluated by hematoxylin and eosin and Van Gieson’s stain. We observed typical vascular lesion of chronic rejection or graft vascular disease as described previously (Figure 3).⁶

Discussion

Dambrin and associates speculated that size discrepancy between thoracic and abdominal aortas led to a high thrombosis incidence and seriously hindered use of a heterotopic aortic transplant model.³ However, in this study, we used a sleeve technique and found that the incidence of thrombosis was not as high as they reported. Complications such as postoperative hemorrhage are rare when using the sleeve technique. Because of effective anticoagulation, the incidence of thrombosis was low in group 1, although there was size discrepancy between thoracic and abdominal aortas. In addition, we found that the mismatching of sleeve diameter greatly increased the risk of thrombosis. On the other hand, although we protected the intestines well during operative procedures, the occurrence of intestinal obstruction could not be avoided.

Our results show that a mouse heterotopic aortic transplant model using a sleeve technique can be established efficiently and has some advantages: it takes only 5 to 10 minutes to separate thoracic aorta, and 15 to 25 minutes to separate infrarenal aorta. The thoracic aortas do not perceptibly deform or shorten after removal from the donor mice. The thoracic aorta is long enough, and each donor artery (2 cm) can be divided into 2 segments to provide separate grafts, thus reducing the number of donors required.

Cho and associates have reported successfully establishing a rejection model by using BALB/c (H-2d) mice as donors and C57BL/6 (H-2d) mice as recipients of aortic allografts.⁷ Chronic rejection models have been used widely to investigate the pathogenesis of chronic vascular rejection.^8-13

In conclusion, size discrepancy between donor and recipient aortas is not in obstacle in a mouse heterotopic aortic transplant if the sleeve technique is used. The approach we report in this study will help develop a better heterotopic aortic transplant model for basic and translational research on chronic rejection.

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