EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: The murine cervical heterotopic heart transplant model was initially designed to test the immune response to third-party allografts, modified by cuff techniques. While cuff techniques simplify the execution of this procedure, cutting of the carotid artery and the external jugular vein alters the blood supply to central nervous system and makes it difficult to achieve long-term graft survival. In the present study, we describe modified techniques that preserve the continuity and function of blood vessels and improve transplant integrity.

Materials and Methods: The modified techniques in this study comprise the following aspects: (1) Preservation of the sternal head of the right sternocleidomastoid muscle, (2) use of the donor’s intrathoracic inferior vena cava for anastomosis and (3) preservation of the function of the recipient’s carotid artery and external jugular vein and thus, continuity of blood flow to the central nervous system.

Results: Stable, long-term, disease-free allograft survival has been achieved with syngeneic transplants (> 200 days), whereas allografts from fully major histocompatibility complex-mismatched donors were acutely rejected in a time similar to the traditional abdominal heterotopic heart transplant model (8.2 ± 1.3 vs 8.4 ± 1.4 days; P = .73 in the Mantel Cox test, and P = .61 in the Gehan-Breslow-Wilcoxon test). Similar alloresponses could be induced in these 2 models.

Conclusions: It is possible and feasible to achieve long-term graft survival in the mouse cervical heart transplant model using the modified procedures described in the present study.

Key words : Cervical cardiac transplant, Modified techniques, Continuity, Function

As more than 1300 strains of genetically modified mice are currently available, mouse transplant models have greatly advanced the progress of transplant immunology during the past decades, more so than solid organ transplant in the rat.¹ Mouse models of transplant offer a valuable platform to study the biology of a spectrum of diseases, particularly those of the immune system.^{2, 3} Furthermore, the high degree of similarity between the mouse H-2 major histocompatibility complex (MHC) complex, and the human leukocyte antigen MHC complex provides clinically relevant mechanistic insights.

Since the abdominal heterotopic heart transplant model in mice was first reported by Drs. Corry and Russell in 1973,⁴ it has been widely used in research centers. However, some disadvantages of the abdominal model linger, such as abdominal infection, postoperative paralysis of the hind limbs, difficulty handling lumbar vessels, and time limitations associated with obstructing the recipient’s blood flow. Moreover, the relatively large area exposed during abdominal surgery elicits fluid loss,⁵ and use of ice-cold saline solution in the abdomen during vascular anastomosis can decrease body temperature.

Additionally, the deep location of the transplanted heart engenders a degree of difficulty in monitoring graft function by palpation. Heterotopic heart transplant in the cervical area has been recognized as an alternative technique, often meeting a need for testing third-party heart transplant survival in mice that have already undergone abdominal heart transplant. To reduce the surgical difficulties of the cervical procedure, the common carotid artery and external jugular vein of the recipients are usually cut and cuffed for subsequent reconstruction of blood flow^6-10; however, this procedure alters hemodynamics and disrupts blood supply to the central nervous system, creating high blood pressure in the transplanted graft. Moreover, the cuff technique has a notable pitfall of thrombosis,¹¹ which affects long-term graft survival.

We introduce modified techniques to establish heterotopic cervical heart transplant by preserving the continuity and function of the carotid and external jugular vein. By means of this reliable approach, a high surgical success rate and long-term graft survival have been achieved.

Materials and Methods

Mice
Inbred wild-type C57BL/6 (H-2^b) and BALB/c (H-2^d) male mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) at 8 to 12 weeks of age and housed under pathogen-free conditions at Beth Israel Deaconess Medical Center (Boston, MA, USA). Before the study, all protocols were approved by the institution's animal welfare regulatory committee, and all protocols were in conformity with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health 86-23, revised in 1985. Syngeneic heterotopic heart transplants were performed using C57BL/6 (H-2^b) donors and C57BL/6 (H-2^b) recipients; allogeneic transplants were performed using BALB/c (H-2^d) donors and C57BL/6 (H-2^b) recipients.

Surgical instruments
Surgical procedures were performed under a desktop operating microscope (Carl Zeiss Operating Microscope, f170, Opmi pico, Germany) at magnifications of × 4 to × 25. Microsurgical instruments for this study included straight supergrip forceps, micro serrefine clamps, micro-dissecting scissors (Fine Science Tools, Germany), and a curved needle holder (World Precision Instruments, Sarasota, FL, USA). For vessel anastomosis, 10-0 swaged microsurgical sutures were used (Fine Science Tools, Inc., Foster City, CA, USA). Retractors manufactured in-house were used to expose the surgical field. A low-temperature Cautery (World Precision Instruments) was used to coagulate the recipients’ small vessels, such as tributaries of the external jugular vein.

Surgical procedure
Anesthesia
Mice were anesthetized using an intraperitoneal injection of a ketamine (60 mg/kg) (Butler Animal Health Supply, Dublin, OH, USA) and xylazine (10 mg/kg) (Akorn Inc., Decatur, IL, USA) and then placed in a supine position under an operating microscope (Carl Zeiss Meditec Inc).

Donor graft procurement
After somatic heparinization (100 U/mL) (Heparin Sodium, APP Pharmaceuticals, LLC, Schaumburg, IL, USA) was performed via the inferior vena cava (IVC), a bilateral thoracotomy was performed to better expose the heart. First, the intrathoracic IVC was carefully dissected and transected in close vicinity to the diaphragm. Then, the ascending aorta was freed from the surrounding tissue and then transected close to the innominate artery. The other vessels, including the pulmonary vessels and the left and right superior vena cava, were ligated using 6-0 silk sutures. The en bloc heart graft was then freed, removed, and stored in preservation solution (normal saline) at 4°C.

Donor heart implantation
The recipient was anesthetized as described for the donor and laid supine, with the caudal part toward the surgeon, and the limbs were held in place with a piece of adhesive tape. Hair was shaved, and the surgical field of skin was cleaned with 70% ethanol and iodine. An oblique longitudinal incision was made from the sternum to the right mandibular angle. A modified approach was devised for the recipients’ vascular preparation, by which the right submaxillary gland, external jugular vein, and the right carotid artery were kept intact to maintain adequate blood flow to the central nervous system. The tributaries of the external jugular vein were carefully electrocauterized instead of using ligatures. The clavicular head of the right sternocleidomastoid muscle was cut for better exposure of the right carotid artery. The distal and proximal parts of carotid artery were tied with 6-0 silk sutures, while their counterparts of the external jugular vein were clamped. An elliptical arteriotomy and venotomy, equivalent to the section size of the donor aorta, and the intrathoracic IVC, was made under the guidance of a 10-0 suture on the recipient’s vessels. Flushing thoroughly with heparinized saline solution was performed to clean intraluminal blood clots and prevent thrombosis after surgery.

The donor heart was covered with an ice-cold normal saline pad placed in the surgical region. Warm ischemia time starts when the graft is positioned in the recipient. After 2 stay stitches were placed at the caudal and cephalic corners, the donor aorta was anastomosed end-to-side to the carotid artery of the recipient by continuous suturing with 10-0 nylon sutures in a clockwise direction. The donor heart was flipped from the right side of the recipient to the left. The donor’s intrathoracic IVC was anastomosed end-to-side to the external jugular vein of the recipient by a running stitch with a 10-0 nylon suture in a clockwise direction. To minimize bleeding, a cotton ball was applied around anastomotic sites before ties or microvascular clamps were released. The proximal clip on the external jugular vein was released first, then unclamped at the distal clip, and then the distal and proximal knots of the carotid artery were untied (in that order). The heart graft was immediately reperfused and turned red in color. After dropping warm water (30°C to 37°C) onto the donor heart, the graft began to contract in sinus rhythm. The incision was closed with a 5-0 absorbable suture, and the recipients were placed on a water-circulating heat pad until conscious. Warm physiological saline was sometimes given subcutaneously, depending upon blood loss during surgery. The graft function was assessed daily by observation of donor heartbeat palpation. Rejection was defined as the date on which the donor heartbeat ceased completely.

Flow cytometry
At day 4, spleens of naive C57BL/6 mice, transplanted recipients with modified techniques and recipients with traditional techniques were recovered. All samples were stained with anti-mouse fluorophore-labeled antibodies (clones) as follows: CD3-PE-Cy7, CD4-FITC, and CD8-PE from eBioscience, Inc. (San Diego, CA, USA). Three-color flow cytometry was performed on a FACSCalibur dual-laser cytometer (BD Biosciences, Mountain View, CA, USA) to determine the phenotype and number of lymphocytes in the spleen.

Congenic mouse strains are genetically identical except for an MHC allele (class I and/or class II). The MHC-disparity between the donor and the recipient induce allogeneic response, leading to various graft survivals. In our modified cervical heterotopic heart transplant model, stable long-term, disease-free allograft survival has been achieved with syngeneic transplants (n=6; > 200 days), whereas allografts from fully MHC-mismatched donors (n=10) were acutely rejected similar to the traditional heterotopic abdominal heart transplant model (n=9; 8.2 ± 1.3 vs 8.4 ± 1.4 days; P = .73 in the Mantel-Cox log-rank test, P = .61 in the Gehan-Breslow-Wilcoxon test) (Figure 1). Furthermore, the allogeneic responses induced in the spleens in the modified and traditional models revealed similar results, showing that immune responses of acute rejection generated an evident shift of CD4+/CD8+ T cells ratio (Figure 2). This dominant amount of CD8+ T cells in the spleen contributed to the failure of graft survival.

Our modified transplant model is both practical and stable enough for transplant immunology research and has several unique advantages. First, posttransplant surgical thrombus formation was not seen in any of our transplanted recipients. This stands in contrast to the use of the traditional cuff technique, which sometimes results in thrombotic complications, even in the bigger animal models such as rats and rabbits.¹¹ Second, unlike the use of the donor pulmonary artery, use of the donor intrathoracic IVC for vessel reconstruction creates more ideal surgical exposure and favorable manipulation space. Third, the intrathoracic IVC is of sufficient length to avoid being adjacent to the ascending aorta of the donor, which is beneficial for vascular anastomosis owing to the large existing distance between the external jugular vein and the carotid artery. Fourth, the function of the external jugular vein and carotid artery is preserved. This feature not only ensures continuity of the blood supply to the central nervous system, but also avoids direct irrigation of the engrafted heart by the entire blood volume from the carotid artery, which negatively affects graft function by creating hypertension. Finally, and most important, is the long-term graft survival achieved using our modified techniques. This long-term survival allows for studies that induce permanent transplant acceptance. One caveat to our system, however, is that care should be taken to prevent twisting of vessels before venous reconstruction.

The first animal model of vascularized heterotopic cardiac transplant was established by Mann and associates in 1933.⁹ Although the transplanted heart is de facto quiescent and nonfunctional, this approach to vascular reconstruction is still used, particularly in the cervical model, which uses cuff techniques, as well as the traditional abdominal model. However, in our modified cervical model of the heterotopic heart transplant, we use an approach that is distinct from the aforementioned models in which the blood circulation in the graft is altered.

Blood initially flows from the recipient’s carotid artery (Figure 3, A1 and B1) into the donor’s ascending aorta (Figure 3, B2) and then enters the coronary arteries of the graft. Partial blood leaks into the cavities of a quiescent heart by insufficient valves and high blood pressure although aortic valves were closed. After the myocardium of the graft is supplied with blood from the coronary arteries, venous blood flows into the right atrium via the coronary sinus, and eventually drains into the recipient’s external jugular vein (Figure 3, A2 and B4) through the donor’s intrathoracic IVC (Figure 3, B3) rather than the pulmonary artery. As a result, the tonus of the left ventricle is in equilibrium with that of the right, which achieves harmonious contraction of both ventricles. This modified technique leads to improvement in long-term graft survival in the mouse cervical heart transplant model, thereby allowing for the more-extensive use of the cervical location as a primary or secondary site of engraftment.

Volume: 10 Issue: 2 April 2012

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