Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.
With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.
Over the past six decades, solid organ transplantation has evolved from an experimental procedure to a standard of care for the treatment of end-stage organ failure1. Due to the improvement of antimicrobial agents, surgical techniques and advancement in immunosuppressive regiments, the early success rate of solid organ transplantation have significantly increased over the past decades2.
However, long-term graft survival rates have not significantly improved in the same manner3. The development of CAV is the major factor limiting long-term survival4,5,6. This pathology is characterized by the formation of a concentric neointimal layer consisting of smooth muscle cells, leading to progressive narrowing of the vessel and consecutive malperfusion of the transplanted solid organ. In heart transplant recipients, CAV lesions can be diagnosed in up to 75% of patients 3 years after transplantation7.
The pathophysiology of CAV is not fully understood yet. It seems to be related to numerous immunological and non-immunological factors, leading to endothelial damage with subsequent endothelial activation and dysfunction8. Thus far, no causal treatment option exists for the prevention of CAV, emphasizing the need for a reproducible small animal model in order to study the formation and potential therapy of CAV.
With the use of murine aortic transplantation models, CAV like lesions can be seen 4 weeks after transplantation. Those lesions consist mainly of vascular smooth muscle cells, thereby, resembling the human pathology. Because of a wide variety of transgenic and knock out mice, the use of mouse models in transplant associated pathologies offers a unique opportunity to identify new therapeutic options and understand their development. Due to the small diameter of the transplanted vessels however, the use of mouse models is commonly associated with long learning curves and an initial high complication rate9. With the introduction of the non-suture cuff technique, this most challenging part of the operation can be facilitated and the diameter of the anastomosis is kept constant10,11.
All experiments were performed according to the guidelines of the German animal welfare act (TierSchG.) (AZ: 55.2-1-54-2532.Vet_02-80-2015).
1. Animal housing
2. Recipient preparation
3. Donor operation
4. Implantation
5. Postoperative care
6. Aortic graft explanations
In the fully MHC-mismatch transplantation model, a concentric neointimal layer can be seen 4 weeks after transplantation (Figure 2). This layer consists primarily of vascular smooth muscle cells as immunohistological staining for SM22 (a selective marker for mature vascular smooth muscle cells) revealed. As stated before, these vascular smooth muscle cells are pathognomonic for lesions seen in chronic allograft vasculopathy. For further analyses, aortic segments should be sectioned and stained by Elastica van Gieson-staining. Here, the neointimal layer can easily be differentiated to the elastic fibers of the internal elastic membrane, dividing the Tunica intima from the Tunica media.
In order to evaluate a potential therapeutic effect in this model, the neointima-media ratio, as well as the luminal cross-sectional area narrowing, can be measured in those sectioned samples13,15. In our described modified model of non-suture aortic transplantation, a technical success rate of >91% could be achieved in over 300 aortic transplantations performed. This high success rate could be accomplished by using a cuff made from polyimide tubing with an outer diameter of 0.610 mm and a wall thickness of 0.0254 mm.
Figure 1: Intraoperative pictures. (A) Cutting the ligated carotid artery. (B) Clamped carotid end after removing the ligature and flushing the end with heparinized saline. (C) Cuffing procedure. (D) Completed recipient preparation (both carotids end cuffed). (E) Transplanted aortic segment before and (F) after reperfusion. Please click here to view a larger version of this figure.
Figure 2: Histological specimen of transplanted aortic segments 4 weeks after transplantation. (A) Representative immunohistological staining with SM22 (green fluorescence) and DAPI (blue fluorescence) showing the thick neointimal layer consisting of vascular smooth muscle cells. The elastic fibers are shown in red fluorescence (20x magnification). (B) Elastica-van-Gieson staining (10x magnification). (C) Syngeneic transplanted aortic segment 4 weeks after transplantation (10x magnification). Please click here to view a larger version of this figure.
Chronic allograft vasculopathy is the major cause of late graft loss after solid organ transplantation of the heart and likely renal and lung allografts8. Thus far, no causal therapeutic regimen could be developed in order to prevent the formation of CAV.
The pathophysiology of CAV is multifactorial and involves immunological and non-immunological aspects16. The use of rodent models in transplantation have been essential in understanding the underlying pathophysiology of allograft rejection processes in solid organ transplantation and helped to identify novel therapeutic approaches that prevent rejection17. CAV is characterized by the formation of a neointimal layer consisting of vascular smooth muscle cells leading to consecutive narrowing of the vessel and malperfusion of the transplanted organ with subsequent deterioration of organ function7.
In the described murine aortic transplantation model, the concentric neointimal formation can be observed in fully MHC (H-2d to H-2b) mismatch thoracic aortic grafts 4 weeks after transplantation. Those lesions primarily consist of vascular smooth muscle cells (Figure 2). Shi et al. described the first mouse model of transplant arteriosclerosis in 199418. They grafted a carotid artery segment to the carotid artery by end to side suturing. In 1996, Koulack et al. established the first abdominal mouse aortic transplantation model by grafting an aortic segment to the infrarenal aorta by end-to-end anastomosis19. Dietrich et al. first described the use of a non-suture cuff technique for the transplantation of a thoracic aortic segment to the carotid artery in 200011.
In comparison to mouse models using microvascular anastomosis to transplant aortic segments, the cuff technique offers several benefits. First, the procedure is simpler and easier to learn. Second, the ischemic time of the grafted aortic segment is constant, since the recipient animal is prepared first for the anastomosis before the procurement of the aortic segment of the donor is performed, minimizing cold and warm ischemia time. Third, the diameter of the anastomosis is kept constant due to the rigid character of the polyimide cuff. Thus, strictures of the anastomotic region can be neglected.
Furthermore, the surgical procedure is less traumatic for the recipient animal as compared to the technique with the abdominal incision. In addition, the implementation of the cuff technique offers the possibility of various different types of solid organ transplantation while using the same technique in the recipient animal11,20,21.
Even though we are convinced that this microsurgical technique is easier to learn than other aortic transplant models described in the literature there are some possible pitfalls during the procedure. During the recipient operation, be sure to properly cauterize the sternocleidomastoid muscle before cutting it. The muscle is well vascularized and severe bleeding can occur if the accompanying vessels are not thoroughly coagulated. These bleedings are hard to control as the muscle will retract once fully dissected. In addition, while mobilizing the common carotid artery be sure not to directly grab the artery itself.
The cuff procedure itself is the most challenging part of the operation by far and most susceptible to failure. It is, therefore, vital to work with the right length of the carotid artery. In the beginning, surgeons tend to prepare too little length of the carotid artery, which makes the procedure a lot harder to complete. Moreover, in the beginning, surgeons tend to set the dividing ligature around the common carotid artery right in the middle of the operating field. This can lead to difficulties while performing the more cranial located cuff as this carotid end segment is quite rigid and difficult to mobilize. Meanwhile, a significant portion of the common carotid artery can be mobilized by slight tension from the area below the clavicle. Therefore, we suggest ligating the carotid artery slightly more proximally to leave more length to the cranial part of the common carotid artery. Once the common carotid artery is dissected and the ends are passed through the cuffs and fixed with the vascular clamps, dilatation of the carotid artery must be performed. In this part of the operation, it is very important not to overstretch the vessel as this may damage the vessel wall, leading to failure during the cuffing procedure. Rotating the whole operative field so that the traction is in line with the alignment of the arterial clamp on the cuff facilitates the procedure.
While procuring the aortic segment, be sure not to rip out any intercostal arteries or other branches of the thoracic aorta. On the other hand, take care not to cauterize too close to the thoracic aorta in order to prevent damage of the graft with increased risk of thrombosis of the graft after transplantation.
While implanting the aortic segment, be sure that both ends are properly aligned in order to prevent torsion of the graft. In addition, the aortic segment should be shortened to the correct length to prevent kinking during reperfusion. When reperfusing the graft, be sure to always open the more cranial clamp first to observe hemostasis. Small hemorrhages of not completely coagulated intercostal arteries can be controlled by applying gentle pressure with a small cotton swab.
The whole transplantation should take less than an hour with maximum of 30 minutes for the recipient preparation and maximum of 15 minutes each for the donor operation and implantation.
The most discussed disadvantage of this method is that the cuff will persist in the anastomosis during the length of the experiment. This could lead to a certain foreign body reaction and a possible higher risk of thrombosis. However, histopathological analyses of specimens transplanted with the cuff technique revealed only mild foreign body reaction of the graft and the tubing22. Another discussed limitation of the procedure is the heterotopic placement of the thoracic aorta into the common carotid artery. Due to the differing vessel diameters between the thoracic aorta and the common carotid artery, one could expect a more turbulent flow in the transplanted aortic segment in comparison to an orthotropic aortic interpositioning. This could lead to methodical based intimal changes. However, syngeneic transplanted segments revealed only little neointima formation ruling out a methodical based bias (see Figure 2).
This paper aims to facilitate the implementation of this model by other researchers in their laboratories. With the above-mentioned modifications, this mouse model of aortic transplantation can be accomplished with basic microsurgical skills, while achieving a high success rate.
The authors have nothing to disclose.
None.
Balb-c Mice (H2-d) | Charles River | Strain# 028 | Donor animal |
Bipolar cautery system | ERBE | ICC 50 / 20195-023 | Bipolar cautery |
C57BL/6J (H-2b) | Charles River | Strain# 027 | Recipient animal |
Halsey Needle Holders | FST | 12501-12 | Needle Holder |
Halsted-Mosquito Forceps | AESCULAP | BH111R | Curved Clamp |
Medical Polyimide Tubing | Nordson MEDICAL | 141-0031 | Cuff-Material |
Micro Serrefines | FST | 18055-04 | Micro Vessel Clip |
Micro-Adson Forceps (serrated) | FST | 11018-12 | Standard Forceps |
Micro-Serrefine Clamp Applying Forceps | FST | 18057-14 | Clipapplicator |
S&T Forceps – SuperGrip Tips (Angled 45°) | S&T | 00649-11 | Fine Forceps |
S&T Vessel Dilating Forceps – Angled 10° (Tip diameter 0.2 mm) | S&T | 00125-11 | Vesseldilatator |
Schott VisiLED Set | Schott | MC 1500 / S80-55 | Light |
Stereoscopic microscope | ZEISS | SteREO Discovery.V8 | Microscope |
Student Fine Scissors / Surgical Scissors – Sharp-Blunt | FST | 91460-11 / 14001-12 | Standard Sissors |
Vannas-Tübingen Spring Scissors (curved, 8.5 cm) | FST | 15004-08 | Microsissors (curved) |
Vannas-Tübingen Spring Scissors (straight, 8.5 cm) | FST | 15003-08 | Microsissors (straight) |