We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.
Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.
Over the past 30+ years, advances in immunosuppressive drugs have diminished graft rejection due to acute rejection but chronic rejection remains a main challenge. The main manifestation of chronic heart transplant rejection is transplant arteriosclerosis (TA) 1,2. This condition is characterized by intimal hyperplasia and vasomotor dysfunction of allograft arteries and develops as a result of immunological targeting of endothelial and smooth muscle cells by the recipient immune system. The specific targeting of the graft vasculature due to recognition of foreign peptide-major histocompatibility complex (MHC) is highlighted by the development of TA exclusively in graft arteries while sparing host vessels 3. In keeping with this is the observation that TA does not occur experimentally when the recipient is genetically identical to the donor or when the recipient lacks T and B cells 4. Immune-mediated vascular injury and dysfunction causes the development of intimal thickening and fibrosis, as well as the aberrant accumulation of lipids and ECM proteins, in TA 5. Intimal thickening tends to be concentric throughout the entire arterial tree 4-6. Graft loss and death usually occur as a result of progressive ischemia resulting from luminal occlusion of allograft arteries 4.
In 1991, Mennander et al. 7 pioneered an aortic interposition model in rats to model TA. Several groups have subsequently adapted this procedure for use in mice. In this model, allograft aortic segments develop lesions that have features comparable to TA observed in clinical transplants. This includes intimal thickening characterized by the accumulation of smooth muscle-like cells and recipient leukocytes 7. Over the past 2 decades this model has been used to generate important insight into the mechanisms of vascular injury, rejection and TA. It can be used to examine questions related to immune and vascular responses during arterial pathology. The choice of antigen mismatch impacts the ability to appropriately address these questions.
Transplantation across complete MHC barriers permits a comprehensive evaluation of immune responses that are known to be involved in organ transplant rejection. This includes direct CD4 and CD8 T cell recognition and targeting of foreign peptide-MHC presented by graft-derived cells, indirect CD4 (and possibly CD8) T cell recognition and targeting of graft-derived alloantigens presented by recipient antigen presenting cells, and antibody-mediated recognition of alloantigens on vascular cell surfaces 8. However, the vascular response to injury in complete MHC-mismatched experiments may be different than that observed clinically. Johnson et al. 9 showed that, in aortic interposition grafts transplanted across a complete MHC mismatch barrier, most of the neointimal cells are of recipient origin and not of donor origin. This is different than that observed in human transplants where most intimal smooth muscle cells are of donor origin 9,10. To account for this limitation, alternate experimental models that involve grafting across minor histocompatibility antigen mismatches have been developed that trigger vascular responses that more closely resemble those observed in clinical transplantation 11. While these alternate models allow for important conclusions to be made regarding the vascular responses that drive the development of TA, the immunological processes that cause vascular rejection in minor histocompatibility antigen mismatched grafts do not completely re-capitulate those which occur in the clinical setting. For instance, minor histocompatibility antigens are recognized poorly by graft reactive antibodies 12. Given the above considerations, it is important to consider the pathological question being examined when choosing the type of antigen mismatch used in an aortic interposition model. Here we describe a detailed protocol for murine aortic interposition grafting. We describe interposition grafting between complete MHC-mismatched mice but the same protocol is used for grafting across other antigen mismatched mouse strains.
All the protocols in this study were reviewed and approved by the Simon Fraser University animal care ethics committee. Use Balb/cYJ (H2d) donor mice and C57Bl/6 (H2b) recipient mice to examine allogeneic reactions. Mice are used for experiments between the ages of 8 to 12 weeks. Use either female or male mice. Syngeneic controls consist of aortic segments from C57Bl/6 donors into C57Bl/6 recipients.
1. Donor and Recipient Preparation
Note: Both the donor and recipient are anesthetized and prepared before the surgery to minimize ischemia of the graft. Injectable anesthetics are used in the protocol to prevent obstruction of the animal by equipment needed for the delivery of inhaled anesthetics. However, if desired, inhaled anesthetic is an appropriate alternative. From the initial injection of the anesthetics, the entire procedure takes approximately 90 min to complete. Ischemic time of the graft is less than 30 min.
2. End-to-end Anastomosis Procedure
3. Tissue Collection
Note: Pre-determined end points range from 3-60 days depending on the type of analysis desired and the nature of the antigen mismatch. Generally, intimal thickening is robust at day 30 after transplantation across complete MHC mismatched mouse strains.
4. Morphological Analysis of Grafts
Note: TA is characterized by intimal thickening 13. In this model, intimal thickening and a resultant reduction in the size of the lumen are reflective of the severity of immune-mediated vascular injury. Syngraft controls are used to determine the baseline characteristics of vessels in the absence of allogeneic immune responses. These controls also permit evaluation of arterial damage that occurs as a result of the surgical procedure. There should be no intimal thickening in syngrafts.
In this model, the abdominal aorta from a Balb/cYJ mouse is interposed into the infrarenal aorta of a C57Bl/6 recipient. This permits a comprehensive evaluation of alloimmune responses that target allograft arteries. Immune-mediated vascular injury in this model initiates vascular reparative responses that culminate in intimal thickening, luminal narrowing and recruitment of immune cells (Figures 1 and 2). These criteria then serve as a read-out for the severity of alloimmune responses, vascular rejection, and TA. Success of the procedure can be assessed by the development of robust intimal thickening of allograft artery segments and the absence of intimal thickening in syngraft controls. Clinically relevant immunosuppression may be used with this model to more closely resemble clinical transplantation. This model may also be used with transgenic animals to study the effect of specific proteins/pathways in alloimmune responses as we have done 14.
Allograft aortic segments develop intimal thickening
The amount of immune-mediated allograft vascular damage was examined by quantifying intimal thickening and luminal narrowing in grafted aortic segments at day 30 post-transplantation. Allograft artery segments develop significant intimal thickening and luminal occlusion and no changes are observed in syngraft control artery segments (Figure 1).
Accumulation of leukocytes in allograft aortic segments.
The accumulation of leukocytes in allograft arteries was examined by quantifying the number of CD4 T cells, CD8 T cells, and macrophages (Mac-3)15 by immunohistochemistry. Mac-3 was used to stain for macrophages in this study although other markers, such as F4/80, can also be used 16. CD4 T cells, CD8 T cells and macrophages were detected in the intima of allograft arteries (Figure 2)
Figure 1: Histological analysis of grafted arteries. (A) Abdominal aorta segments from syngeneic and allogeneic mice were interposed into the resected abdominal aortas of C57Bl/6 mice. The grafted arteries were harvested at day 30 post-transplant. Representative photomicrographs of elastic van Giesen stained arteries are shown. Scale bar = 0.1 mm = 100 µm. (B) Diagram depicting how the different layers of the vessel are measured, L: lumen, E: endothelial layer, I: intima, M: media, A: adventitia. (C) Quantification of intima/media ratio and % luminal narrowing from 30 day syngeneic (n = 3) and allogeneic (n = 6) transplants, *P <0.05. Please click here to view a larger version of this figure.
Figure 2: Immune cell accumulation in allograft arteries. Representative photomicrographs of allograft aortic segments immunohistochemically stained for (A) CD4, (B) CD8, and (C) Mac-3 and counterstained with hematoxylin are shown. Scale bar = 0.1 mm = 100 µm. Please click here to view a larger version of this figure.
We have described a protocol for aortic interposition grafting in mice that is useful for studying immune-mediated vascular rejection and TA. This model can be used to investigate the causes of TA as well as the development of novel therapeutic strategies. It has been used in the past to establish an essential role of adaptive immunity, cytotoxic T cell responses, cytokine-mediated CD4 T cell effector responses, and antibody-mediated graft damage in TA 14,17-21. Artery transplantation in mice is difficult due to the obvious small size of the animal; however, with practice and due diligence, successful surgeries can be accomplished. Success depends on the patency of the vessel. This involves ensuring that the graft is not damaged by improperly handling the vessel with forceps, constriction of the vessel lumen, suturing the back wall of the vessel, and repetitive suturing of the same site. Leakage of the vessel is also problematic and requires care to ensure that the number and position of the stitches are divided evenly around the vessel wall. It is also essential that graft ischemia is minimized to less than 30 minutes. With this, a success rate of greater than 95% can be attained.
The aorta is the largest artery in the mouse so this procedure is the simplest microsurgical approach for investigating vascular rejection in this model animal. Also, the aortic segment is grafted in a physiologically relevant location, experiences normal blood flow, and intimal thickening develops rapidly. The other main model used for the assessment of vascular rejection and TA is heterotopic heart transplantation, which has the advantage of examining the development of TA in coronary arteries and in the context of heart transplantation that is the most relevant clinical scenario for TA. However, heterotopic heart transplants are placed in a non-physiological location within the body (usually anastamosed to the vena cava and aorta in the abdomen) and the heart does not pump blood through the ventricles due to the retrograde nature of the blood supply into the transplanted heart. As such, the nature of blood flow through the coronary tree may be different from what is normally experienced by the coronary vasculature 22,23. Also, strategies to overcome acute rejection of the heart must be incorporated in order to evaluate arterial changes. In both models, it is important to carefully choose the type of antigen mismatch utilized in order to be able to appropriately address specific questions related to vascular rejection and TA.
In summary, aortic interposition grafting is a powerful technique for investigating immune-mediated arterial damage and TA. It can be routinely mastered with practice and diligence on the part of the researcher. Once mastered, this procedure can be modified for the interposition grafting of other arterial segments, such as the carotid artery, that may enable the examination of additional scientific questions. Also, the use of this model can be extended beyond the study of transplantation. Interposition grafting of arteries can be used to introduce arterial segments from modified (e.g. transgenic or lipid-fed) mice into non-modified counterparts, or vice versa, to isolate the biological effects of molecules to artery wall cells versus non-arterial wall cells 24,25.
The authors have nothing to disclose.
This work was supported by grants from the Canadian Institutes of Health Research and Heart and Stroke Foundation of BC & Yukon (JCC).
Name | Company | Catalogue | Comments |
C57BL/6J (H-2b) | Jackson Laboratories, Bar Harbour ME | Strain# 000664 | |
Balb/cBYJ | Jackson Laboratories, Bar Harbour ME | Strain# 001026 | |
Ketamine Hydrochloride Injection USP 100 mg/ mL | Ketalean | DIN 00612316 | |
Xylazine Injection 20 mg/mL | Rompum | DIN 02169592 | |
Ketoprofen Injection 100 mg/mL | Anafen | DIN 01938126 | |
Butorphanol Tartrate injection 10 mg/mL | Torbugesic | DIN 008450000 | |
Buprenorphine Injection 0.3 mg/mL | Reckitt Benckiser | B.N. 5241 | |
Atipamezole hydrochloride sterile injectable solution | Antisedan | DIN 02237744 | |
Heparin Sodium Injection, USP, 1000 units/mL | McKesson Distribution | DIN 02264315 | |
Tears naturale ophthalmic ointment | Alcon | DIN 02082519 | |
Stereomicroscope | Leica | M80 | |
0.9% Sodium Chloride, sterile | Baxter Corporation | ||
Lactated Ringer’s solution, sterile | Baxter Corporation | ||
0.9% Sodium Chloride Injection, sterile, 10 mL | Baxter Corporation | ||
Alcohol Prep Pads | Loris | ||
Povidone Iodine | Betadine | ||
Chlorohexidine Gluconate 4% w/v | Germi-Stat | ||
Black Polyamide Monofilament | AROSurgical Instruments | T4A10Q07 | |
Suture, 10-0 suture, 70 microns | Corporation | ||
Blue monofilament suture 5-0, P3 needle | Ethicon | 8698G | |
1 ml Syringe | BD | REF 309659 | |
10 ml Syringe | BD | REF 309604 | |
1cc TB insulin syringe with 28G 1/2 | BD | REF 309309 | |
25G 7/8, hypodermic needle | BD | REF 305124 | |
27G 1/2, hypodermic needle | BD | REF 305109 | |
Colibri Retractor- 1.5cm spread 4cm | Fine Science Tools | 17000-04 | |
S&T CAF-4 Clip applying forceps, without lock | Fine Science Tools | 00072-14 | |
Supergrip forceps, S&T | Fine Science Tools | 00632-11 | |
Medical No.5 forceps | Fine Science Tools | 11253-20 | |
Lexer Baby Scissors | Fine Science Tools | 14078-10 | |
Micro Adson forceps serrated | Fine Science Tools | 11018-12 | |
Vannas-Tubingen microscissors | Fine Science Tools | 15003-08 | |
Micro clamps, b-1; 3.5mm x 1mm; 7mm length | Fine Science Tools | 00396-01 | |
Graefe-forceps, 10cm 1×2 teeth | Fine Science Tools | 11054-10 | |
Castroviejo with lock and tungsten jaws | Fine Science Tools | 12565-14 | |
Hot glass bead sterilizer | Inotech 250 | IS-250 – Steri-250 | |
Non-woven gauzes | Progene | ||
Cotton Tipped Applicators | Puritan | ||
Beard Trimmer | Wahl | ||
Heating pad | Sunbeam |