We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.
Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner’s time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.
1. Presurgical Preparation
2. Donor Operation
3. Recipient Operation
Figure 1 shows an aortic graft. The white arrows denote the suture lines. A patent graft will show a visible pulse. Figure 2 indicates a typical experiment in which recipient survival was followed for a period of 56 days. One group consisted of wild-type (C57BL/6 x FVB) recipient mice transplanted with BALB/c aorta. The other group, designated “KO” consists of recipients (C57BL/6 x FVB) deficient in expression of heme oxygenase-1, which results in thrombosis of the grafts within 24 hr. Notably, this results in the death of all recipients as shown in the figure. Figure 3 shows echo measurements of the IVC and abdominal aorta in a normal animal and in a transplant recipient. Note that the graft is patent and similar in appearance to the non-transplanted aorta.
Figure 1. A view of a transplanted aorta. The white arrows denote the suture lines.
Figure 2. Kaplan-Meier depictions of survival after aortic transplantation in two groups of mice transplanted with aorta from a BALB/c mouse. “KO” designates recipients deficient in the expression of heme oxygenase-1, which results in thrombosis of the aortic grafts within 24-48 hr. “WT” designates the wild-type littermates. Reprinted from: Carbon monoxide rescues heme oxygenase-1-deficient mice from arterial thrombosis in allogeneic aortic transplantation, Chen B, Guo L, Fan C, Bolisetty S, Joseph R, Wright MM, Agarwal A, George JF. Am J Pathol. 2009 Jul;175(1):422-9 with permission from Elsevier.
Figure 3. Echo imaging of the inferior vena cava (IVC) and abdominal aorta in vivo in a normal mouse (left panel) and in an aortic transplant recipient (right panel). The images were produced with a Visualsonics Vevo 660 instrument. Asterisks denote the lumen of the vessels.
Mouse models of aortic transplantation provide a number of advantages because mice are very well defined immunogenetically9,12,13, and they can be easily manipulated to alter their expression of specific genes, if desired. As noted in the introduction, vascular surgery in the mouse is more difficult than most models because of the size of the vessels. Even the great arteries, such as the aorta are usually no more than 100-200 μm in inner diameter; so manipulating these vessels requires a significant amount of skill and dexterity8,14. The most common complication observed in this surgery by those just beginning to implement the system is hind-leg paralysis, usually caused by thrombosis resulting from injury of intima by rough handling or clamping. Most of the clamps marketed by companies that sell tools suitable for microsurgery create too much pressure to be used for mice, with pressures usually greater than 25 gm/cm2. The clamps we use are smooth, with a pressure of 2 gm/cm2, a pressure that is just enough to achieve hemostasis and not damage the vessels. Some surgeons prefer to use suture rather than clamps. We do not recommend this practice because it is a method that is highly dependent on the ability of the surgeon to gauge the correct pressure and, in our view, can contribute to a lower success rate. With practice, survival rates of 90% or better should be expected.
Suturing technique for the end-to-end anastomoses is a critical skill that is acquired with experience. Depending on prior experience with surgical techniques and manual dexterity, full proficiency may be achieved after 50-100 procedures. More recent studies suggest alternative technologies for anastomoses may become available in the future15.
The primary limitation of this procedure is, like most microsurgical procedures in mice, that a high level of skill is required for successful execution and individuals without excellent fine motor skills may never achieve a high level of proficiency. However, with practice, most individuals can achieve an acceptable survival rate. The small size of the tissues also results in additional constraints because the size of the graft is very small, so the amount of material for subsequent analysis is limiting. The largest advantage of the model is the well characterized immunogenetics of mice, the wide availability of numerous inbred strains, transgenic mice, and knockout mice, allowing for very useful experiments to address molecular mechanisms in vivo.
The authors have nothing to disclose.
This work was funded by the core resource of the NIH P30 O’Brien center (DK 079337).
Item | Company | Catalog No. |
Vascular Clamps | Fine Science Tools | 00396-01 (Size B-1) |
Dumont Forceps | Fine Science Tools | 11293-00 |
10-0 Needled microsuture | AROSurgical | TK-107038 |
Straight scissors | Roboz Surgical Instrument Co. | RS-5620 |
Low temperature cauterizer | Beaver-Visitec International | 8441000 |
Self retaining retractor | World Precision Instruments | 14240 |