Although the orthotopic rat hind limb model has been performed with great success over the past decades, several attempts have been made to make it less time consuming and technically demanding. Since the suture connection of blood vessels seemed to be the surgically most demanding step in the whole procedure, more feasible anastomosis techniques have been applied. One such technique uses a polyimide or polyethylene cylinder, slightly larger in diameter than the blood vessel itself, which is pulled over the donor vessel so that the vessel wall can finally be everted over the cuff. For anastomosis , the recipient’s blood vessel is simply pulled over the donor vessel and cuff, which finally results in an end-to-end anastomosis of both vessels (Fig. 5.1). This delicate technique, which establishes endothelial contact between both donor and recipient vessel, was first applied by Alexis Carrel, a French surgeon and biologist who was awarded the Nobel Prize in Physiology or Medicine in 1912 for his pioneering vascular suturing techniques [20]. For investigation of ischemia-reperfusion-related injuries, where prolonged cold storage leads to extremely fragile vessel walls and renders the conventional suture anastomosis nearly impossible, the cuff technique was able to simplify and shorten the whole surgical procedure and thus provide a rapid and reliable surgical approach to hind limb transplantation in rats [21].

Since orthotopic hind limb transplantation is a traumatic surgical procedure that includes the resection of the recipient limb to make room for an allograft, there are multiple concerns, including intra- and postoperative bleeding, insufficient fixation and stabilization of the graft bone to the recipient bone, and loss of normal gait; these concerns have fueled the development of less traumatic and less time-consuming VCA models. The most frequently used alternatives to the orthotopic hind limb model are heterotopic transplants of myocutaneous or osteomyocutaneous flaps to the recipient’s groin [22]. One of the unique biologic features of a VCA is that its composition as a whole determines immunological outcome [8]. Therefore, heterotopic transplant models bear the advantage that relative sizes of individual graft tissue components can be easily manipulated in order to examine their impact on overall immunogenicity. One example of a heterotopic transplant model using a hind limb that contains the entire femur bone was described by Ulusal et al. [23]. In this case, an epigastric skin flap was used to cover the surgical defect at the recipient site. The authors also reported a decreased risk for postoperative bleeding and embolism since the integrity of the graft’s femur bone could be preserved when compared to the “original” orthotopic hind limb model where osteotomy at the mid-femur level has to be performed using an intramedullary splint to connect both the donor with the recipient femur bone. A similar model containing a smaller allograft which only consisted of the lower leg was developed by Nazzal et al. [24]. A third modified model was described by Tobin et al. [25] in which the osteomyocutaneous graft consisted of the distal femur and proximal tibia and used the femoral artery and vein as the vascular pedicle. Since the recipient’s femoral vessels were used for graft reperfusion, the animal’s foot was solely perfused by its deep femoral vessels. Using this approach, the authors reported a significant reduction in operation time and recipient mortality. These heterotopic allografts are extremely well suited for immunological and ischemia-reperfusion-related studies; however, their functional assessment after transplantation is severely limited because they are not connected to afferent nor efferent nerves.

When it comes to the study of immunologic outcomes after VCA besides skin and muscle, it is crucial to include other tissue components like vascularized bone marrow that may have a substantial impact on the posttransplant immunological behavior of the graft. Zamifrescu et al. provided evidence that the bone component of a VCA, which comprises a permanent source of vascularized bone marrow, is capable of inducing cellular microchimerism at postoperative days 30 and 60 in a heterotopic rat hind limb model. In contrast, animals which only received intravenous suspensions of foreign bone marrow cells were unable to generate a stable donor cell population together with its own [26]. A similar study by Kubitskiy et al. confirmed improved survival rates of hind limbs transplanted along with vascularized bone marrow ; however, their experimental setting was unable to generate stable chimerism in the recipient [27].

Another cellular-based approach to impact on the recipient’s immune system is achieved by a repeated intravenous administration of donor adipose-derived stem cells. In combination with transient conventional immunosuppression, these immunomodulatory cells are capable of suppressing alloreactive T cells while increasing the CD4/CD25/Foxp3 T regulatory cell population in vitro and in vivo, resulting in a significant prolongation of rat hind limb allograft survival [28]. In addition, the authors report significantly elevated levels of donor cell chimerism and upregulation of transforming growth factor-β and interleukin-10 levels, all accounting for the beneficial outcome.

Transplantation of foreign tissues is inevitably paired to a transient stop of blood and nutrient supply to the grafted tissue. Paradoxically, the restoration of circulation results in a profound inflammatory response is commonly referred to as IRI . Another interesting scientific emphasis is therefore centered around the investigation of IRI-related injuries since it might critically influence the outcome of graft and patient survival after reconstructive transplantation [29]. Using the cuff technique for vascular anastomosis , Sucher et al. have established a reliable rat hind limb transplant model to study different aspects of IRI [21]. However, more detailed information about how IRI might contribute to the immunological and functional outcome of a VCA are still under intensive investigation [see also Chapter 22].