Improved Cuff Technique and Intraoperative Detection of Vascular Complications for Hind Limb Transplantation in Mice

Abstract

Background: Vascularized composite tissue allotransplantation (VCA) from a cadaveric donor has now become a clinical reality and the treatment modality of choice for patients with devastating injuries, deformities, and complex tissue defects. However, many VCA patients experience severe toxicities due to the strong immunosuppression required secondary to high antigenicity of the grafts. To improve immunosuppressive protocols for VCA, feasible and reliable preclinical models are necessary. The purpose of this study was to introduce new techniques to an established preclinical VCA model to accelerate future investigations.

Methods: C57BL/6 (H-2^b) and BALB/c (H-2^d) mice were used to perform VCA as recipients and donors, respectively. Surgery time, success rate, associated complications, and mortality were analyzed. Blood flow in grafts was interrogated with laser speckle image (LSI).

Results: A nonsuture cuff technique was used with the abdominal aorta for end-to-end anastomosis. The cuff technique demonstrated efficiency for donor surgery (52 ± 10 minutes for donor vs. 45 ± 8 minutes for recipient surgery). Successful revascularization was achieved in 27 (90%) of 30 transplants. The majority of surgical complications occurred within 48 hours including artery occlusion, venous occlusion, cerebral stroke, and minor bleeding without mortality. LSI was useful in detecting intraoperative vascular complications with display patterns predictive of complication type.

Conclusions: The described techniques may facilitate a more efficient heterotopic hind limb transplantation mouse model of VCA.

FIGURE 1

Representative photomicrograph for a donor’s surgery via a dissecting scope (magnification, ×10-15) shows the AA (A) and anchored with two 6-0 silk sutures (B). C, D, Inferior mesenteric artery was identified and cauterized. D, E, Right common iliac artery was anchored with 9-0 nylon suture and ligated. F, Left femoral vein was anchored with 6-0 black silk, and left internal iliac (arrow) and medial femoral (arrowhead) arteries were identified. G, A whole arterial segment of the donor. H, The AA was dissected with microscissors. I, J, Middle caudal artery (arrow) was identified, and ligated with 9-0 nylon suture tie.

FIGURE 2

Surgical procedures of nonsuture cuff technique. A, The femoral vein (arrow) was cut near left inguinal ligament. B, Microtubes for femoral vein (v) and the AA (a). C, Femoral vein was mounted and secured with 11-0 nylon suture ties. D, Heparin solution was perfused through the AA stump. E, The AA was pulled through a microtube, and then (F) the AA was everted around the microtube. G, The AA was secured with 11–0 nylon suture ties. H, The completed donor graft including the AA (a), femoral vein (v), skin, fat, muscle, and femur (arrow).

FIGURE 3

Representative photomicrograph for the recipient procedure via a dissecting scope (magnification, ×15-20) shows left EJV (arrow) and CCA (arrowhead) (A). B, End-to-end anastomosis between the donor AA and the recipient CCA secured with a 9-0 nylon suture tie. C, A cuff of the donor femoral vein was inserted to the recipient’s EJV after applying a vascular clamp, and secured with a 9-0 nylon suture tie. D, Revascularization after removing clamps. E, The viable transplanted graft. F, Skin repair with interrupted 6-0 black silk sutures, and a mouse nipple (arrow) was recognized.

FIGURE 4

H&E characteristics and gross findings of grafts 8 days after VCA. A-C, Severe allogeneic graft rejection was identified with aggressive lymphocytes infiltration, vacuolization of muscle fiber, and skin color change. D-F, Well-tolerated allogeneic graft with tacrolimus showed minimal lymphocytes infiltration in the graft and normal hair growing in the skin. G-I, Syngeneic graft showed intact skin and muscle.

FIGURE 5

LSI device setup (A) Platform position and computer attachment. Gross speckle image and LSIs in left groin skin before VCA (B), in the AA (a) and the femoral vein (v) just after revascularization (C), and in the skin after the completion of VCA (D). E, Quantitative assessment of relative the graft blood flow before and after VCA (n = 4, P = 0.79). ns, not significant.

FIGURE 6

Intraoperative detection of vascular (a; AA, v; femoral vein) complications, gross speckle image and LSIs. A, No blood flow in “a” and “v” was detected in a case of arterial obstruction. B, No blood flow in the skin 1 day after arterial obstruction. C, The skin necrosis from ischemia after VCA. D, Blood flow was measured in “a” and the graft but “v” in a case of venous obstruction. E, Blood flow was detected in “v” after fixing venous anastomosis. F, 1 day after VCA.