Cryopreservation and replantation of amputated rat hind limbs

Abstract

Background: In spite of the relatively high success rate of limb replantation, many patients cannot undergo replantation surgery because the preservation time of an amputated limb is only about six hours. In addition, although allotransplantation of composite tissues is being performed more commonly with increasingly greater success rates, the shortage of donors limits the number of patients that can be treated. So the purpose of this study is to examine the feasibility of cryopreservation and replantation of limbs in a rat model.

Methods: Twelve five-month-old Sprague-Dawley rats were divided evenly into group A (above-knee amputation) and group B (Syme's amputation). One hind limb was amputated from each rat. The limbs were irrigated with cryoprotectant, cooled in a controlled manner to -140°C, and placed in liquid nitrogen. Thawing and replantation were performed 14 days later.

Results: In group A, the limbs became swollen after restoration of blood flow resulting in blood vessel compression and all replantations failed. In group B, restoration of blood flow was noted in all limbs after replantation. In one case, the rat chewed the replanted limb and replantation failed. The other five rats were followed for three months with no abnormalities noted in the replanted limbs.

Conclusions: Limbs with a minimal amount of muscle tissue can be successfully cryopreserved and replanted.

Figure 1

Limb harvesting. (A) Preoperative preparation. (B) Amputated limb. (C) Amputated limb in cryopreservative before cooling.

Figure 2

Cooling curve.

Figure 3

Scatter plot of double-parameter flow cytometry. Human SV40 transfected osteoblasts; dots at lower left quadrant (H3) were viable cells (FITC-/PI-); dots at upper right quadrant (H2) were non-viable cells, which were necrotic (FITC+/PI+); dots at upper left quadrant (H3) were apoptotic cells (FITC+/PI-).

Figure 4

Intra- and postoperative images. (A-C) Above-knee amputation group; (A) Limb post freezing and rewarming is swollen compared to the healthy limb. (B) Immediately after anastomosis and (C) attempted restoration of blood flow marked muscle swelling was noted. (D-G) Syme’s amputation group; (D) Immediately after replantation the limb is pink indicating adequate blood flow. (E) Ten days after replantation no signs of necrosis were noted. (F) At two months after replantation the limb is viable. (G) Angiography at two months after surgery showed blood flow in the transplanted limb, though a reduced number of blood vessels.

Figure 5

Electron microscopy. (A) Normal skeletal muscle obtained after severing the rat limb but before freezing. Regularly arranged myofibrils, clear z-lines, dense mitochondrial cristae, and intact mitochondrial membranes without swelling were seen. (B) Skeletal muscle of a failed replantation after cryopreservation. Images were obtained four hours after reperfusion. Some myofibrils were ruptured with unclear z-lines, the mitochondria exhibited maximal swelling, crests were disarrayed, and parts of the myofibrils showed vacuolation.