Subzero nonfreezing preservation in a murine limb replantation model

This study was designed to examine the most effective temperature for hypothermic storage, without freezing, to prolong ischemic tolerance in an amputated murine hindlimb model. We measured freezing points in the calf muscle and the subcutaneous tissue of the foot in the amputated limbs of Fisher 344 strain male inbred rats. The highest freezing point was -1.5°C, which was recorded in the calf muscle. To prevent freezing in any of the tissues in the amputated limb, the temperature for the lowest nonfreezing preservation was defined as −1°C. The amputated limbs were preserved at subzero nonfreezing temperature (−1°C) and at 4°C for 4, 8, 12, 24, 48, and 72h, and were then transplanted to other inbred rats by microsurgical techniques. We evaluated the vascular patency of the anastomoses by direct observation and performed histological examinations on the seventh day after replantation. Subzero nonfreezing preservation of a limb at −1°C for 72h was significantly superior to hypothermic preservation at 4°C for 72h in terms of anastomotic patency rates (P < 0.05). The histology of skeletal muscles preserved at 1°C for 8h showed greater similarity to the normal situation than the histology of those preserved at 4°C for 8h. Bone viability with osteoblastic activity was maintained in grafted limbs preserved at −1°C for 72h, but in the limbs preserved at 4°C for 72h the bone was not viable, showing no osteoblastic activity. Clinically, the period of ischemia in major limb replantation at normal ambient temperatures is limited to about 6h. In this study, the maximum ischemia time for replantation of a limb containing muscle tissue was prolong to 8h at −1°C, but the maximum ischemia time at 4°C could not be prolonged to 8h. Our results suggest that, in the major replantation of a limb containing muscular tissue, hypothermic preservation at −1°C would be more useful than preservation at 4°C.