Background
Tissue ischemic changes after amputation of limbs are dependent on time and can determine the success or failure of replantation and/or allotransplantation. The aim of this study was to elucidate changes that occur in skeletal muscle cells during ischemic conditions imparted by common tissue storage practices.
Methods
A total of 23 porcine forelimbs were amputated (mean weight 3 kg per limb) and stored under 4 different conditions for 12 h, as follows: a) storage on ice slurry at 4C b) storage on ice slurry at 4C ice after flushing with University of Winsconsin solution (UW), c) ex vivo perfusion with acellular solution at 10C, and d) storage at room temperature. Biopsies of the muscle tissue were taken every 2 h during storage and assessed for histomorphologic changes, apoptosis, glycogen and ATP storage.
Results
Histopathology did not show significant differences in cellular and tissue structure after 12 h of storage, nor did markers for apoptosis. Staining for intracellular glycogen revealed complete loss of glycogen in groups (a), (b) and (d) after 6 h of storage, whereas glycogen stores remained unchanged in the perfusion group. Total ATP depletion in cold storage samples was noted after 2 h.
Conclusion
Hypoxia of a duration of up to 12 h does not seem to be immediately lethal for skeletal muscle tissue. Early loss of ATP and depletion of intracelullar glycogen stores were observed even when metabolism was slowed down by cooling to 4C; these may lead to cellular and molecular changes that might be responsible for subsequent damage, injury and cell death after reperfusion.
Figure 1. Intracellular glycogen staining with PAS (Periodic acidðSchiff stain) of muscle tissue at the time of amputation (0h) and after 6 h of preservation on ice or in our perfusion device. After 6 h on ice, de-coloration indicates consumed intracellular glycogen and therefore loss of cellular energy supply.
