2537: The ischemic limb? A timeline of pathophysiological processes in amputated porcine forelimbs

2537: The ischemic limb? A timeline of pathophysiological processes in amputated porcine forelimbs Nicco Krezdorn, Rachel Lopdrup, Mario A. Aycart, Muayyad Alfhefzi, Harriet Kiwanuka, Thet-Su Win, Ericka Bueno, and Bohdan Pomahac Brigham and Women’s Hospital, Boston, MA, USA 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 whenmetabolismwas 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 acid+Schiff 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. CONTACT Nicco Krezdorn nkrezdorn@bwh.harvard.edu Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kvca. © 2016 Nicco Krezdorn, Rachel Lopdrup, Mario A. Aycart, Muayyad Alfhefzi, Harriet Kiwanuka, Thet-Su Win, Ericka Bueno, and Bohdan Pomahac. Published with license by Taylor & Francis. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. VASCULARIZED COMPOSITE ALLOTRANSPLANTATION 2016, VOL. 3, NOS. 1–2, 45 http://dx.doi.org/10.1080/23723505.2016.1234245