*Purpose: The purpose of this study was to evaluate cellular injury and myocyte function of amputated limbs following ex vivo normothermic limb perfusion (EVNLP) compared to traditional cold storage.

*Methods: Twenty human upper extremities were procured from organ donors after brain death. Ten were perfused with an oxygenated colloid solution containing packed red blood cells at 38°C for 48 hours or until the termination criteria were met (maximum arterial pressure >115 mmHg, weight increase >20%, compartment pressure >30 mmHg, tissue oxygen saturation decrease >20%). Electrolyte derangements were managed with partial perfusate exchanges every 3 hours beginning at hour 6 of perfusion. Contralateral upper limbs (controls) were preserved at 4°C. Limb viability was assessed through contractility testing, tissue oxygen saturation, infrared thermography, and indocyanine green (ICG) angiography. H&E and caspase-3 staining were performed. Myocyte injury scores (MIS) and caspase-3 positivity were assessed using the Aperio ImageScope software by two investigators blinded to the experimental group and time. Myocytes were classified as damaged if there was nuclei extravasation, disrupted cell membrane, or distinct fissures within the cytoplasm. Statistical analysis was performed with Kruskal-Wallis and paired t-test.

*Results: Limbs were perfused for 41.6 ± 9.4 hours. For the EVNLP limbs, contractility was observed for a median of 30.5 hours (range 16-40 hours), and maximum contractility was maintained for a median of 8 hours (range 1-15 hours). Control limbs had a contractility score of 0 after procurement. The final weight change was +0.4 ± 12.2% and 0 ± 0% in the EVNLP group and control group, respectively. One limb developed compartment syndrome after 6 hours. Thermography and ICG angiography demonstrated uniform peripheral perfusion of the experimental limbs throughout. Average MIS was 24.7 ± 13.0% and 44.3 ± 30.6% for the EVNLP and control groups, respectively. In the EVNLP group, MIS did not significantly differ among time-points 0, 12, 24, 36, and 48 hours (p=0.46). In the control group, MIS was significantly higher at end time-points (mean = 44.8 ± 4.8 hours) compared to time-point 0 (p=0.009).

*Conclusions: In contrast to hypothermic preservation, EVNLP can halt the progression of myocyte injury for up to 48 hours and maintain normal muscle function beyond 24 hours.

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