5. 24-Hour Ex-Vivo Perfusion in the Swine Total Hindlimb Model

Introduction: Vascularized Composite Allotransplantation (VCA) is expanding worldwide. Currently, static cold storage at 4°C is the standard preservation method for large extremity segments in transplantation and trauma but has limitations in preservation time. Ex vivo perfusion techniques are gaining interest in VCA preservation. This study presents a 24-hour sub-normothermic perfusion protocol in a swine total hindlimb model, using autologous red blood cells (RBCs) as oxygen carriers. Methods: Five consecutive 24-hour ex vivo perfusions were compared to 14 cold storage controls (12 hours SCS). Limbs were flushed with low potassium dextran solution (LPD). The perfusate included LPD enriched with 2.5 g/dL BSA and supplemented with heparin, methylprednisolone, dextrose, insulin, L-alanyl L-glutamine, and sodium bicarbonate. Washed RBCs were added to achieve a hematocrit of 10–15%. Perfusion pressure was maintained at 60–65 mmHg, and muscle temperature between 28–32ºC. Hourly perfusate analysis and compartment pressure measurements were performed. Perfusate free hemoglobin was measured every 4 hours, and muscle biopsies from the thigh(proximal) and foot(distal) were taken every 6 hours for morphologic assessment and ATP measurement. At 24 hours, neuromuscular stimulation and ICG angiography were performed. Limb weight was recorded at baseline and endpoint. Results: The average animal weight was 39.38 kg, with limb weights of 4.06 kg. Washed RBC volume was 1.1 L, and ischemia time averaged 68.00 minutes. Mean percent weight change was -0.62% in controls and -0.53% in the perfusion group, with no significant differences between groups. In proximal muscles, baseline injury score was 3.03 in perfusion limbs and 2.78 in SCS limbs. At 24 hours, scores were 3.26 and 3.73, respectively, with a significant increase in cold storage controls (p = 0.0240) but no change in the perfusion group (p = 0.9916). In distal muscles, baseline scores were 2.90 (Perfusion) and 2.91 (SCS), increasing to 4.63 and 3.97 at endpoint, with a significant rise in cold storage controls but no change in perfused limbs (p = 0.3533). ATP levels significantly dropped in cold storage controls. In proximal muscle, ATP decreased from 0.573 to 0.18 nmol/μL (p < 0.001), and in distal muscle, from 0.458 to 0.12 nmol/μL at 12 hours (p < 0.001). In contrast, perfusion maintained ATP levels, with no significant decline at 24 hours (proximal: 0.416 to 0.367 nmol/μL, p = 0.1727; distal: 0.315 to 0.267 nmol/μL, p = 0.9759). Limbs retained contractility at 24 hours, though two limbs showed patchy distal ICG signal. Conclusions: We successfully demonstrated the feasibility of 24-hour whole hindlimb perfusion while maintaining stable physiological parameters, preserved muscle morphology, conserved energy stores, sustained contraction response, and no weight change. No significant myocyte damage was observed in both biopsy sites, indicating consistent perfusion throughout the limb. ATP levels remained stable, unlike cold storage controls, which declined. These findings suggest perfusion may extend preservation time 2- to 4-fold compared to cold storage however reperfusion study is needed.Figure 1.: Muscle Analyses. A) HISS scores for proximal muscle and representative images (10x) B) HISS scores for distal muscle and representative images (10x) C) ATP content in proximal and distal muscles (10mg), Asterisk: Hypoxic myocytes, Error bars represent standard deviation.

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