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Background Revascularization of amputated extremities after prolonged ischemia is complicated by reperfusion injury. We assessed ischemia/reperfusion (I/R) injury of porcine extremities after prolonged preservation using extracorporeal circulation (ECC). Methods Forelimbs of 32 pigs were divided into four groups based on ischemia times: group I: 6 h, group II: 12 h, group III: 0 h plus replantation, and group IV: 6 h plus replantation. Limbs were perfused with autologous blood using ECC for 12 h except group II with only 5 h perfusion. Limbs from groups III and IV were heterotopically replanted with a 7-d follow-up. Contralateral limbs served as controls in all groups. Tissue, plasma, and serum were analyzed for the extent of I/R injury. Results No significant differences in tissue wet/dry ratios were found within or between groups. This finding was confirmed by histology, except for an increased damage in group IV muscles compared with baseline (P = 0.016). Complement C3 deposition was only increased in group IV muscle (P = 0.031), group II nerves (P = 0.046), and group II vessels (P = 0.037). Group IV muscle and nerve tissues were the only ones with significant IgM antibody deposition (P = 0.031) at end of perfusion. Values were normal again after replantation. Reduced complement activity and elevated IL-6, IL-8, MCP-1, VEGF, PDGF-bb, bFGF, and complement split products were found during perfusion but were normal again after replantation. Staining for heparin sulfate proteoglycans and von Willebrand factor confirmed minimal activation of endothelial cells. Conclusion The results demonstrate that prolonged limb preservation using ECC has minimal impact on I/R-induced tissue injury. Extracorporeal perfusion is a potential limb-preserving technique encouraging further studies for use in limb revascularization.

Stereology is an essential method for quantitative analysis of lung structure. Adequate fixation is a prerequisite for stereological analysis to avoid bias in pulmonary tissue, dimensions and structural details. We present a technique for in situ fixation of large animal lungs for stereological analysis, based on closed loop perfusion fixation.Twenty anesthetized ventilated pigs (30 ± 3 kg) underwent cannulation of the pulmonary artery and ligation of the right hilus. Following circulatory arrest a continuous positive pressure of 12 mbar was applied to the airways and lung perfusion started with the fixative solution (1.5% paraformaldehyde; 1.5% glutaraldehyde in 0.15 M HEPES). In five animals, a single-pass perfusion technique was performed, in 15 subsequent animals, the closed-loop technique was applied. Afterwards, lungs were removed, externally postfixed in the recycled fixative solution, and stored at 4 °C. Fifteen lung specimens underwent stereological analysis with volume estimation and subsequent systematic uniform random sampling for light and electron microscopic analysis.Singlepass perfusion did not result in satisfactory fixation. Left lung closed loop perfusion rate was 0.5-0.7 L/min with total median [min-max] perfusion time of 15 min (11-19). Perfusion pressure was 15 mm Hg (9-33). Subsequent lung analysis revealed well-preserved cell and tissue ultrastructure.The closed loop perfusion technique represents a valuable and reproducible fixation method in large animal models. Pressure controlled fixation perfusion results in high-quality preservation of in situ parenchymal architecture of lungs with or without injury, which is ideally suited for quantitative assessment of lung structure by stereology.