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Evaluation Of The Preservation Solution On Ischemia Reperfusion Injury In A Vascularized Composite Allograft Hindlimb Model
Sara Rostami, Postdoctoral research fellow1, Siba Haykal, MD PhD FRCSC1,2.
1Latner Thoracic Surgery Research Laboratories, Division of Thoracic Surgery, University Health Network, Toronto, ON, Canada, 2Division of Plastic and Reconstructive Surgery, University of Toronto, Toronto, ON, Canada.

Purpose: Ischemia is clinically an inevitable factor following harvesting an organ and the role of Ischemia-Reperfusion Injury (IRI) is often underestimated which can determine graft survival, function and rejection rate. Although an optimal preservation solution can play a pivotal role in attenuating ischemia-reperfusion injury, there is no efficient preservation solution for preservation of Vascularized Composite Allotransplantation (VCA) during ischemia. Here, we tend to evaluate tissue-specific damage and the effect of different preservation solutions in an allogeneic rat hind limb.
Methods: To assess ischemia reperfusion injury effects on different tissues in VCA, a modified osteomyocutaneous flap was used. The donor flap composed of various tissues including, muscles, skin, bone, nerve and vessels were harvested between mid-femur to distal tibia. For the ischemia-reperfusion group, twelve Sprague Dawley rats were divided into 4 groups and the harvested donor flaps were flushed with 4 different preservation solutions including, heparinized-saline, heparinized-Perfadex, heparinized-University of Wisconsin (UW) or heparinized-histidine-tryptophan-ketoglutarate (HTK) through the vessels. It was placed at 4C for 6 hours. The donor flap was then hetertopically transplanted into the recipient by incising the skin in the gluteal area and creating a tunnel from gluteal area to groin area of the recipient. Then, the flap was inset and femoral vessels were passed anteriorly through the tunnel and anastomosed to the recipient femoral vessels in groin area. The flap was allowed to reperfuse for a total of two hours after which the rats were euthanized.
For the ischemia group, the contralateral hind limb of the Sprague Dawley rats were procured, flushed and stored in heparinized-saline, heparinized-Perfadex, heparinized-UW or heparinized- HTK for 6 and 12 hours at 4C.
The samples were assessed histologically by utilizing hematoxylin-eosin staining, TUNEL assay and immunohistochemistry for laminin and Cleaved Caspase 3.
Results: The tissues were assessed immunohistochemically for presence of cleaved caspase-3, Tunnel and laminin for evaluation of apoptosis and changes in extracellular matrix respectively.
Histology revealed that after 6-hours of cold ischemia time, the tissues maintain their morphology but at 12-hours of cold ischemia time the muscle especially looks different, therefore, we decided to reperfuse the flap for 6 hours. Although apoptosis was not significant at 6-hours of cold ischemia, changes in structure of the tissues were noticeable including, centralization of nuclei in skeletal muscle cells, loosening of the muscle tissues, mild spongiosis in epidermis and disintegration of basement membrane in skin were seen.
After perfusion, we noticed that the most effected tissues after 6-hours of cold ischemia time were skeletal muscle and then vessels. Immunohistochemistry results revealed that the apoptosis was more significant in IRI group although we noticed some differences in different preservation solutions.
Conclusion: Preservation solution is important to prevent ischemia repetition injury as evidence by markers of ischemia, apoptosis and necrosis.


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