Transplantasyon ve Replantasyon Sırasındaki Uzun İskemi Probleminin Çözümüne Dair Ekstrakorporeal Dolaşım Modeli

Abstract

The standard composite tissue storage and transportation method during limb transplantation and replantation is cold storage. However, although the cold storage method is thought to provide sufficient time for transportation of extremities with large muscle mass, prolonged cold storage cannot prevent ischemia-reperfusion injury. Consequently, function and survival of the replanted or transplanted extremity can be unsuccessful in prolonged cases of cold storage. Alternatively, if vascularized composite tissues are stored in ex-situ perfusion systems, sufficient oxygen is provided to the ischemic tissue, the temperature of the tissue is kept at the desired level, and the metabolic waste products are eliminated. Therefore, it is thought that the inflammatory response will decrease and the function and survival of the replanted extremity will increase. In order to find a solution to the ischemia problem in replantation/transplantation surgery, in this study we aimed to compare results of the cold storage method and the perfusion system storage method. Following the institutional review board approval, an experimental study on 2 groups of rats using the ischemia-reperfusion model was performed. Both groups underwent 1 hour of reperfusion after 6 hours of ischemia. Superoxide Dismutase (SOD), catalase, Total Antioxidant Status (TAS), Total Oxidant Status (TOS) and total thiol levels in gastrocnemius muscle and blood samples were biochemically analyzed using the Enzyme Immun Assay (ELISA) technique to determine oxidative damage levels. In addition, TNF-α, NF-κB and IL-10 levels were measured using the ELISA method in the same samples to evaluate the degree of inflammation. Additionally, biochemical parameters (Na+, K+, LDH, CK, Myoglobin and Lactate) were analyzed to demonstrate the inflammatory changes caused by ischemia-reperfusion injury in the blood. Apoptosis was evaluated by measuring the levels of Bax and Bcl-2 proteins in gastrocnemius muscle samples, using the ELISA method. Muscle fiber damage was evaluated by mitochondrial cytochrome oxidase (cox) staining and hematoxylin/eosin staining methods. Skin findings of ischemia were observed from skin samples taken from the extremity. It was found that the extracorporeal circulation system causes less oxidative damage than the cold storage system. TAS, SOD and Total Thiol levels in muscle homogenate and blood samples were found to be significantly higher in the perfusion system. TOS levels were found to be higher in the cold storage system than in the perfusion system. Inflammatory damage was lower in the perfusion system than in the cold storage system. The amount of TNF-α and NF-κB, which indicate inflammatory damage, was lower in the perfusion system, and the amount of IL-10, which an anti-inflammatory cytokine, was higher in the perfusion system. This study was designed to evaluate the early phase of ischemia reperfusion injury therefore it was found that bax and bcl-2 levels, which are markers of apoptosis, did not differ between the two groups. Considering the biochemical parameters, CK, Myoglobin values, which are muscle breakdown products, LDH and lactate values, which show metabolic load, were found to be higher in the cold storage group than in the perfusion group. No difference was found between the skin findings after ischemia reperfusion between the two groups; however, after Cox staining and hematoxylin/eosin staining of the muscle samples, significant mitochondrial damages were observed only in the cold storage system. In conclusion, the extracorporeal circulation model caused less inflammatory and oxidative damages than the cold storage model in the early phases of ischemia reperfusion injury. Therefore, the use of extracorporeal circulation systems in the replantation or transplantation of extremities with large muscle mass may produce more successful results.