Basic And Translational Research Of Machine Perfusion Intervention On The Reperfusion Injury Of Limb Ischemia

Background:Acute limb ischemia (ALI) is a potentially life-threatening clinical event that occurs when a blood flow of extremity is sudden interrupted due to either embolic vascular occlusion or thrombotic vascular occlusion. Severe acute limb ischemia is a vascular emergency while the early diagnosis and rapid intervention in restoration of perfusion are critical in order to salvage the ischemic extremity, whereas delay may result in morbidity,including limb amputation, and mortality. Despite many surgical improvements during the last decades, the extent of injury associated with re-perfusion after ischemia that results in postreperfusion syndrome remains high. Many basic and clinical researches focus on how to intervene limb ischemic re-perfusion injury and control the initial re-perfusion that modifies the conditions of re-perfusion and re-perfusate composition, this will open the new door that approach to salvage extremities and life-threatening condition from severe complication. However, Machine Perfusion (MP) has regained clinical interest since it reduces ischemic re-perfusion injury significantly and has a high potential for improving clinical outcome. This study is aim to establish limb ischemia perfusion models based on perfusion machine, and then explore the protection function of machine perfusion for limb ischemia in vivo and ex vivo.Methods:The first part is based on the perfusion machine. Establishment of animal limb ischemia perfusion model in vivo and ex vivo. Then taking perfusion liquid for CK, CKMB, LDH, GLU testing.In the second part, the experiments are carried out in isolated perfused limbs of rat and pig. The lower limbs were divided into machine perfusion group and static cold storage group,5 rats in each group. In machine perfusion group, a certain perfusion flow rate and pressure was set, and perfusate samples were taken in Oh, 1h and 3h, after 3h muscle tissue samples, serums and perfusate fluids were stored in liquid nitrogen at-80℃ within 30 minutes while static cold storage group was preserved for 3h. Perfusate samples were collected and tested for CK, CKMB, GLU and LDH. Muscle dried/wet weight ratio (D/W) and MTT were compared for tissue edema and viability. MPO and MDA were evaluated for oxidative status imbalance.In the third part, the in vivo rat models of limb ischemia were used to conduct perfusion experiments. Rats were anesthetized and induced limb ischemia using ligation of femoral vessels by tourniquet at femoral head. After 1h of occlusion, the inferior epigastric artery was selected for re-perfusion via machine perfusion. The protocol included flow-controlled perfusion (lrmp) of the limb with 50mL heparin for 1 hour without additional solution. Perfusion fluid was collected at Omin,30min and 60min and placed into liquid nitrogen at -80℃, while femoral vessels were released after 3h of ischemia. Perfusate samples were tested for CK, CKMB, GLU and LDH. Muscle dried/wet weight ratio (D/W) and MTT were compared for muscle activity. MPO and MDA were evaluated for oxidative status imbalance. All analyses were performed with SPSS, version 22.0. Statistical significance was accepted for values of P<0.05.Results:(1) Based on the perfusion machine, successfully established a kind of limb ischemia perfusion model ex vivo and in vivo, the results of CK, CKMB, LDH, GLU level show that the MP model is feasible and stable.(2) The results of rat limb perfusion ex vivo show that glucose level, LDH level, CKMB level in MP perfusion group were significantly reduced (P< 0.05); MTT results of MP group showed significantly higher than that of SCS group (P< 0.05); D/W rat showed that there is no significant difference in the tissue edema degree of MP and the SCS (P< 0.05).Ex vivo pig ischemia limb perfusion experiment results show that MTT level of MP group is significant higher than SCS group (P< 0.05); D/W rate of MP is significantly higher than that of SCS (P< 0.05). HE results indicate the injury of ischemia limb was reduced by machine perfusion.(3) In the results of machine intervention on acute ischemia re-perfusion injury in rats in vivo, we found that after machine perfusion, the right lower limb ischemia perfused by machine fluid within 1 h perfusion in GLU, LDH, CK, CKMB gradually decline, down significantly at 20 min to 40 min (P<0.05). Sodium, potassium and chloride ions in perfusion liquid has no obvious change; MDA level of MP group was decreased significantly; For the blood tests in rats acute limb ischemia after machine perfusion found decreased blood levels in LDH and CKMB, and glucose levels then controlled group.Conclusion:(1) This project successfully developed the ex vivo model of lower limb acute ischemia by rat and porcine, then developed the in vivo model of acute ischemia in rat lower limbs. All these models were feasible and provided basis for studying the mechanism and transformation research of ischemia reperfusion injury in lower limbs.(2) Through further investigations, our intervention in the limb ischemia by controllable and precise machine perfusion method reduced the ischemia reperfusion injury as proposed. So machine perfusion could be a new key for studying the mechanism and cure of acute ischemia reperfusion injury in lower limbs.(3) Related mechanism study in machine perfusion show that MP can restrain the oxygen free radical damage, and further reducing inflammation cell reaction and cell apoptosis. This research plays an important role on further study in machine perfusion mechanisms for protection of limb ischemia.(4) How to achieve the goal of in vivo model for ischemia and perfusion in lower limbs is the focus of preclinical studies, results of our project showed that machine perfusion could control and reduce acute ischemia reperfusion injury in rat lower limbs. Thus, my research would provide evidence for clinical use of machine perfusion for ischemia in lower limbs.