Effects And Mechanisms Of Ischemia-reperfusion Injury After Lung Transplantation In Mice

Part I: Effects of cold ischemia time on ischemia-reperfusion injury and primary pulmonary dysfunction after lung transplantationPrimary graft dysfunction(PGD)is a major cause of morbidity and mortality after lung transplantation.Ischemia–reperfusion injury(IRI)is a key event that contributes to PGD,though complex interactions affect donor lungs status,such as preceding brain death(BD),hemorrhagic shock(HS),and pre-engraftment lung management,the latter recognized as important risk factors for PGD.We hypothesized that a multi-hit isogenic mouse model of lung transplantation is more closely linked to PGD than IRI alone.Left lung transplants were performed between inbred C57BL/6mice.A one-hit model of IRI was established by inducing cold ischemia(CI)of the donor lungs at 0? for 1,72,or 96 hours before engraftment.Multi-hit models were established by inducing 24 hours of HS and/or 3 hours of BD before 24 hours of CI.The recipients were killed at 24 hours after transplant and lung graft samples were analyzed.In the one-hit model of IRI,up to 72-hour CI time resulted in minimal cellular infiltration near small arteries after 24-hour reperfusion.Extension of CI time to 96 hours led to increased cellular infiltration and necroptotic pathway activation,without evidence of apoptosis,after 24-hour reperfusion.In a multi-hit model of PGD,“HS+BD+IRI” demonstrated increased lung injury,cellular infiltration,and activation of necroptotic and apoptotic pathways compared with IRI alone.Treatment with an inhibitor of receptor-interacting protein kinase 1 kinase,necrostatin-1,resulted in a significant decrease of downstream necroptotic pathway activation in both single-and multi-hit models of IRI.Thus,activation of necroptosis is a central event in IRI after prolonged CI,though it may not be sufficient to cause PGD alone.Pathological evaluation of donor lungs after CI induced IRI,in conjunction with pre-engraftment donor lung factors in our multi-hit model,demonstrated early evidence of lung injury consistent with PGD.Our findings support the premise that pre-existing donor lung status is more important than CI time alone for inflammatory pathway activation in PGD,which may have important clinical implications for donor lung retrieval.Part II: Protective ventilation extends tolerable warmischemic time of donor lungs up to 12 hours after cardiac death The pulmonary donor pool would increase substantially if lungs could be donated after cardiac death(DCD).However,it is characterized by warm ischemic period,which could be involved in severe ischemia reperfusion injury(IRI)with early graft dysfunction,and there have been ethical and legal obstacles since administration of heparin must be done immediately after cardiac death.Objectives: To identify whether ventilation of DCD lungs without administrating heparin after cardiac death could improve graft function and to determine the molecular pathways by which they contributed to IRI.Methods: Firstly,A mouse surgical model of transient unilateral left pulmonary artery clamping(PAC)4 or 5 hours without bronchial involvement was used to create ventilated lung I/R injury for assessing lung function.Then orthotopic left lung transplants(OLT)were performed between inbred C57BL/6 mice.DCD Lungs were randomly assigned to three groups,treated with PAC(PAC-LTx),ventilation(37V4-LTx),or non-touch(37N4-LTx)for 240 minutes WI period.Graft function,proinflammatory cytokines,and pathway activation were measured 24 hours after transplantation.Results: Non-touched lungs developed airway epithelia swelling,had highly impaired blood gas and a significantly increased neutrophil and monocyte infiltration.The ventilated lungs demonstrated excellent blood gas and unchanged inflammatory cell and cytokine(IL-6,HMGB1 and MCP-1)infiltration when compared with grafts from PAC-LTx.These effects were mediated by apoptosis pathway activation according to higher expression of cleaved caspase-3 and caspase-9 in 37N4-LTx group.Donor lung tissue adenosine 5'-triphosphate(ATP)levels were significantly higher in the 37N4-LTx group than in the 37V4-LTx at the end of 240 minutes after WI.Conclusions: Donor protective ventilation strategy can increase warm ischemic time in combination with avoiding heparinization,which facilitate the use of DCD lungs for transplantation...