Feasibility Study Of Evaluating Donor Liver Quality In DCD Rats By High-throughput Detection Of Cytokines In Functional Warm Ischemic Period

[Objectives](1)To establish a rat model of functional warm ischemia time donor after cardiac death(F-WIT-DCD)liver transplantation.(2)To describe the changes of cytokines during ischemia and reperfusion in DCD rats with different functional warm ischemia time.(3)To explore the feasibility of evaluating the quality of donor liver in DCD rats by the changes of cytokines during functional warm ischemia.[Methods] Part I is the establishment of rat model of F-WIT-DCD liver transplantation.Firstly,learning and training rat orthotopic liver transplantation in vivo;secondly,to simulate the operation of Maastricht III DCD liver transplantation in clinic,I induce blood oxygen stagnation in rats by cutting diaphragm muscle,and dynamically monitor the arterial pressure of rats,to obtain the characteristics of blood pressure fluctuation in rats,and record the time point when rats enter the stage of functional warm ischemia;finally,to establish a stable rat model of F-WIT-DCD liver transplantation.Part ? is the changes of cytokines during ischemia and reperfusion in DCD rats.According to the duration of functional warm ischemia time,the experiment was divided into four groups:(1)living donor liver transplantation control group(LT,n = 4);(2)functional warm ischemia 0 minutes group(0 min,n = 10);(3)functional warm ischemia 15 minutes group(15 min,n = 10);(4)functional warm ischemia 30 minutes group(30 min,n = 10).There was no ischemic treatment in LT group,while the other groups underwent different functional warm ischemia time and all groups had reperfusion period for 6 hours after liver transplantation.Samples were obtained in two stages: immediate donor liver acquisition and 6 hours after reperfusion.Donor liver tissues and peripheral blood were obtained(for the third part of the study).The changes of IL-1??IL-1??IL-2?IL-4?IL-5?IL-6?IL-7?IL-10?IL-12?IL-13?IL-17A?IL-18?GCSF?GM-CSF?M-CSF?GRO/KC?IFN-??MCP-1?MIP-1??MIP-3??RANTES?TNF-? and VEGF were detected by Luminex?200,focusing on the changes of cytokines related to ischemia-reperfusion injury.SPSS 22.0 was used to analyze the single factor variance of each cytokine concentration level,P < 0.05 was identified as significant difference between the groups;R 3.5.2 was used to draw a thermogram to analyze the changes of cytokine concentration during ischemia and reperfusion.Part ? is to explore the feasibility of functional warm ischemic cytokines in evaluating the quality of donor liver in DCD rats.Malondialdehyde(MDA)kit and superoxide dismutase(SOD)kit were used to detect the level of them in donor liver tissues,and H&E staining was used in the other part of liver tissues.Serum samples were prepared by low temperature and ultrahigh speed centrifugation.Alanine aminotransferase(ALT)as well as aspartate aminotransferase(AST)levels were measured.Graph Pad Prism 6.0 was used to test ALT,AST,SOD and MDA,and the data were expressed as mean-standard deviation(x±s).Pathological changes of donor liver were observed by H&E staining,and liver injury score was established according to the results.Using SIMCA 14.1 to do principal component analysis(PCA)of cytokines to explore specific cytokines,and combining with liver injury score and specific cytokines to make ROC regression curve to explore the best combination of indicators for evaluating the quality of donor liver.[Results] In the part I,after the diaphragm was cut off,the blood pressure rose by 10 mm Hg,then decreased gradually,and dropped to 50 mm Hg in about 4 minutes and 30 seconds.The time point of blood pressure was selected as a marker of functional warm ischemia in rats.Rats underwent orthotopic liver transplantation after functional warm ischemia for different periods of time.They recovered about 1-2 hours after the operation and they were in good basic condition and had a slightly poor mobility,drinking water freely.In part ?,(1)A total of 17 cytokines showed significant differences(p < 0.05),including IL-1?,IL-2,IL-4,IL-7,IL-10,IL-12,IL-13,IL-17,G-CSF,GM-CSF,IFN-?,M-CSF,MIP-3?,TNF-?,VEGF and MCP-1.These cytokines showed a gradual downward trend with the prolongation of functional warm ischemia time,and the decreasing range was not the same;(2)After 6 hours of reperfusion,all cytokines had no significant difference between groups;(3)The changes of cytokines in the ischemic and reperfusion periods were different.In part ?,(1)Serum AST and ALT increased gradually with the prolongation of functional warm ischemia time,which confirmed that liver injury increased gradually with the prolongation of ischemia time;(2)SOD in liver tissue increased gradually with the prolongation of functional warm ischemia time,and MDA decreased gradually,reflecting that oxidative stress increased gradually in the process of ischemia-reperfusion injury;(3)H&E staining showed that with the prolongation of functional warm ischemia time,the degree of donor liver injury increased gradually.(4)Principal component analysis showed that IL-2,IFN-? and TNF-? were the most representative cytokines in functional warm ischemia;(5)ROC regression curve showed that the sensitivity of combined IL-2,IFN-? and TNF-? in evaluating donor liver quality was higher than any single index.[Conclusions](1)The rat model of F-WIT-DCD liver transplantation is stable and reliable.It can fully simulate the clinical situation of Maastricht type III and is suitable for studying various pathophysiological conditions during heart death liver transplantation.(2)The concentration of cytokines in donor liver tissue change during functional warm ischemia.(3)Combined cytokine markers during functional warm ischemia can be used to evaluate the quality of donor liver.