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Objective:To develop an ex vivo normothermic mechanical perfusion(NMP)and compare the effect of air-oxygenated NMP versus oxygen-oxygenated NMP on reducing renal injury from donor after cardiac death(DCD).Methods:All kidneys from DCD rats were subjected to 30 min in situ warm ischemia after cardiac attest.And harvested kidneys were stored for 8h under static cold preservation after NMP for 2h.In experimental groups, kidneys were subjected to either air-oxygenated NMP(group A, n=6)or oxygen-oxygenated NMP(group O, n=6). Sham operation(group C, n=6)and DCD kidneys under static cold preservation without NMP(group SCS, n=6)were employed as controls.The evaluation parameters included creatinine(Cr), aspartate amino transferase(AST)and lactate dehydrogenase(LDH)in perfusate, pathological changes by hematoxylin-eosin(HE)staining, histological criteria, expressions of myeloperoxidase and intercellular adhesion molecular-1(ICAM-1)by immunohistochemistry and Western blot, tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)by enzyme-linked immunoadsorbent assay and level of malondialdehyde(MDA)by thiobarbital method and activity of superoxide dismutase(SOD)by WST-8 in renal tissues.Differences between two groups were analyzed by two-tailed unpaired Student's test and differences among more than two groups by one-way ANOVA.Results:Renal arterial oxygen tensions in NMP perfusate were(576.3±68.2)mmHg with oxygen-oxygenation and(137.0±39.1)mmHg with air-oxygenation.There was significant difference( P<0.05). The pathological injury scores in groups SCS, O and A by HE staining were(7.0±0.1), (5.0±0.9)and(2.5±0.5); injury scores and the expressions of renal proximal tubular epithelial cell vacuolar degeneration in groups O and A were lower than those in group SCS( P<0.05)and injury score in group A was lower than group O( P<0.05). In perfusate, the levels of △Cr, △AST and △LDH in groups O and A were(43.9±52.8)μmol/L and(12.6±3.5)μmol/L, (532.3±52.8)U/L and(49.1±50.4)U/L and(9998.0±2014.4)U/L and(1477.0±810.4)U/L.There were significant differences( P<0.05). In perfused kidneys, the MDA level and SOD activity in groups O and A were(0.192±0.018)mmol/g, (0.162±0.023)mmol/g, (0.6±0.3)×10 3 U/g, (1.7±0.4)×10 3 U/g; TNF-α and IL-6 levels in groups O and A were(124.376±19.635)and(89.331±13.123)ng/g, and(4.038±1.026)×10 3 and(1.774±0.518)×10 3 ng/g.After air-oxygenated NMP, lower renal damage indices were characterized by a lower MDA level and a higher SOD activity, the lower levels of TNF-α and IL-6 and the lower expressions of MPO and ICAM-1 than those in oxygen-oxygenated NMP( P<0.05). Conclusions:NMP with air-oxygenation mimics renal perfusion under physiological conditions and decreases oxidative stress and inflammation injury.It may confer a better retrieval in DCD kidney against warm ischemia injury.
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Objective:To develop an ex vivo normothermic mechanical perfusion(NMP)and compare the effect of different portal perfusion pressures on attenuating hepatic injury from donor after cardiac death(DCD).Methods:All rat livers were subjected to in situ warm ischemia for 30 min after cardiac attest and thereafter stored for 8 h under cold preservation. Six livers were harvested and regarded as static cold storage(group CS, n=6). In experimental group, liver received an ex vivo dual NMP with oxygenated perfusion via hepatic artery for 2 h after cold storage. Hepatic injury was assessed and compared from perfused livers with full portal vein pressure(group M1, n=6)and low portal vein pressure(group M2, n=6). The evaluation parameters included perfusion flow, liver enzymes of perfusate, pathological changes by hematoxylin-eosin staining, Suzuki histological criteria, expression of activation markers of polymorphonuclear neutrophils and macrophages, myeloperoxidase (MPO)and CD68 by immunohistochemistry, level of malondialdehyde(MDA)and activity of superoxide dismutase(SOD). Results:In experimental group during NMP, perfusion flows tended to increase when portal pressures were stabilized in groups M1 and M2.Perfusion flow during NMP 60~120 min was significantly higher than during NMP 0~20 min.After NMP with full portal pressure, hepatic sinusoidal congestion, hepatocyte necrosis, steatosis and Suzuki criteria were lower in group M1 than those in group CS( P<0.05). Compared with group M1, lower hepatic injury was characterized with a lower change of liver enzymes in perfusate( P<0.05), a better histological evaluation( P<0.05), a lower level of MDA and a higher activity of SOD( P<0.05), lower expressions of CD68 and MPO ( P<0.05)and lower levels of TNF-α and IL-6( P<0.05)in perfused liver. Conclusions:The ex vivo dual NMP with oxygenated perfusion via hepatic artery mimics liver perfusion under the physiological conditions.NMP with a lower portal pressure can attenuate hepatic ischemia-reperfusion injury and confer a better protection against liver damage from DCD.
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Objective To study the effect of bone marrow mesenchymal stem cells (BMMSCs) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) after DCD donor liver transplantation in rats.Methods The third generation of BMMSCs and the BMMSCs modified by Ad/HO-1 (Ad/HO-1/BMMSCs) were cultured,identified and expanded in vitro.To establish a stable NMP system device in vitro.The DCD liver transplantation models were constructed in rats after cardiac ischemia for 30 minutes,220 SD recipient rats were randomly divided into sham operation group (S group,n=44) static cold storage (SCS group,n =44) group,and simple NMP group (P group,n =44),BMMSCs combined with NMP group (BP group,n =44) and BMMSCs modified by Ad/HO-1 combine with NMP group (HBP group,n =44),NMP group,BP group and HBP group were subjected to vitro perfusion for 4h.The group were taken at 0,1,7 and 14 days after transplantation and the relevant indicators were detected,n =6 in each group.The survival rate of the recipient rats,liver function and pathological changes of the bile duct were observed.The expression of cytokeratin 19 (CK19) protein in BEC was detected by immunohistochemistry and Western blot.Apoptotic biliary epithelial cells were detected by TUNEL staining and the expression of apoptosis-related protein caspase-3 was detected by immunohistochemistry.Results The survival time of HBP group was significantly prolonged for (5.6 ±0.8) d in SCS group vs.(18.4 ±2.0) d in NMP group,(20.5 ± 1.5) d in BP group,(82.5 ±3.2) d in HBP group,the differences were statistically significant (all P ＜ O.05).Compared with other groups,the HBP group and the BP group were significantly improved in liver function and biliary pathology,and the expression of CK19 protein in BEC was significantly increased [(0.81 ±0.02) in S group vs.(0.35 ±0.03) in SCS group,(0.47 ±0.02) in NMP group,(0.63 ± 0.02) in BP group,(0.77 ± 0.01) in HBP group on postoperative day (POD) 14],the differences were statistically significant (all P ＜ 0.05).The number of apoptosis and the expression of apoptosis-related protein caspase-3 in HBP group were significantly decreased [(10.0 ± 1.2) in S group vs.(57.3 ±5.5) in SCS group,(40.1 ±4.6) in NMP group,(32.0 ± 2.2) in BP group,(13.7 ± 3.1) in HBP group on POD 14],the difference was statistically significant (all P ＜ 0.05).Compared with the BP group,the protective effect of the HBP group was more obvious,and the difference was statistically significant (P ＜ 0.05).Conclusion By the method of the BMMSCs modified by Ad/HO-1 combined with NMP in vitro preservation of rat,DCD donor liver can significantly improve the effect of BEC on rats and the survival rate after liver transplantation.
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Objective To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation. Methods The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples. Results The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP. Conclusion Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.
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Objective@#To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation.@*Methods@#The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples.@*Results@#The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP.@*Conclusion@#Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.
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Objective To study the repairing effect of bone marrow mesenchymal stem cells (BMMSCs) on the donor liver after cardiac death (DCD) under normal temperature mechanical perfusion (NMP) in rats.Methods BMMSCs of Wistar rats were cultured in vitro,and 45-min warm ischemia after cardiac death model was established.The 30 Wistar rats were randomly divided into NMP,NMP + BMMSCs (N + B),cold storage (CS) groups,and the parameters were detected at 2 h and 4 h (n =10).Results N + B group was superior to NMP group and CS group in repairing the liver function and liver pathology including ultrastructure,improving the perfusate acidic environment,and increasing adenosine triphosphate level (P ＜ 0.05).The oxygen consumption of NMP group and N + B group were significantly different after 2h [2 h:(24.35 ±0.64) ml/min vs.(29.33 ±0.47) ml/min;3 h:(25.33 ±0.86) ml/min vs.(30.34 ± 0.49) ml/min;4 h:(26.88 ± 1.07) ml/min vs.(31.76 ± 0.96) ml/min;P ＜ 0.05],suggesting that the liver condition in N + B group was significantly better than that in the other two groups.Conclusion Bone marrow mesenchymal stem cells could obviously repair the DCD grafts under normal temperature mechanical perfusion.