The Establishment Of Hypothermic Machine Perfusion System For Rat Livers And Research Of Molecular Mechanisms Linking With The System To Improve The Quality Of Livers Donated After Cardiac Death

Liver transplantation has been considered as the most effective solution to various end-stage liver problems.The persistent shortage of suitable brain-dead donors has prompted a reexamination of donors after cardiac death(DCD)to extend the pool of organs for transplantation.However,DCD is considered as a primary risk factor to inferior liver transplantation outcomes and complications like primary non-function and early allograft dysfunction due to the higher vulnerability to ischemia/reperfusion injury(IRI).Therefore,novel strategies that improve the preservation conditions of DCD organs and retain their vitality are needed.Hypothermic machine perfusion(HMP)is a dynamic hypothermic technique for preserving transplantation organs.It simulates the physiological conditions of organs by supplying them with oxygen and/or key nutrients with the removal of the waste products.Many experimental and clinical reports indicated the effectiveness of HMP in improving DCD liver preservation when compared to cold storage(CS)through the attenuation of IRI.However,the underlying molecular mechanism associated with HMP remains unclear.Sirtuin-1(SIRT-1),a member of the sirtuin family,is a deacetylase that was reported to reduce IRI in a nicotinamide adenine dinucleotide(NAD+)dependent manner.Given the protective role of SIRT-1 and HMP in IRI,we speculated that HMP may have influence on SIRT-1 to reduce IRI to DCD livers.Therefore,we firstly established a novel HMP system based on Lifeport kidney transporter for preservation of rat liver.Then,using this HMP system,we studied the influence of heterogeneous hypothermic machine perfusion on molecular markers of IRI in different regions of DCD rat livers.Finally,based on these findings,we investigated the modulatory effects of HMP on SIRT-1 in regulating NF?B-p65 activity which in turn protects DCD livers from IRI.Part ?A Novel Hypothermic Machine Perfusion System Based on Lifeport Kidney Transporter for Preservation of Rat LiverObjective The protective mechanisms for liver preservation linking with hypothermic machine perfusion(HMP)remain unclear.However,the lack of a common and portable HMP system for rat livers limits the study of HMP.Our work aimed to develop a novel,modified HMP system based on Lifeport Kidney Transporter(Lifeport)for rat liver.Methods A simple "Y" shunt combined a pressoreceptor for flow and pressure regulation was adapted to perfuse rat livers via the portal vein continuously by Lifeport under prime mode.An electronic scale was installed under the liver container to calculate the portal inflow according to the relationship within weight,density and volume.Ten adult male Sprague Dawley rat livers underwent 6h of HMP using HTK solution enriched with acridine orange(AO)and propidium iodide(PI).Fluorescence microscopy was used to assess perfusion scope by AO+PI positive cell count(APPCC)in core region(CR)and peripheral region(PR)of rat liver.The dynamics were assessed to identify whether this system met the demands for HMP of rat liver.Biochemical and histological parameters were tested to determine cellular damage associated with HMP.Results No significant difference was observed between CR and PR according to the comparison of APPCC,indicating a complete perfusion was achieved.Intrahepatic resistance remarkably decreased during the first 3 hours and remained stable till the end of HMP.The release of alanine transaminase and lactate dehydrogenase significantly increased from 34.75± 17.96 and 422.65 ± 167.97 to 145.14 ± 13.13 and 1865.59 ±160.50 U/L,respectively.The levels of endothelin and oxygen consumption were constant throughout HMP.No histological changes were observed after 6h of HMP.Additionally,the results of ATP test were 638.47±50.41 nmol/L and bile production were 4.37±0.77 ?L/h/g liver at the end of HMP.Conclusion We demonstrated the modified HMP system met the demands suitable for a rat liver preservation with minimal cellular injury associated with HMP while retaining the dependability and portability of Lifeport.Part II The Influence of Heterogeneous Hypothermic Machine Perfusion on Markers of Ischemia Reperfusion Injury in Different Regions of Rat Livers Donated after Cardiac DeathObjective The aim of the study was to explore the different influences of Heterogeneous hypothermic machine perfusion(HMP)on molecular markers of ischemia reperfusion injury(IRI)in different regions of rat livers donated after cardiac death(DCD).Methods 10 adult male Sprague Dawley rats' livers suffered from 30min warm ischemia were randomly divided into cold storage(CS)or HMP group(n=5 per group).Livers were preserved for 3h with HTK solution followed by acridine orange(AO)and propidium iodide(PI)staining in CS group or HTK solution enriched with AO+PI in HMP group.Fluorescence microscopy was used to assess perfusion scope by AO+PI positive cell count(APPCC)in core region(CR)and peripheral region(PR)of rat livers in the two groups.Heterogeneous HMP resulted in only a part of livers without congestion.Therefore,samples in regions with or without congestion were obtained followed by assessment of morphology,superoxide dismutase(SOD)activity,malondialdehyde(MDA),adenosine triphosphate(ATP)level,NF-icB-p65,Kruppel like factor 2(KLF2),IL-1 ? and TNF-? protain expressions.Results No significant difference was observed within CS-CR,CS-PR,HMP-CR and HMP-PR subgroups according to comparisons of APPCC,indicating complete perfusion was achieved after 3h of HMP.However,KLF2,an indirect indicator for perfusion resistance,was significantly up-regulated by HMP,particularly in regions without congestion where sinusoidal endothelial cells were exposed to the largest perfusion volume.Meanwhile,regions with or without congestion in HMP group both showed significant improvement of morphology,SOD activity and ATP level,with respect to CS group.In addition,the upregulation of other IRI molecular markers such as MDA,the expression of proinflammatory cytokines(IL-1?,TNF-?)and NF-KB-p65 were significantly attenuated by HMP,especially in the regions without congestion.Conclusion HMP is enable for reducing IRI molecular makers of DCD livers more effectively than CS.Moreover,the heterogentity of HMP due to microcirculatory dysfunction of DCD livers resultes in regions with or without congestion and has the largest effect on the makers of IRI in regions without congestion.Part ? Hypothermic Machine Perfusion Ameliorates Inflammation during Ischemia/Reperfusion Injury through Sirtuin 1-Mediated Deacetylation of Nuclear Factor-KB-p65 in Rat Livers Donated after Cardiac DeathObjective To explore cellular mechanisms linking HMP to ameliorate ischemia-reperfusion injury(IRI)induced inflammatory response.Methods Adult male Sprague Dawley rat livers were exposed to 30 min of warm ischemia after cardiac arrest,and then preserved by CS or HMP for 3h(n=10 per group).The severity of IRI was assessed thereafter on normothermic reperfusion(NR)in vitro for 2h.During NR,intrahepatic resistance(IR)was calculated.The perfusate enzyme activities,O2 consumption rate and bile production of livers were evaluated.After NR,livers were obtained for histological study,measurement of adenosine triphosphate(ATP)level,malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,nicotinamide adenine dinucleotide(NAD+)level and ratio of NAD+/NADH.Finally,protein expressions of Sirtuin-1(SIRT-1),acetylated(K310)nuclear factor-?B-p65(NFKB-p65)and NFKB-p65 were detected by Western blotting and mRNA expressions of the inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factor-a(TNF-a)were measured by Semi-quantitative RT-PCR.Results Compared with CS,HMP resulted in significantly lower IR,enzyme release and MDA levels,higher O2 consumption rate,ATP levels,SOD activity as well as better preserved morphology,with the exception of bile production.The protein expressions of NFKB-p65,acetylated(K310)NF?B-p65 and mRNA expressions of TNF-a and IL-6 significantly decreased in HMP group compared with CS group(P<0.05).Under HMP preservation,SIRT-1 activity and protein expression were increased while the expression of acetylated(K310)NFKB-p65 was decreasedConclusion Our results indicate that HMP reduced inflammatory response during IRI through SIRT-1-mediated deacetylation of NFKB-p65.