The Role And Mechanisms Of TLR3 On Warm Ischemia Injury Preconditioned With Hepatic Hypoperfusion In Mice

After half a century of development, liver transplantation(LT)has become a effective life-saving treatment for patients with end stage liver diseases.While the successful development of LT,burgeoning waiting lists with insufficient donation rates makes a lot of patients in the waiting for death.To expand the donor pool,living donor liver transplantation, split liver transplantation as well as donation after cardiac death(DCD) have been deeply researched and widely performed through the wordwild.Among them,DCD is one of most important ways to expand the donor supply at present,at the earliest,DCD kidney was usded in transplantation in 1950s, it was replaced by DBD grafts then due to the high incidence of complication and mortality rate.The renewed interest in DCD started in 1990s following the limited success of the transplant community to expand DBD organ supply.After decades of development,although DCD solid organ transplantation has achieved positive results at present,the outcome of DCD LT is superior to DBD.The incidence of grafts primary nonfunction(PNF), ischemic type biliary lesion(ITBL) and hepatic artery thrombosis (HAT) after DCD LT was significantly higher than DBD.Retransplantation has been performed once early graft function failure occurred,it caused more expensive costs and higher risk of operation.Thus,to reduce the incidence of complications and increase patient and graft survival rates after DCD LT have draw more and more attention through the worldwide.The hypoperfusion and warm ischemia injury occurred in DCD liver before transplant is main risk factors which influence DCD LT outcomes.In DCD,donor death is diagnosed on the basis of irreversible cessation of cardio-pulmonary function instead of conventional neurologic criteria. In this process,the donor was inevitablely subjected to a period of hypotension and hypoxia which caused hepatic hypoperfusion.Liver as a metabolic organs is sensitive to hypotension, hypoxia and highly susceptible to hypoxia injury. Although vasoactive agents and ventilator can improve the situation, microcirculation disturbance or even failure in liver were unable to effectively reverse. Besides, hepatic sinusoid as a umen of half open was easily susceptible to ischemia damage. After withdrawal of both ventilator and cardiac support,DCD liver undergoed a period of warm ischemia time, duration of warm ischemia time significantly affected tissue viability and graft function after transplantation. Therefore,to research the mechanisms of warm ischemia injury in DCD liver is key to improve the outcomes of DCD LT.The phenomenon that outcomes of DCD LT was superior to DBD did not happen in DCD kidney LT. It indicated that,the mechanisms of hepatic hypoperfusion injury was different from other organs. Traditional view suggested liver was a metabolic organ,it was demonstrated now liver was also a immune organs which innate immune played a predominant role.NK and NKT cell account for more than half of lymphocytes in liver.The regulation and reply of innate immune to warm ischemia injury is the main difference from DCD kidney LT.Toll-like receptors(TLRs) is one of the most important PRRs(patten recognition receptors)in innate immune,it can not only identificate exogenous ligands such as bacteria and virus,bus also can identificate v arious of the dangerous signals in the body.TLRs extensively participated in the local immune response and regulation in liver,and play an important role in ischemia-reperfusion(IR) injury.TLRl-9 Widely expressed in the cell surface,TLR3 expressed in hepatocyte, biliary epithelial cell,kupffer cell,DC cell and hepatic lymphocyte.Rerearchs indicated,TLR3-/-,TLR4-/-,TLR9-/- can reduce IR injury in liver and kidney.It further indicated that,TLRs might play a important role in hepatic local innate immume response and regulation. So we suspected,innate immune of which TLRs were as main receptors mediated warm ischemia injury in DCD liver.There are no stable animal models mimicing the clinical condition of DCD liver at present.The sustained hepatic hypoperfusion model in mouse we have constructed through portal vein constriction can mimic the hypoperfusion condition of DCD liver in some degree, especially for Maastrich categorie III donor.We use liver function test and histopathological examination to evaluate hepatic injury in hypoperfusion model constructed with TLRs knock-out mice and use Agilent expression spectrum analysis and flow cytemetry analysis to research the underlying mechanisms of innate immune mediating and regulating warm ischemia injury.The research to the mechanisms of warm ischemia injury in mice provides meaningful guidance to clinical DCD LT.Part 1.A sustained hepatic hypoperfusion model in mouse to mimic the clinical condition of liver graft in donation after cardiac deathObjectives:To construct a stable mouse model of sustained hepatic hypoperfusion to mimic the clinical condition of liver graft in donation after cardiac death(DCD), and to explore the influence of sustained hepatic hypoperfusion on warm ischemic-reperfusion(IR) injury in mouse.Methods:C7BL/6 mice of 6-8 weeks were chosen for model construction, whose portal vein was constricted to the diameter of lmL syringe needle. Liver function(ALT/AST) and histopathology(HE) were tested on 3,7,14 and 21 days after constriction. Hepatic warm IR injury of 70%　was performed using stable hepatic hypoperfusion model. Afterwards, liver function and histopathology were tested on 3,24, and 48 hours after reperfusion. Wild type C7BL/6 mice were used for control.Results:After portal vein constriction, ALT and AST significantly rose in varying degrees (F=68.9, p<0.001), which peaked on 7d (ALT:60.8±6.2U/L　vs.25.5±2.8 U/L, p<0.001; AST:74.9±6.1 U/L vs.39.1±3.2 U/L, p<0.001). Meanwhile, HE stain showed the most serious damage on hepatocytes and more inflammatory cell infiltration on 7d after constriction. On 21 d, ALT basically returned to normal(p=0.12), but AST was still higher than the base line(p=0.03). Compared with the control group, damage of hepatocytes was more severe in group of 7d hypoperfusion precondition after warm hepatic IR, whose transaminase level peaked at 3h after reperfusion (ALT:8217.0±1111.8 U/L vs.557.4±1015.3 U/L, p=0.004; AST:8548.2±1155.4 U/L vs 5765.4±956.9 U/L, p=0.003). At 48 hours after reperfusion, ALT and AST were back to normal in the control group, but still higher in the hypoperfusion precondition group (ALT:608.8±442.9 U/L vs 47.4±20.1 U/L, p=0.008; AST:861.8±442.8 U/L vs.70.8±68.3 U/L, p=0.008)Conlusions:We successfully constructed a stable mouse model of sustained hepatic hypoperfusion and found the tolerance for warm IR injury significantly decreased in hypoperfusion precondition liver. This model can mimic the clinical condition of liver graft in donation after cardiac death.Part 2. The influence of TLR3 deficiency on the warm ischemia injury induced by sustained hepatic hypoperfusion and the underlying mechanisms in miceObjective:on the basis of the sustained hepatic hypoperfusion model mimicing the clinical condition of liver graft in donation after cardiac death in mouse,to further research the influence of toll-like receptor 3 deficiency on the warm ischemia injury induced by sustained hepatic hyoperfusion and the underlying mechanisms in mice.Methods:as previously mentioned, TLR3-7-、TLR4-/-、TLR9-/- and MyD88-/- mice with same age and weight were chosen for constructing the sustained hepatic hypoperfusion model respectively to mimic the clinical condition of liver graft in donation after cardiac death,model constructing with wild type C57BL/6 mice was as control group.Liver function(ALT/AST) and histopathology(HE) were tested to compare the degree of warm ischemia injury induced by hepatic hypoperfusion among the groups then;Other TLR-3-/- and C57BL/6 mice were chosen for model construction as experimental group and control group respectively,hepatic warm ischemia-reperfusion(IR) injury of 70% was performed using stable hepatic hypoperfusion model.Then liver function and histopathology were tested on 3,24 and 48 hours after reperfusion and tolerance for warm IR injury in hypoperfusion liver were compared;the liver lymphocytes of hepatic hypoperfusion moedel and the peripheral blood monouclear cells of IR injury liver preconditioned with hepatic hypoperfusion in WT were isolated for Agilent expression spectrum analysis; liver lymphocytes of sustained hepatic hypoperfusion model in WT and TLR3-/- mice were isolated for flow cytemetry analyse to test the T/B lymphocytes changes.Results:ALT and AST level of TLR4-/-、TLR9-/-、MyD88-/- hypoperfusion liver were no significantly different from WT mice, hepatic enzyme level was significantly lower than WT mice(ALT:35.0±10.7 vs.68.0±8.8, p<0.001;AST:39.9±9.4 vs.75.1±63, p< 0.001), HE stain showed lighter damage on hepatocytes and less inflammatory cell infiltration in TLR3-/-;IR injury of TLR3-/- liver preconditioned with hepatic hypoperfusion was significantly decreased compared with WT mice, transaminase level of TLR-37-/- and WT mice were icked at 3h hours postreperfusion(ALT:4519.6±1025.7 U/L vs.7634.4± 742.U/L, p<0.001; AST:4009.2±1201.3 U/L vs.7961.4±977.2U/L, p<0.001),,at　48 hours after reperfusion,though,ALT and AST were still not back to normal in TLR-3-/-,but it was significant lower than WT mice (A ALT:129.4±114.3U/L vs.669.4±201.8 U/L,p=0.001;AST:175.4±156.8 U/L vs.653.8±251.6 U/L,p=0.007),HE stains showed lighted hepatocytes damage in TLR-3-/-, especially there was almost no lymphocytes infiltration; Agilent expression spectrum analysis of hypoperfuson liver in WT mice showed,the expression of B lymphocyte specific surface antigen CD19/CD20 and B-cell activating factor Baff was upregulated,at the same time,the expression of chemotactic factor CCL7/CCL2/CXCL4 increased and endogenous danger signal(HSPA4) was also expressed-Analyse for peripheral blood monouclear cells in IR liver preconditioned with hepatic hypopusion showed abnormal activation in innate immune downstream signaling pathways (interferon stimulated gene and cytokines),especially the expression of IP 10 explosivey rised;flowcymetry analyse showed B lymphocytes were deeply infiltrated in hypoperfusion liver in WT mice,and on the constract,the expression of B lymphocytes in TLR3-/- hepacit hypopersion liver was significantly decreased.Conlusions:TLR3 deficiency can significantly reduce warm ischemia injury induced by sustained hepatic hypoperfusion and increase the tolerance for warm IR injury.The underlying mechanism could be that,TLR3 defficiency block signaling which depend on MyD88,down-regulated the expression of cytokines and chemokines which reducing the inflammatory injury caused by B lymphocytes infiltration.