The Effect Of Portal Blood Stasis On Hepatic Ischemia Reperfusion Injury

Part One The effect and mechanism of portal blood stasis on intestinal endotoxemia and hepatic ischemia reperfusion injury in a rabbit modelObjectiveA rabbit hepatic ischemia reperfusion injury model was established by in situ hypothermic irrigation for different time to observe the influence of portal blood stasis removal on intestinal endotoxemia and hepatic ischemia reperfusion injury.The purpose was to find an ideal method for portal blood stasis removal and provide the experimental proof for clinical application of liver transplantation.MethodsEighty New Zealand white rabbits of both genders,weighing 2.0-2.5 kg,purchased from the Laboratory Animal Center of Second Military Medical University,were randomly divided into control group(group C) and experimental groups.The rabbits of experimental groups were divided into three groups equally by different time of the portal vein occlusion for 20,30,40 minutes and then each group was re-divided into 3 subgroups by different amount of portal blood stasis removal:group A₀ no blood removal,group A₅ 5ml blood removal,group A₁₀ 10ml blood removal.Each group had eight rabbits,nine groups in all. The other eight rabbits(group C) did not receive hepatic portal occlusion and hypothermic irrigation.Their abdomens were opened and only served as control group.Endotoxin content both in serum and in portal blood stasis,alanine aminotransferase(ALT), hyaluronic acid(HA),tumor necrosis factor-α(TNF-α),hepatic pathology,content of malondialdehyde(MDA) and activity of superoxide dismutase(SOD) and activation of nuclear factor-κB(NF-κB) in liver tissue were examined respectively after reperfusion 4h.ResultsThe levels of serum endotoxin were significantly increased by extension of portal vein occlusion.At the same time of portal vein occlusion the level of serum endotoxin in portal blood stasis significantly decreased with each 2.5ml blood removal,subsequently reaching a minima at the 7.5ml blood removal.In groups of portal vein occlusion for 30 and 40 minutes,removing portal blood stasis ameliorated hepatic ischemia reperfusion injury as shown by ALT,HA,TNF-α,MDA,SOD,NF-κB and the pathology.Compared with the non-removal group,the effect was significant,while the effect of removing portal blood stasis in the group of portal vein occlusion for 20 minutes was not significant.DiscussionLong-time occlusion of the portal vein in the process of liver transplantation results in gastro-intestinal congestion,barrier failure,insufficient blood and oxygen supply,and increasing gut mucosal permeability,which leads to obvious increase of intestinal endotoxin in portal blood,especially portal blood stasis.The levels of serum endotoxin in portal blood stasis were significantly increased by extension of portal vein occlusion.The first 5ml portal blood stasis contains high volume of endotoxin which may be responsible for hepatic reperfusion injury.The alteration of ALT,HA,TNF-α,MDA,SOD,NF-κB and the pathology can reflect hepatic reperfusion injury in all ways.Removing portal blood stasis also ameliorated hepatic ischemia reperfusion injury as shown by those.ConclusionThe levels of serum endotoxin in portal blood stasis were significantly increased by extension of portal vein occlusion.The first 5ml portal blood stasis contains high volume of endotoxin which may be responsible for hepatic reperfusion injury.Removal of portal blood stasis before the resume of splanchnic circulation may ameliorate hepatic ischemia reperfusion injury.The possible mechanism may be that portal blood stasis removal reduces endotoxin absorption,and this decreases production of serum TNF-αas well as hepatic activation of NF-κB.Part Two The effect of different dosage of portal blood stasis removal on endotoxemia and hepatic ischemia reperfusion injury ObjectiveThe blood volume of rabbit is about equal to one fourth that of human body and 5ml blood volume is equivalent to 200ml that of human.A rabbit hepatic ischemia reperfusion injury model was established by in situ hypothermic irrigation for different time to observe the influence of portal blood stasis removal on intestinal endotoxemia and hepatic ischemia reperfusion injury.The purpose was to find an safe and effective method for clnical liver transplantation.MethodsEighty-eight healthy New Zealand white rabbits were randomly divided into a control group(group C) and experimental groups.The rabbits of experimental groups were divided into two groups equally by different time of the portal vein occlusion for 30,40 minutes and then each group was re-divided into five subgroups by different amount of portal blood stasis removal:group A₁ 2.5ml blood removal,group A₂ 5ml blood removal, group A₃ 10ml blood removal,group A₄ 15ml blood removal,and group B no blood removal.Each group had eight rabbits.The other eight rabbits did not receive hepatic portal occlusion and hypothermic irrigation,whose abdomens were opened and only served as control group.Serum endotoxin content,ALT,HA and TNF-αwere examined respectively after reperfusion 1h,2h,and 4h.Hepatic tissues were sampled to determine the pathology,content of MDA and activity of SOD and activation of NF-κB in liver tissue were examined respectively after reperfusion 4h.ResultsRemoving portal blood stasis also ameliorated hepatic ischemia reperfusion injury as shown by ALT,HA,MDA,SOD and the pathology.5ml and 10ml blood removal had the maximal favorable effect,while the effect of 2.5ml or 15ml blood removal was not significant.DiscussionThe levels of serum endotoxin in portal blood stasis were significantly increased by extension of portal vein occlusion.Portal blood stasis removal could not only reduce the total amount of endotoxin absorbed into liver and systemic circulation,but also reduce the damage of some important organs such as liver,lung,gastrointestinal tract.That the effect of 2.5ml or 15ml blood removal was not significant was correlated with that the first portal blood stasis contains high volume of endotoxin were not removed completely.The levels of serum endotoxin tended to rise with more portal blood stasis removal.The main reason was sequence portal blood stasis removal could not improve the protective effect because the first portal blood stasis containing high volume of endotoxin had been removed. Secondly the increase of portal blood stasis removal led to insufficient blood supply which worsened organ injury and endotoxemia.ConclusionAppropriate quantity of portal blood stasis removal can protect the liver against ischemia reperfusion injury.The possible mechanism may be that portal blood stasis removal reduces endotoxin absorption,and then decreases production of serum TNF-αas well as hepatic activation of NF-κB.But too much or too little of portal congestion blood released could not improve the protective effect.Part Three The effect of portal blood stasis on lung and renal injury induced by hepatic ischemia reperfusion in a rabbit modelObjectiveThe portal blood stasis removal can protect the liver against ischemia reperfusion injury,but there was no experimental proof whether it might ameliorate the lung and renal injury induced by hepatic ischemia reperfusion.A rabbit hepatic ischemia reperfusion injury model was established by in situ hypothermic irrigation to observe the effect of portal blood stasis on lung and renal injury induced by hepatic ischemia reperfusion.The purpose was to provide the experimental proof for protecting lung and renal and reducing postoperative complications.MethodsFifty-six healthy New Zealand white rabbits were randomly divided into control group and experimental groups.The rabbits of experimental groups were divided into two groups equally by different time of the portal vein occlusion for 30,40 minutes and then each group was re-divided into three subgroups by different amount of portal blood stasis removal:group A₀ no blood removal,group A₅ 5ml blood removal,group A₁₀ 10ml blood removal.Each group had eight rabbits.The other eight rabbits did not receive hepatic portal occlusion and hypothermic irrigation,whose abdomens were opened and only served as control group. After reperfusion 4h serum endotoxin content,TNF-α,urea nitrogen(BUN),creatinine (Cr),and wet to dry weight ratio,broncho-alveolar lavage fluid protein content in lung tissues were examined respectively.Meantime,lung and kidney tissues were sampled to determine the content of MDA,SOD and the pathology.ResultsRemoving portal blood stasis ameliorated lung and renal injury as shown by serum endotoxin,TNF-α,BUN,Cr,wet to dry weight ratio,broncho-alveolar lavage fluid protein content,MDA in lung and kidney tissue being significantly reduced,SOD in lung and kidney tissue being significantly increased and the pathology becoming better.DiscussionLong-time occlusion of the portal vein resulted in obvious increase of endotoxin in portal blood stasis.At reperfusion the endotoxin in portal blood would activate Kupffer cells,which might contribute to produce a great deal of cytokine,reactive oxygen species, and proteolytic enzyme.Those things not only produced liver injury and microcirculation disturbance,but also,through systemic circulation,resulted in lung and kidney injury by activating capillary endothelium and neutrophil.It showed the pressure of systemic circulation decreased and the injury of extra-hepatic organs,serum endotoxin,TNF-α, BUN,Cr,wet to dry weight ratio,broncho-alveolar lavage fluid protein content,MDA, SOD and the pathology objectively reflect the severity of systemic inflammatory response syndrome and the degree of lung and kidney injury.Those index ameliorated showed that the removal of portal blood stasis before the resume of splanchnic circulation may ameliorate the lung and renal injury induced by hepatic ischemia reperfusion.ConclusionRemoval of portal blood stasis before the resume of splanchnic circulation may ameliorate the lung and renal injury induced by hepatic ischemia reperfusion.The possible mechanism may be that portal blood stasis removal reduces endotoxin absorption,and then decreases production of serum TNF-α.Part Four Effects of different dosage of portal blood stasis removal on liver function during early stage after liver transplantationObjectiveThe pricking blood therapy has been accepted by clinician because it would reduce theoretically endotoxin entering into liver and protect the liver against ischemia reperfusion injury.How much portal blood stasis should be removed in clinic liver transplantation was empirical and there was no related report about it.Forty-seven patients who received liver transplantation in our hospital were divided into 2 groups according to different dosage of portal blood stasis removal during operation:group A 50ml and group B 200ml portal blood stasis removal respectively.The levels of plasma endotoxin, inflammatory factors and the restoring of liver function after liver transplantation was observed in order to provide the experimental proofs for clinical liver transplantation.MethodsClinical data:A total of 47 cases undergoing orthotopic liver transplantation were selected from Eastern Hepatobiliary Surgery Hospital,Second Military Medical University from January 2006 to October 2007.In 47 cases,43 males and 4 females,age from 33 to 67(average 49.9±8.5) years,were randomly divided into two groups by different dosage of portal blood stasis removal:group A(n=26) 50ml blood removal and group B(n=21) 200ml blood removal.Measurements:(1) The patients of sex,age,primary liver diseases and Child-pugh's classification,donor liver of cold ischemic time and total operation and anhepatic time, operation methods,volume of blood loss and transfusion were recorded before and in operation.(2) Samples from venous blood were obtained at beginning of surgery(T₀), 60min after graft reperfusion(T₁),at the end of operation(T₂),and 24h after surgery.The levels of plasma endotoxin,D-lactate,TNF-α,Interleukin-6(IL-6) were measured respectively.(3) The levels of plasma alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),direct bilirubin(DBIL),albumin(Alb), prealbumin(PAB),prothrombin time(PT) activated partial thromboplastin time(APTT) and fibrinogen(FIB) were examined in the day before operation and 1,3,7 d following surgery.ResultsThere was no significant difference in sex,age,primary liver diseases and Child-pugh's classification,cold ischemic time,total operation and anhepatic time, operation methods,volume of blood loss and transfusion,plasma endotoxin,D-lactate, TNF-α,IL-6,liver function and blood coagulation parameters before operation.Most of observations showed the restoration in group B was better than that in group A.The plasma levels of endotoxin,D-lactate,TNF-α,IL-6,ALT,AST,PT,APTT in group B were significantly lower than those in group A(P＜0.05).The level of plasma PAB in group B was significantly higher than that in group A(P＜0.05).DiscussionThe plasma levels of endotoxin,TNF-α,IL-6 are very sensitive markers of the degree of hepatic and body injury.The plasma levels of D-lactate response gut mucosal permeability.The plasma levels of aminotransferase,bilirubin,prealbumin,blood coagulation are very sensitive markers of liver functional restoration after liver transplantation.Our study showed that the levels of serum levels of endotoxin,D-lactate, TNF-α,IL-6,ALT,AST,PT,APTT after operation in group of 200ml portal blood stasis removal were significantly lower than those in group of 50ml blood removal.Meantime, the level of serum PAB was significantly higher.This study showed 200ml portal blood stasis removal before resuming of splanchnic circulation may ameliorate hepatic ischemia reperfusion injury,ruduce inflammatory reaction,protect intestinal mucosal barrier and liver functional restoration would be more effective than that of 50ml.The reason was that the first 200ml portal blood stasis containing high volume of endotoxin which might be responsible for hepatic reperfusion injury had been removed completely. ConclusionThis study showed the effect of 200ml portal congestion blood removal was better than that of 50ml,which contributed to relieving endotoxemia and restoring liver function after liver transplantation.