The Protective Effect Of Hypothermic Oxygenated Machine Perfusion(HOPE) On Resuscitating Steatotic Liver And Its Underlying Mechanism

Part ? The modification of rat orthotopic liver transplantation modelObjective The rat orthotopic liver transplantation(ROLT)model plays a crucial role in organ quality evaluation.Traditional reconstruction via microscopic suture for vessels and bile duct takes long,accompanied by high technical requirements,is not conducive to promoting.The Kamada's two cuff technique and its modification simplified and stabilized this model.Based on this method,the study further discussed the potential risk factors during operation in detail,analyzed the death reason,improved the anastomosis technique of suprahepatic inferior vena cava(SHVC),in order to obtain stable postoperative survival.Methods Adult SPF Sprague-Dawley rats,male,weighing 220 ± 10 g.Three groups were involved:Group 1(n=60):practice stage of ROLT;Group 2(n=60):stabilizing ROLT stage;Group 3(n=60):ROLT model improved phase(60).During operation,the time cost in each part,including anhepatic period,the reconstruction of SHVC,portal vein,infrahepatic inferior vena cava and bile duct were recorded.Postoperative condition was observed every day,1-day and 7-day postoperative survival were included.Detailed analysis of the died cases.Results 90 cases of ROLT were performed.The time cost in anhepatic period of stage? was shortened by 40%compared with stage ?,the number in stage ? was shortened by 64%compared with stage ?.The success rate of ROLT in the stage I(Group 1)is 20%(6/30);the success rate of ROLT in the stage ?(Group 2)is 86.7%(26/30);the success rate of ROLT in the stage ?(Group 3)is 100%(30/30).The postoperative survival was 10%(3/30),60%(18/30)and 93%(28/30),respectively?Conclusions The devil is in the details.The anastomosis of SHVC should be finished quickly and skillfully.Surgery safety can be secured by shorten the anhepatic peroid,which depends on the operation proficiency and technical stability.The improvement of anastomosis technique of SHVC shortened the anhepatic time and guaranteed the rats safety.The intraoperative risk factors can be reduced,perioperative and postoperative survival could be ensured after handling the internal environment disturbance and hemodynamic changes during anhepatic phase,which lay a solid foundation for future experiments.Part ? The establishment of Hypothermic oxygenated machine perfusion(HOPE)systemObjective The study try to establish a stable and effective hypothermic oxygenated machine perfusion(HOPE)system based on refined perfusion parameter,which realized a complete and elaborate perfusion,to rescue and refine the liver quality,which will provide theoretical basis for clinical application of HOPE and expand donor pool.Methods Adult SPF Sprague-Dawley rats,male,weighing 220 ± 10 g.Five groups were involved depended on the portal vein perfusion pressure(PVP):Group A(n=6):PVP = 0 mmHg(no perfusion);Group B(n=6):PVP = 2 mmHg;Group C(n=6):PVP = 4 mmHg;Group D(n=6):PVP = 6 mmHg;Group E(n=6):PVP = 8 mmHg.Wet weight of livers were recorded after three hours perfusion.ALT,AST and LDH in perfusate were detected.MDA,glycogen and lactate were examined in tissue.Results The wet weight of liver increased in all groups.With the PVP grew,ALT,AST,LDH in perfusate,MDA and lactate in liver tissue were elevated,the glycogen deceased in group B and group C while increased in group D and group E.Conclusions HOPE may prolong the preservation time and improve the organ quality.However,the perfusion system parameter must be strictly controlled in case of machine perfusion injury.The adequacy of perfusion and its adverse effect of injury can be balanced using the controlled PVP which no more than 4 mmHg.Part ? Rat model of steatotic liver for transplantationObjective This study aimed at establishing a stable rat model of hepatic steatosis,imitating the pathogenesis in human beings,for rescuing the marginal liver(steatotic liver)in liver transplantation.Methods Adult SPF Sprague-Dawley rats,male,n = 90,weighing 220 ± 10 g.Ten rats were sacrificed 1 week after their arrival to set the baseline.The residual 80 rats were randomly assigned to two groups:Group A:n= 40,High-fat diet,HFD.The HFD consisted of 60%lipid,20.6%carbohydrate and 19.4%protein(kJ).The lipids included in HFD consisted of 90%lard and 10%soybean oil;Group B:n= 40,Standard chow diet,SCD.Histology and serology changes was evaluated every week.Results The values of ALT and AST showed no significant difference between goup A and B in each week.TG rose up quickly in the first two weeks,over doubled of the baseline and declined gradually to the baseline during the latter 6 weeks.The plasma glucose,insulin and lipid content of liver revealed significant difference between group A and B(P<0.05,P<0.05,P<0.01,respectively).The content of MDA in liver showed no significant difference between two groups.Microvesicular steatosis was predominately detected in the previous three weeks,however,macrovesicular steatosis was observed primarily in the latter five weeks.Conclusions According to the experiment design,particular extent of steatosis can be achieved based on feeding time.This steatotic liver model verified the efficacy and stability of the high-fat diet,which provided a reliable model for studying marginal liver.Part IV The protective effect of Hypothermic oxygenated machine perfusion(HOPE)on resuscitating steatotic liver and its underlying mechanismObjective The discrepancy between donor organs supply and demand has increased dramatically.Refinement of steatotic liver can raise its utilization,expanding the donor pool.Aim of this work was to analyze the impact of steatotic livers with conventional cold storage(CS)and hypothermic oxygenated perfusion(HOPE)approaches,and further to investigate the rescue of steatotic livers applied by HOPE and elucidate the potential mechanism.Methods Sprague-Dawley rats,male,weighing 220 ± 10 g.44 rats were randomly divided into 4 groups:group A,control.Non-steatotic livers were procured from SCD rats and preserved at University of Wisconsin(UW)solution(0-4 ?)for 45 min ex-vivo.Group B:cold storage(CS).Steatotic livers were excised and cold stored in UW(0-4 ?)for 4 hours.Group C:HOPE.Steatotic livers perfused with 3-h HOPE followed by 1-h CS.Group D:DF.On the basis of Group C,defatting cocktail were added to the perfusate.All the livers were then taken for orthotopic liver transplantation(OLT)after being preserved.The expression of KLF-2,eNOS,ICAM-1,HMGB1,TLR4,PRDX6,Caspase-3,PP2A,PPY,GRP78,CHOP and ATF-6 in liver were examined by Western blot.Apoptosis was assessed using Tunel.ATP and glycogen were detected by ELISA kits.The post-transplantation survival was assessed in all groups.Results According to the macroscopic appearance during transplantation,the blood reperfusion revealed uneven.The worst reperfusion manifestation of theses groups was group B,while the best one was group A.Compared with group B,the expression of microcirculation related proteins KLF2 and eNOS were increased in group C and group D(p<0.05);accompanying with the deceased expression of ICAM-1.Expression of HMGB1,PRDX6 and TLR4 had statistical significance between CS group and HOPE group.Expression of endoplasmic reticulum stress proteins(ATF-6,PPY,GRP78,and CHOP)were down-regulated in group C compared to group B(p<0.05),the proteins expression was further being down regulated in group D.The PP2A expression showed opposite trend.According to Tunel,the hepatocytes apoptosis was obveiously being inhibited in group C and group D compared with group B.The contents of ATP in group C was obviously increased,the differences revealed statistical significance when comparing to group B(p<0.05).Meanwhile,the overall post-transplantation survival rates in group C and group D significantly improved comparing to group B.Conclusions The mechanisms of protection of steatotic donor livers by hypothermic oxygenated machine perfusion refer to protect from microcirculation disturbance and endoplasmic reticulum stress and then promote ATP and glycogen synthesis to improve mitochondrial function.Meanwhile,under the ischemia reperfusion injury conditions,prevents nuclear injury and endothelial damage through suppressing the release of inflammatory mediator.The HOPE approach dramatically boosts long-term survival rate,which appears to be a very promising and powerful theoretical foundation to improve steatotic liver in clinical transplant and explores an effective therapeutic target for assessment of donor quality.Defatting cocktail cannot accelerate the lipidolysis of steatotic liver,but it can further improve the microcirculation,inhibit the release of inflammatory mediators,consequently improve the steatotic livers.Lay theoretical foundation for application of steatotic liver in clinical practice.