OBJECTIVE: To investigate the efficacy of a novel artificial perfusate based on oxygen-carrying perfluoronaphthalene-albumin nanoparticles in normothermic machine perfusion (NMP) for preservation of porcine liver donation after cardiac death. METHODS: Artificial perfusate with perfluoronaphthalene-albumin nanoparticles was prepared at 5% albumin (w/v) and its oxygen carrying capacity was calculated. The livers of 16 Landrace pigs were isolated after 1 h of warm ischemia, and then they were divided into 4 groups and preserved continuously for 24 h with different preservation methods: cold preservation with UW solution (SCS group), NMP preservation by whole blood (blood NMP group), NMP preservation by artificial perfusate without nanoparticles (non-nanoparticles NMP group) and NMP preservation by artificial perfusate containing nanoparticles (nanoparticles NMP group). Hemodynamics, tissue metabolism, biochemical indices of perfusate and bile were monitored every 4 h after the beginning of NMP. Liver tissue samples were collected for histological examination (HE and TUNEL staining) before preservation, 12 h and 24 h after preservation. RESULTS: The oxygen carrying capacity of nanoparticles in 100 mL artificial perfusate was 6.94 μL/mmHg (1 mmHg=0.133 kPa). The hepatic artery and portal vein resistance of nanoparticles NMP group and blood NMP group remained stable during perfusion, and the vascular resistance of nanoparticles NMP group was lower than that of blood NMP group. The concentration of lactic acid in the perfusate decreased to the normal range within 8 h in both nanoparticles NMP group and blood NMP group. There were no significant differences in accumulated bile production, alanine aminotransferase and aspartate aminotransferase in perfusate between nanoparticles NMP group and blood NMP group (all P>0.05). After 24 h perfusion, the histological Suzuki score in blood NMP group and nanoparticles NMP group was lower than that in SCS group and non-nanoparticles NMP group (all P<0.05), and the quantities of TUNEL staining positive cells in blood NMP group and non-nanoparticles NMP group was higher than those in nanoparticles NMP group and SCS group 12 h and 24 h after preservation (all P<0.05). CONCLUSION Artificial perfusate based on oxygen-carrying nanoparticles can meet the oxygen supply requirements of porcine livers donation after cardiac death during NMP preservation, and it may has superiorities in improving tissue microcirculation and alleviating ischemia-reperfusion injury.
Swine ; Animals ; Liver Transplantation ; Organ Preservation ; Liver ; Perfusion ; Death ; Oxygen/metabolism*

Objective To establish a porcine model of liver failure after different percent hepatectomy.Methods The porcine models of liver failure 75%,85%,95% hepatectomy were developed and the living conditions and survival time were recorded.The blood samples of pre-surgery,post-hepatectomy d1,d3,d5 and post-hepatectomy 1 week,2 weeks,and 3 weeks were collected for hepatic function analysis.Histological examination of liver tissues was performed using HE staining.Liver injury histology was interpreted and scored in the terminal samples.Results The average survival time of pigs with post-hepatectomy liver failure after 75%,85%,95% hepatectomy was 19.0±5.6 days,17.3±5.5 days,1.3±1.5 days,respectively.Their pathological scores were 5.67±0.52,8.17±0.82 and 8.50±0.71,respectively.With the increase of percent hepatic resection,the incidence of hepatic failure was increasing.ALT,AST,ALP,LDH and TBA were dramatically increased in the pigs after 85% hepatectomy.Conclusions The pig model of acute liver failure by 85% hepatectomy is successfully established,which can cause typical acute liver failure in Bama miniature pigs.

Objective To investigate the clinical value of preoperative nutritional support (PNS) therapy in the hepatectomy of patients with nutritional risk.Methods The prospective study was conducted.The clinical data of 133 patients with nutritional risk who were admitted to the Drum Tower Hospital Affiliated to Nanjing University Medical School from August 2012 to June 2016 were collected.All the patients undergoing PNS and traditional therapy were divided into the PNS group and the control group by random number table method,respectively.Observation indicators:(1) comparisons of laboratory indexes between groups;(2) comparisons of postoperative situations between groups;(3) comparisons of postoperative complications between groups.Measurement data with normal distribution were represented as-x±s.Comparisons between groups were evaluated with the independent-sample t test.Comparisons of count data were analyzed using the chi-square test,and repeated measures data were analyzed by the repeated measures ANOVA.Results All the 133 patients were screened for eligibility,including 68 in the PNS group and 65 in the control group.(1) Comparisons of laboratory indexes between groups:alanine transaminase (ALT),aspartate transaminase (AST),total bilirubin (TBil),cholinesterase,albumin (Alb),prealbumin,transferrin and C-reactive protein (CRP) in the PNS group were respectively (36± 13) U/L,(29± 10) U/L,(18.5±2.4) mmol/L,(5 738± 1 824) U/L,(37.4±5.1) g/L,(155±48) mg/L,(2.2±0.5)g/L,(10±4) g/L at admission and (33 ± 9) U/L,(27 ± 8) U/L,(17.9± 1.8) mmol/L,(5 796± 2 016) U/L,(38.5 ± 4.7) g/L,(181 ± 40) mg/L,(2.4± 0.5) g/L,(8± 4) g/L before operation and (285±100)U/L,(218±93)U/L,(33.5±6.3)mmol/L,(4 847±1 044)U/L,(32.6±3.8)g/L,(105±34)mg/L,(1.3±0.4) g/L,(55±28) g/L at 1 day postoperatively and (149±84) U/L,(76±42) U/L,(22.7±4.9) mmol/L,(3 866±893) U/L,(34.2±2.4) g/L,(125±30) mg/L,(1.6±0.4) g/L,(51±34) g/L at 3 days postoperatively and (64±33) U/L,(44±18) U/L,(19.4±2.8) mmol/L,(4 257± 1 032) U/L,(37.0±2.1) g/L,(148±42) mg/L,(1.9±0.4)g/L,(16±11)g/L at 7 days postoperatively;ALT,AST,TBil,cholinesterase,Alb,prealbumin,transferrin and CRP in the control group were respectively (36± 15)U/L,(31± 12)U/L,(18.3±2.9)mmol/L,(5 762±1 693)U/L,(37.3±6.1)g/L,(162±51)mg/L,(2.3±0.5)g/L,(10±4)g/L at admission and (36±11)U/L,(30±11)U/L,(18.2±2.8)mmol/L,(5 789±1 673)U/L,(37.8±7.1)g/L,(166±57) mg/L,(2.3±0.6) g/L,(9±5) g/L before operation and (305±127) U/L,(246± 104) U/L,(34.2±7.8) mmol/L,(4 842±1 173)U/L,(32.0±4.1) g/L,(83±32) mg/L,(1.2±0.4) g/L,(61 ±31) g/L at 1 day postoperatively and (163±104)U/L,(82±62)U/L,(23.1±6.0)mmol/L,(3 672±937) U/L,(33.8±3.6) g/L,(106±30)mg/L,(1.4±0.4)g/L,(61±40)g/L at 3 days postoperatively and (77±48) U/L,(52±27) U/L,(20.2±3.5) mmol/L,(3 925±987) U/L,(36.6±2.8) g/L,(125±40) mg/L,(1.7±0.4) g/L,(22± 12) g/L at 7 days postoperatively,showing no statistically significant difference in changing trends of above indicators between groups (F =1.007,2.223,0.579,0.014,0.235,3.533,2.970,2.143,P＞0.05).Results of further analysis showed that there were statistically significant differences in the levels of ALT,AST and cholinesterase at 7 days postoperatively between groups (t=1.832,2.073,1.899,P＜0.05),and in the levels of prealbumin before operation and at 1,3 and 7 days postoperatively between groups (t =1.698,3.738,3.625,3.178,P＜0.05) and in the levels of transferrin and CRP at 3 and 7 days postoperatively between groups (t=2.917,2.709,1.667,2.990,P＜0.05).(2) Comparisons of postoperative situations between groups:time to initial exsufflation,time of initial defecation,infused volume of exogenous albumin and duration of postoperative hospital stay were respectively (46± 15)hours,(64±16)hours,(23±10)g,(9.2±2.6)days in the PNS group and (55±18)hours,(78±21)hours,(39±25)g,(11.7±5.3) days,with statistically significant differences in the above indicators between groups (t =2.830,4.157,5.044,3.497,P＜0.05).(3) Comparisons of postoperative complications between groups:23 and 33 patients in the PNS and control groups had postoperative complications,showing a statistically significant difference between groups (x2=3.915,P＜0.05).Eight and 17 patients in the PNS and control groups were respectively complicated with peritoneal effusion,with a statistically significant difference between groups (x2 =4.508,P＜ 0.05).Conclusion PNS therapy in the hepatectomy of patients with nutrition risk can effectively improve pre-and post-operative nutrition statuses,reduce liver damage,accelerate recoveries of liver and gastrointestinal functions,reduce complications,shorten duration of postoperative hospital stay and accelerate patients' recovery.

The main features of liver failure are extensive necrosis of hepatocytes,rapid disease progression,and poor prognosis,and at present,there are no effective drugs and methods for the treatment of liver failure.This article summarizes four treatment methods for liver failure,i.e.,medical treatment,cell transplantation,liver transplantation,and artificial liver support therapy,and elaborates on the existing treatment methods.The current medical treatment regimen should be optimized;cell transplantation has not been used in clinical practice;liver transplantation is the most effective method,but it is limited by donor liver shortage and high costs;artificial liver can effectively remove toxic substances in human body.Therefore,this article puts forward artificial liver as a transition for liver transplantation;artificial liver can buy time for liver regeneration or liver transplantation and prolong patients' survival time and thus has a promising future.The new treatment modality of bioartificial liver combined with liver transplantation may bring good news to patients with liver failure.

Objective To study the synergetic effect and possible mechanism of transplanting mesenchymal stem cells (MSCs) in combination with interleukin-1 receptor antagonist (IL-1Ra) on acute liver failure (ALF).Methods MSCs transplantation combined with IL-1Ra was used for a swine model of ALF induced by 85％ total hepatectomy.The living conditions,blood samples and survival time were recorded or collected for analysis of hepatic function.Liver injury histology was analyzed.Hepatic cell regeneration and apoptosis were studied by immunohistochemistry staining of Ki67 and TUNELassays respectively.The expression levels of AKT and NF-κB were analyzed by Western blotting.Results The difference on the survival time between the model group and combined therapy group was statistically significant (P ＜ 0.05).Combined therapy displayed improvement not only in the serum biochemical conditions but also in the serum inflammatory cytokines.Furthermore,the observed hepatic histopathological score was significantly less compared to model group.In addition,the combined therapy group significantly inhibited the liver cell apoptosis and increased hepatic cell regeneration.Finally,a significant increase in AKT expression and decrease of NF-κB expression (P ＜ 0.05) were observed,which was consistent with their important roles in liver regeneration.Conclusion The combined therapy displayed a synergistic effect on liver regeneration,by promoting restoration and reconstruction of ALF,through regulation of inflammation and apoptosis signaling network.

Objective To explore the most effective route of mesenchymal stem cell transplantation (MSCs) in D-galactosamine (D-gal) induced porcine model with acute liver failure (ALF) and the potential mechanism.Methods BA-MA mini-pigs with D-gal-induced ALF were transplanted with porcine mesenchymal stem cells (MSCs) through four routes:intraportal injection (InP),peripheral intravenous injection (PV),hepatic intra-arterial injection (AH) and intrahepatic injection (IH).The survival time was recorded.The blood samples before and after MSC transplantation were collected for detecting liver function.Liver histology was interpreted and scored.Hepatic apoptosis and regeneration were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay and Ki-67 assay.The protein expression of cleaved caspase-3,survivin,AKT and ERK were analyzed by Western blot.Results The average survival time in each group was (10.7 ±1.6) days (InP),(6.0 ±0.9) days (AH),(4.7 ±1.4) days (PV),(4.3 ± 0.8) days (IH) when compared with D-gal group [(3.8 ± 0.8) days].The histopathological scores revealed a significantly decrease in InP group (3.17 ± 1.04,P ＜0.05) and AH group (8.17 ± 0.76,P ＜ 0.05) when compared with that in D-gal group (11.50 ± 1.32).The apoptosis rate in InP group (25.0 ± 3.4％,P ＜ 0.05) and AH group (40.5 ± 1.0％,P ＜ 0.05) was lower than that in D-gal group (70.6 ± 8.5％).The expression of active caspase-3 was inhibited,while the expression of survivin,AKT and ERK was elevated in InP group.Conclusions The intraportal injection was superior to other pathways for MSCs transplantation.Intraportal MSC transplantation could improve liver function,inhibit cell apoptosis,promote cell proliferation and prolong the survival in porcine ALF model.

ABSTRACT:Objective To investigate changes in the neutrophils in rats with D-galactosamine (D-GalN)-induced acute liver failure (ALF)and to explore the therapeutic effect of interventions treatment of neutrophils on ALF.Methods Liver function,the expressions of inflammatory cytokines TNF-αand IL-1β,and the changes of neutrophils in the peripheral blood and the liver were observed in rats with D-GalN (intraperitoneal injection)-induced ALF.SD rats were randomly divided into three groups when treated with intervention of neutrophils:control group,ALF group (intraperitoneal injection of D-GalN),and treatment group (intravenous injection of anti-PMN serum via tail vein 24 h before modeling).Biochemical analysis was used to detect serum ALT,AST, TBIL and blood ammonia.Hematology analyzer was applied to analyze the number and percentage of peripheral blood neutrophils.The number of neutrophils in the liver was evaluated by immunohistochemistry.Liver RT-PCR was adopted to detect the mRNA expression of inflammatory cytokines TNF-αand IL-1β.Results We found that 6 h after D-GalN injection,serum ALT,AST,TBIL and blood ammonia in ALF rats were significantly increased (P <0.05).The mRNA expression levels of inflammatory cytokines TNF-αand IL-1βin the liver reached the peak at 6 h after modeling (P <0.001),and it was still notably higher at 24 h than before modeling (P <0.001 ).The number and percentage of peripheral blood neutrophils and the number of neutrophils in the liver were all markedly increased 12 h after modeling (P <0.001 ),and the increase continued at least until 24 h (P <0.001 ).24 h after intravenous injection of anti-PMN serum via tail vein,ALF rats had a distinct decrease in the number of peripheral blood neutrophils and neutrophils in the liver 24 h after modeling (P <0.001).Meanwhile,serum ALT,AST,TBIL and blood ammonia were all greatly decreased compared with those in ALF group (P <0.05);a significant reduction of hepatocyte apoptosis was observed.Also,the expressions of TNF-α and IL-1β in the liver were remarkably decreased after treatment (P <0.05).Conclusion Neutrophils accumulated in peripheral blood and liver of rats with D-GalN-induced ALF.The treatment of anti-PMN serum may have a therapeutic effect on liver function and immune microenvironment in ALF rats.

Surgery is so far the most widely used and effective treatment of neoplastic diseases.However,residual tumour cells during surgery remain a major trigger of cancer recurrence and matastasis.Although intraoperative rapid pathological R0 resection can be achieved based on preoperative imageological examination,but for small satellite lesions and the naked eye can not find the error quickly and so often cause pathological presence of residual tumour cells.Thus,quick and accurate identification of residual cancer cells is crucial for prognosis of cancer patients.Indocyanine green (ICG) is a new type of fluorochrome that can stain tumours under the near-infrared fluorescence during surgery,the paper will be reviewed latest developments in the reagent for fluorescence in tumours.

In the practice of building its humanistic environment of a research hospital,Nanjing Drum Tower hospital adheres to such humanistic characteristics of the hospital as humanistic concept,planning, environment,management,service,and quality.Furthermore,the hospital upholds such keys as learning, innovation, cooperation, undertaking, competition and development. High focus, high starting point planning,persistence,and down-to-earth efforts,effectively promoting hospital development.

Objective To investigate the therapeutic effect of bone marrow mesenchymal stromal cell (BMSC) transplantation on D-galactosamine-induced acute liver failure in Sprague-Dawley (SD) rats,as well as the mechanism of neutrophils in this process.Methods A total of 39 male SD rats were divided into control group (8 rats,intraperitoneal injection of isotonic saline),model group (10 rats,intraperitoneal injection of D-galactosamine),solvent group (9 rats,tail vein injection of isotonic saline at 2 hours after intraperitoneal injection of D-galactosamine),and treatment group (12 rats,tail vein injection of MSCs at 2 hours after intraperitoneal injection of D-galactosamine).The rats were sacrificed at 24 hours after the model of D-galactosamine-induced acute liver failure was established,and the blood and liver tissue were harvested.The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TBil) were measured,and blood analysis was performed to measure the number and percentage of neutrophils in peripheral blood.Immunofluorescence assay was used to measure the expression of the neutrophil marker Ly6g in the liver,the myeloperoxidase (MPO) kit was used to measure the activity of MPO in liver,and RT-PCR was used to measure the mRNA expression of inflammatory cytokines and chemokines in the liver,i.e.,tumor necrosis factor-α (TNF-α),interleukin-1β (IL-1β),interferon-γ (IFN-γ),interleukin-10 (IL-10),CXC chemokine ligand 1 (CXCL1),and CXC chemokine ligand 2 (CXCL2).Another 64 male SD rats were randomly divided into groups,and the survival rates of rats in each group were observed for 7 days.The independent samples t-test was used for comparison between any two groups (Levene homogeneity test of variance,and the corrected t-test was used for a P value of ＜ 0.05),and the log-rank test was used for comparison of survival rates between any two groups.Results At 24 hours after acute liver failure was induced by D-galactosamine in the SD rats,there were significant increases in the liver function parameters (ALT:2884.1±541.0 U/L vs 45.4±11.0 U/L,P ＜ 0.001;AST:3634.9±755.9 U/L vs 143.9±23.7 U/L,P ＜ 0.001;TBil:44.4±8.4 μmmol/L vs 0.9±0.2 μmmol/L,P ＜ 0.001) and the number and percentage of peripheral blood neutrophils [number:(4.7±1.1)×109 vs (1.4±0.4)× 109,P ＜0.001;percentage:44.9％±8.0％ vs 18.3％±4.4％,P ＜ 0.001].A large number of neutrophils aggregated in the liver tissue,and there were significant increases in the MPO activity (4.72±1.09 U/g vs 1.13±0.24 U/g,P ＜ 0.001),inflammatory cytokines,and chemokines.Compared with the model group,the treatment group showed significant improvements in liver function (ALT:1 823.9a389.2 U/L vs 2 884.1±541.0 U/L,P ＜ 0.001;AST:2173.0±567.3U/L vs 3634.9±755.9 U/L,P ＜ 0.001;TBil:30.9±6.5 μmmol/L vs 44.4±8.4 μmmol/L,P ＜ 0.001) and survival rate (50％ vs 12.5％,P=0.023).Meanwhile,the treatment group also showed significant reductions in the number and percentage of peripheral blood neutrophils [number:(3.5±1.0)× 109 vs (4.7±1.1)×109,P =0.012;percentage:35.9％±8.9％ vs 44.9％±8.0％,P =0.021],number of neutrophils in the liver,and MPO activity (3.52±1.03 U/g vs 4.72±1.09 U/g,P =0.040),as well as significantly inhibited expression of inflammatory cytokines and chemokines (TNF-α:2.458±0.762 vs 3.778±1.046,P =0.005;IL-1β:2.498±0.547 vs 4.065 ± 0.953,P =0.002;IFN-γ:3.977±1.039 vs 5.418±1.255,P =0.025;IL-10:6.056±1.542 vs 3.368±0.952,P=0.001;CXCL1:7.988±1.911 vs 10.366±1.239,P =0.010;CXCL2:3.441±1.005 vs 4.847±1.113,P=0.019).Conclusion BMSC transplantation has a therapeutic effect on D-galactosamine-induced acute liver failure in rats,and this process is accompanied by reduced aggregation and activity of neutrophils in peripheral blood and liver.Inflammatory cytokines and chemokines may be involved in the mechanism of regulation of these two aspects.