Rat Orthotopic Liver Transplantation Research Articles

Treprostinil Improves Hepatic Cytochrome P450 Activity during Rat Liver Transplantation

Cytochrome P450 (CYP450) activity is an important indicator of liver graft function. CYP450 activity is altered by pro-inflammatory cytokines, which are associated with ischemia-reperfusion (I/R) injury during orthotopic liver transplantation (OLT). Treprostinil, an FDA-approved prostacyclin analog, ameliorated cold I/R injury during rat OLT. We hypothesized that treprostinil would improve CYP450 activity in liver graft during cold I/R injury post-OLT. OLT was performed in syngeneic male Lewis rats with 18h graft preservation in cold UW solution. Donor and recipients received treprostinil (100ng/kg/min) or matching placebo for 24h before and up to 48h post-OLT. Liver graft mRNA and protein expression of CYP450 isoforms were analyzed by qRT-PCR and Western blot analysis, respectively. The formation rates of 1-hydroxymidazolam and 6β-hydroxytestosterone, 6-hydroxychlorzoxazone, 2α- and 16α-hydroxytestosterone in liver graft microsomes served as markers for CYP3A, CYP2E1, and CYP2C11 activity, respectively, and were measured by LC-MS. Treprostinil significantly decreased serum ALT and AST levels at 6-48h after OLT, compared to placebo. The expressions of TNFα and IFNγ mRNA in the liver graft were significantly inhibited in the treprostinil-treated group at 1h post-reperfusion. Treprostinil restored CYP2E1 protein expression to that of normal liver and significantly improved CYP3A activity to more than two-fold of placebo early post-OLT. Treprostinil significantly ameliorated hepatic injury, reduced expression of pro-inflammatory cytokines, and improved CYP450 activity in liver graft early post-OLT. These findings suggest that treprostinil has the potential to serve as a therapeutic option to protect liver graft function against I/R injury during clinical OLT.

17β-Estradiol protects the liver against cold ischemia/reperfusion injury through the Akt kinase pathway

BackgroundHepatic ischemia–reperfusion (IR) injury occurs during liver resection and transplantation. Recent studies have shown that 17β-estradiol (E2) can protect the heart and liver against warm IR. The present study focused on the cytoprotective effects of E2 on cold IR injury to the liver. Materials and methodsSprague-Dawley male rats were randomly divided into three groups: sham, IR, and IR plus E2. The model of rat orthotopic liver transplantation was used. The rats in the IR plus E2 group were intraperitoneally injected with E2 (100 μg/kg/d) for 7 d before surgery. The sham and IR group received the same quantity of saline. The donor livers were then orthotopically transplanted into rats after cold ischemia preservation for 4 h at 4°C lactated Ringer's solution. After 6 h reperfusion, liver function, bile flow volume, hepatocyte apoptosis, and activation of Akt, glycogen synthase kinase-3β, and Bcl-2-associated death promoter were assessed. The survival rate of the rats was also investigated. ResultsThe administration of E2 significantly prolonged the survival of liver grafts by improving liver function and decreasing hepatocyte apoptosis. Rats undergoing E2 demonstrated a greater level activation of Akt in the liver compared with the IR group. In addition, E2 also inhibited the activities of glycogen synthase kinase-3β, Bcl-2-associated death promoter, and caspase-3–induced by IR injury. ConclusionsE2 pretreatment attenuated the hepatocellular damage caused by hepatic cold IR injury through the Akt pathway. Estrogen therapy might be important in clinical settings associated with cold IR injury during liver transplantation.

Protective effect of nitric oxide on hepatopulmonary syndrome from ischemia-reperfusion injury.

To evaluate immunological protection of nitric oxide (NO) in hepatopulmonary syndrome and probable mechanisms of ischemia-reperfusion (IR) injury in rat liver transplantation. Sixty-six healthy male Wistar rats were randomly divided into three groups (11 donor/recipient pairs). In group II, organ preservation solution was lactated Ringer's solution with heparin 10, 000/μL at 4 °C. In groups I and III, the preservation solution added, respectively, L-arginine or N(G)-L-arginine methyl ester (L-NAME) (1 mmol/L) based on group II, and recipients were injected with L-arginine or L-NAME (50 mg/kg) in the anhepatic phase. Grafted livers in each group were stored for 6 h and implanted into recipients. Five rats were used for observation of postoperative survival in each group. The other six rats in each group were used to obtain tissue samples, and executed at 3 h and 24 h after transplantation. The levels of alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α and NO metabolites (NOx) were detected, and expression of NO synthase, TNF-α and intercellular adhesion molecule 1 (ICAM-1) was examined by triphosphopyridine nucleotide diaphorase histochemical and immunohistochemical staining. By supplementing L-arginine to strengthen the NO pathway, a high survival rate was achieved and hepatic function was improved. One-week survival rate of grafted liver recipients in group I was significantly increased (28.8 ± 36.6 d vs 4 ± 1.7 d, P < 0.01) as compared with groups II and III. Serum levels of ALT in group I were 2-7 times less than those in groups II and III (P < 0.01). The cyclic guanosine monophosphate (cGMP) levels in liver tissue and NOx in group I were 3-4 times higher than those of group II after 3 h and 24 h reperfusion, while in group III, they were significantly reduced as compared with those in group II (P < 0.01). The levels of TNF-α in group I were significantly lower than in group II after 3 h and 24 h reperfusion (P < 0.01), while being significantly higher in group III than group II (P < 0.01). Histopathology revealed more severe tissue damage in graft liver and lung tissues, and a more severe inflammatory response of the recipient after using NO synthase inhibitor, while the pathological damage to grafted liver and the recipient's lung tissues was significantly reduced in group I after 3 h and 24 h reperfusion. A small amount of constitutive NO synthase (cNOS) was expressed in liver endothelial cells after 6 h cold storage, but there was no expression of inducible NO synthase (iNOS). Expression of cNOS was particularly significant in vascular endothelial cells and liver cells at 3 h and 24 h after reperfusion in group II, but expression of iNOS and ICAM-1 was low in group I. There was diffuse strong expression of ICAM-1 and TNF-α in group III at 3 h after reperfusion. The NO/cGMP pathway may be critical in successful organ transplantation, especially in treating hepatopulmonary syndrome during cold IR injury in rat orthotopic liver transplantation.

Correlation between augmenter of liver regeneration and IFN-γ expression in graft after rat orthotopic liver transplantation

BackgroundPrevious data suggested that augmenter of liver regeneration (ALR) has immunomodulation function by suppressing liver-resident NK cell activity and reducing IFN-γ expression in human liver diseases. The correlation between ALR and IFN-γ expression in graft after rat orthotopic liver transplantation remains uncertain. MethodsA Lewis-to-BN (allograft group) and BN-to-BN (isograft group) rat liver transplantation model was used to investigate the ALR and IFN-γ expression in liver graft. Graft recipients were sacrificed at days 1, 3, 5, and 7 posttransplantation. The histopathologic changes of grafts were observed under light microscope and the intragraft expression of ALR and IFN-γ mRNA and protein was determined by real-time PCR and immunohistochemistry staining, respectively. Correlation between ALR and IFN-γ expression in graft was evaluated by Spearman rank correlation analysis. ResultsThe light microscope inspection revealed severe acute rejection in the allograft group but not in the isograft group at day 7 after liver transplantation. The intragraft ALR showed slight protein expression at day 1 after liver transplantation in both groups and it was significantly increased at days 3, 5, and 7 (P < 0.05). There was no significant difference in ALR mRNA expression between the allograft group and isograft group at day 1 (1.09 ± 0.12 and 1.13 ± 0.10, respectively; P > 0.05, n = 3). The ALR mRNA level was slightly reduced at day 3 in both groups compared with that at day 1 (0.81 ± 0.11 and 0.59 ± 0.10, respectively, P > 0.05). However, it was markedly increased at day 5 (2.86 ± 0.37) and day 7 (3.19 ± 0.33) in the isograft group and was 1.57 ± 0.27 and 1.98 ± 0.13 in the allograft group at days 5 and 7, respectively. IFN-γ protein and mRNA expression in the allograft group was increased at day 1 posttransplantation and reached a peak at day 3, and then it had a slight tendency of decline at day 5 and day 7. And they in the isograft group were at a low level at all times. The levels of ALR mRNA showed a negative correlation with levels of IFN-γ mRNA in the allograft group (r = −0.86, P < 0.05, y = −0.241x + 0.586), whereas there is no correlation between ALR and IFN-γ mRNA expression in the isograft group. ConclusionThese data revealed an obviously negative correlation between ALR and IFN-γ levels intragraft, which indicated that ALR may participate in immunoregulation of acute rejection.

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5. In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

The Effect of Perfusion Speed on Graft Reperfusion Injury after Rat Orthotopic Liver Transplantation

ObjectiveThe objective of this study was to investigate the effects of various perfusion speeds on reperfusion injury of grafts after liver transplantation. MethodsLiver transplantation was performed from Sprague-Dawley (SD) to SD rats. Recipients were divided into 4 groups according to perfusion speed: 50 mL/h for group A, 100 mL/h for group B, 150 mL/h for group C, and 200 mL/h for group D. Peripheral blood was collected from the caudal vein. All survivors were humanely killed at 24 hours posttransplantation. The morphological changes in grafts were evaluated using light microscopy, serum tumor necrosis factor-α (TNF-α), and endothelial nitric oxide synthase (eNOS) proteins as well as their messenger RNA (mRNA)-intragraft levels. ResultsThe pathohistological damage of grafts in the 150 mL/h and 200 mL/h groups were compared with the 50 mL/h and 100 mL/h groups. TNF-α in serum was increased at 6 hours, reaching a peak at 12 hours posttransplantation. The TNF-α levels in the 50 mL/h and 100 mL/h groups were significantly lower than the 150 mL/h and 200 mL/h groups; the 50 mL/h group was greater than the 100 mL/h group as well as the 200 mL/h group was greater than the 150 mL/h group. The expressions of eNOS protein and mRNA intragraft in the 150 mL/h and 200 mL/h groups were significantly reduced compared with the 50 mL/h and 100 mL/h groups, and the 50 mL/h group was decreased when compared with the 100 mL/h group. There was no significant difference between the 150 mL/h group and the 200 mL/h group. ConclusionThe optimal perfusion speed for harvesting rat liver graft is 100 mL/h. High-speed perfusion may impair sinusoid endothelial cells and low-speed perfusion may extend the warm ischemia time and increase microthrombus formation.

The use of a reversible proteasome inhibitor in a model of Reduced-Size Orthotopic Liver transplantation in rats

Ischemia/reperfusion injury (IRI), inherent in liver transplantation (LT), is the main cause of initial deficiencies and primary non-function of liver allografts. Living-related LT was developed to alleviate the mortality resulting from the scarcity of suitable deceased grafts. The main problem in using living-related LT for adults is graft size disparity. In this study we propose for the first time that the use of a proteasome inhibitor (Bortezomib) treatment could improve liver regeneration and reduce IRI after Reduced-Size Orthotopic Liver transplantation (ROLT). Rat liver grafts were reduced by removing the left lateral lobe and the two caudate lobes and preserved in UW or IGL-1 preservation solution for 1h liver and then subjected to ROLT with or without Bortezomib treatment. Our results show that Bortezomib reduces IRI after LT and is correlated with a reduction in mitochondrial damage, oxidative stress and endoplasmic reticulum stress. Furthermore, Bortezomib also increased liver regeneration after reduced-size LT and increased the expression of well-known ischemia/reperfusion protective proteins such as nitric oxide synthase, heme oxigenase 1 (HO-1) and Heat Shock Protein 70. Our results open new possibilities for the study of alternative therapeutic strategies aimed at reducing IRI and increasing liver regeneration after LT. It is hoped that the results of our study will contribute towards improving the understanding of the molecular processes involved in IRI and liver regeneration, and therefore help to improve the outcome of this type of LT in the future.

Hyperbaric oxygen therapy reduces the severity of ischaemia, preservation and reperfusion injury in a rat model of liver transplantation

BackgroundApproaches to increase organ availability for orthotopic liver transplantation (OLT) often result in the procurement of marginal livers that are more susceptible to ischaemia, preservation and reperfusion injury (IPRI). MethodsThe effects of post-OLT hyperbaric oxygen (HBO) therapy on IPRI in a syngeneic rat OLT model were examined at various time-points. The effects of IPRI and HBO on hepatocyte necrosis, apoptosis, proliferation, and sinusoidal morphology and ultrastructure were assessed. ResultsPost-OLT HBO therapy significantly reduced the severity of IPRI; both apoptosis [at 12h: 6.4±0.4% in controls vs. 1.6±0.7% in the HBO treatment group (p < 0.001); at 48h: 2.4±0.2% in controls vs. 0.4±0.1% in the HBO treatment group (p < 0.001)] and necrosis [at 12h: 18.7±1.8% in controls vs. 2.4±0.4% in the HBO treatment group (p < 0.001); at 48h: 8.5±1.3% in controls vs. 3.4±0.9% in the HBO treatment group (P= 0.019)] were decreased. Serum alanine transaminase was reduced [at 12h: 1068±920IU/l in controls vs. 370±63IU/l in the HBO treatment group (P= 0.030); at 48h: 573±261IU/l in controls vs. 160±10IU/l in the HBO treatment group (P= 0.029)]. Treatment with HBO also promoted liver regeneration [proliferation at 12h: 4.5±0.1% in controls vs. 1.0±0.3% in the HBO treatment group (p < 0.001); at 48h: 8.6±0.7% in controls vs. 2.9±0.2% in the HBO treatment group (p < 0.01)] and improved sinusoidal diameter and microvascular density index. ConclusionsHyperbaric oxygen therapy has persistent positive effects post-OLT that may potentially transfer into clinical practice.

A Technique of Recipient Portal Venoplasty and Cuff Insertion for Portal Revascularization in Orthotopic Rat Liver Transplantation

Rat orthotopic liver transplantation (ROLT) remains one of the most challenging animal procedures, requiring significant technical expertise and practice. In a paper recently published in the Journal of Surgical Research, Li et al. describe a technique of recipient portal venoplasty and cuff insertion during ROLT [1]. A review of the literature reveals numerous fundamental techniques as well as a host of variations on those techniques [2–4]. While the ultimate goal of these myriad variations is to decrease the anhepatic phase and time for graft revascularization as well as to improve rat survival, the wide successes and failures of these techniques hint at more influential factors than simply the method used.

Establishmert of rat model for orthotopic liver transplantation by double naked eye

Objective To study the learning curve of establishing the model of othotoptic liver transplantation in the rat by a single operator with naked eyes.Methods Generalized 600 cases of surgical data in different stages,and compared the differences in the donor time,cold perfusion time,anhepatic phase,the success rate of surgery,one week survival rate,long-term survival rate and the surgical complications.Results The operation times in stage one were longer than the other three stages,the differences were statistically significant (P ＜ 0.01 ).Success rate had gradually risen from 26.7％ in stage one to almost 100.0％ in stage four,one week survival and long-term survival rate were both improved steadily.Surgical complications mainly included vascular complications in early period and biliary complications in the later period.Surgical mortality in the late three stages had a sharp decline compare to stage one.Most of surgical complications had gradually decreased,and the modeling technology was gradually stabilized.Conclusion Beginners can gradually master to establish the model of rat orthotopic liver transplantation through systemic and persistent training. Key words: Animal model; Rat orthotopic liver transplantation (ROLT) ; Learning curve

A Technique of Recipient Portal Venoplasty and Cuff Insertion for Portal Revascularization in Orthotopic Rat Liver Transplantation

In addition to suprahepatic vena cava anastomosis in two-cuff rat liver transplantation, recipient portal vein revascularization is one of the most difficult procedures that must be performed, especially for beginners. A total of 43 cases of liver transplantation were performed. Rats in Group 1 and Group 2 were subjected to transplant procedures that used the conventional and portal venoplasty techniques, respectively. The portal vein anastomosis duration, anhepatic phase length, portal vein surgical complications, and 1 wk post-transplant survival rates were recorded for each group. The portal revascularization duration was statistically significantly less for Group 2 versus Group 1 (1.50 ± 0.61 min and 4.32 ± 0.67 min, respectively, P < 0.05). The anhepatic phase length of Groups 1 and 2 were 21.79 ± 1.27 min and 18.55 ± 1.47 min, respectively (P < 0.05). No significant differences between the groups were observed in relation to either portal vein surgery complications or 1-week survival rates. The recipient portal venoplasty and cuff insertion technique is a safe and fast alternative surgical option for portal revascularization in two-cuff rat liver transplantations performed by a single trainee.

NF-κB induced the donor liver cold preservation related acute lung injury in rat liver transplantation model.

BackgroundWe have observed at our clinical work that acute lung injury (ALI) often occurs in patients transplanted with donor livers persevered for long time. So, we conducted this study to investigate the influence of cold preservation time (CPT) of donor liver on ALI induced by liver transplantation (LT), and further study the role of nuclear factor-κB (NF-κB) in the process.MethodsWistar rats were used as donors and recipients to establish orthotopic rat liver transplantation models. Donor livers were preserved at 4°C for different lengths of time. The effect of NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC), on ALI was detected. All samples were harvested after 3 h reperfusion. The severity of liver injury was evaluated first. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in liver tissue and liver outflow serum were measured respectively. The severity indexes of ALI, the activity of NF-κB and inhibitor-κBα (I-κBα) in lung/liver were measured accordingly.ResultsWith the prolonged liver CPT, the liver damage associated indexes and ALI-related indexes all increased significantly. TNF-α and IL-1β in liver outflow serum increased accordingly, and the activity of NF-κB in liver/lung increased correspondingly. All these ALI-associated indexes could be partially reversed by the use of PDTC.ConclusionsExtended CPT aggravates the damage of donor liver and induces the expressions of TNF-α and IL-1β in liver. These inflammatory factors migrate to lung via liver outflow blood and activate NF-κB in lung, inducing ALI finally. NF-κB may play a critical role in LT-related ALI. Patients with or at risk of ALI may benefit from acute anti-inflammatory treatment with PDTC.

Advantages of Promoting Interleukin-10 by Silence of Histone Deacetylase 11 in Inducing Tolerance in Orthotopic Liver Transplantation in Rats

Aims The aims of this study were to study the role of histone deacetylase 11 (HDAC11) in tolerance induction in orthotopic liver transplantation (OLT) in rats and to assess the advantages of gene therapy over the immunosuppressant FK506. Methods Recipients were assigned to an acute rejection group (AcR; group I), an FK506 intervention group (group II), and a tolerance group (group III). Acute rejection (AcR) was graded by the Banff scheme and we examined postoperative survival. The messenger RNA (mRNA) and protein expressions of histone deacetylase 11 (HDAC11) and interleukin (IL) 10 in liver tissue were detected using real-time polymerase chain reaction (PCR) and Western blots, respectively. Plasma levels of tumor necrosis factor (TNF)-α, IL-2, and IL-10 were measured using enzyme-linked immunosorbent Assays. Results Group I displayed severe, Group II had less, and Group III had no evidence of AR. The survivals among Group III were longer than those in Group I and Group II. IL-10 expression was promoted by HDAC11-shRNA at 7 days after OLT. Serum IL-2 and TNF-α levels were significantly lower among Group III compared with Groups I and II, whereas IL-10 showed the opposite result. Conclusions Silence of HDAC11 promotes IL-10 expression and leads to tolerance following OLT in rats. Thus HDAC11 is a promising target for gene therapy to induce tolerance with advantages over immunosuppressive drugs.

The Pathological Characteristics of Acute Antibody-Mediated Rejection in DA-to-Lewis Rat Orthotopic Liver Transplantation

The category of acute antibody-mediated rejection (AMR) is not included in the Banff classification of liver transplantation pathology. We investigated the pathology of acute AMR using an orthotopic rat liver transplantation from DA-to-Lewis rats without immunosuppression. We studied liver graft samples at days 5, 7, and 9 to 11, focusing on the pathological characteristics of acute AMR. Progressive acute cellular rejection and AMR led to irreversible graft failure by day 11 ± 2. At day 5 immunoglobulin G (IgG) was deposited on endothelial cells in the portal veins and small arteries. Thereafter, at day 7 to day 11 the IgG deposition expanded on endothelial cells in portal veins and hepatic arteries, epithelial cells in bile ducts, sinusoids and hepatic cells in lobules. Light microscopic studies during the development of acute AMR showed interstitial edema in portal areas with neutrophilic infiltration. Rejecting grafts revealed congestion and/or thrombi in portal veins and hepatic arteries with neutrophil infiltration and fibrinogen deposition, severe degeneration of epithelial cells in bile ducts with periductal edema, intralobular edema, and hemorrhage with neutrophil infiltration and fibrinogen deposition, as well as hepatic cell degeneration and necrosis. In conclusion, acute AMR that developed in liver transplantation was characterized by endothelial cell injuries in microvasculature of portal veins, hepatic arteries, and sinusoids, accompanied by congestion, hemorrhage, thrombus formation, and neutophilic infiltration, as well as by bile duct and hepatic cell degeneration and necrosis.

Effects of hypoxia inducible factor-1α with high expression mediated by hypoxia precondition on the JNK signaling pathway in rats received orthotopic liver autotransplantation

Objective To investigate the expression of hypoxia inducible factor-1α(HIF-1α)in the transplanted liver in rats and the role of HIF-1α in the JNK signaling pathway.Methods Ninety-six SD rats were randomly divided into normal control(NC)group,autotransplantation(AT)group,hypoxia preconditioning (HP)+ AT group according to simple random sampling method.No treatment was applied to the rats in the NC group except for blood vessel separation.Stable rat models of 70％ AT was established in the AT group.Rats in the HP + AT group were given 8％ oxygen mixed gas for 90 minutes before the operation.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected at 1,2,12,24 hours after operation,and the expression of HIF-1α in the hepatic tissue,mRNA expression of c-Jun,and protein expression of Cleaved Caspase-3 were detected by immunohistochemical staining,reverse transcription-polymerase chain reaction and Western blot,respectively.All data were analyzed using the analysis of variance or t test.Results The levels of ALT and AST in the HP + AT group were significantly lower than those in the AT group,while higher than those in the NC group at 1,2,12 and 24 hours after the operation(F=2631.371,1177.642,810.383,682.848;743.618,1095.522,375.995,580.613,P ＜0.05).The protein expression levels of HIF-1α in the AT group were significantly lower than those in the HP + AT group,but higher than those in the NC group at 1,2,12 and 24 hours after operation(F = 191.737,284.482,459.419,213.782,P ＜ 0.05).The mRNA expression levels of c-Jun in the HP + AT group were significantly lower than those in the AT group,while higher than those in the NC group at 1,2 and 12 hours after operation(F = 66.211,53.169,9.645,P ＜ 0.05).There was no significant difference in the mRNA expression of c-Jun among the 3 groups at 24 hours after operation(F = 1.100,P ＞ 0.05).The protein expressions of Cleaved Caspase-3 in the HP + AT group were significantly lower than those in the AT group,while higher than those in the NC group at 1,2,12 and 24 hours after the operation(F =23.133,31.158,14.347,29.043,P ＜ 0.05).Conclusion High expression of HIF-1α after HP inhibits apoptosis of hepatic cells and alleviates ischemia reperfusion injury of hepatic tissues by suppressing JNK activation,down-regulating protein expression of Cleaved Caspase-3 after orthotopic liver transplantation in rats. Key words: Liver transplantation,autologous; Hypoxia inducible factor-1α; c-Jun N-terminal kinase; Caspase-3; Ischemia reperfusion injury

Indoleamine2, 3-dioxygenase enzyme activity detected by in vivo liver microdialysis after rat ortho topic liver transplantation

Objective To observe the activity of indoleamine 2, 3-dioxygenase (IDO) enzyme in vivo after rat liver transplantation, and the effects of liver transplantation rejection on IDO enzyme activity.Methods Ten SD rats were subjected to othotopic liver transplantations respectively in allogenic OLT group (SD→SD) and heterogenic OLT group (Wistar→SD). In the other 10 SD rats, the blood supply to the liver was obstructed for 15 min for simulating the anhepatic period in the OLT group ( sham group). An in vivo rat liver microdialysis-HPLC system was established on the day I after OLT. The concentrations of kynurenine and tryptophan in dialysate samples were continuously monitored quantitatively and reproducibly for more than 2 weeks in the same animal. Results The average concentrations of tryptophan in dialysate were 97. 14, 85.71, 53.07 nmol/L respectively in the sham, allogenic OLT and heterogenic OLT groups respectively. The concentrations of kynurenine were 3.46, 14. 58, 46.79 nmoL/L in the sham, allogenic OLT and heterogenic OLT groups respectively. The concentration of kynurenine was increased significantly on the day 7 after surgery in heterogeneic OLT group (70. 75 nmol/L). Conclusion The transplantation surgery can induce the activity of IDO enzyme in vivo and accelerate the metabolism of tryptophan in the liver allografts. Key words: Liver transplantation; Acute rejection; Immune tolerance; Tryptophane; Indoleamine 2,3-dioxygenase

High incidence of biliary complications in rat liver transplantation: can we avoid it?

To investigate how to reduce the incidence of biliary complications in rat orthotopic liver transplantation. A total of 165 male Wistar rats were randomly divided into three groups: Group A, orthotropic liver transplantation with modified "two-cuff" technique; Group B, bile duct was cut and reconstructed without transplantation; and Group C, only laparotomy was performed. Based on the approaches used for biliary reconstruction, Group A was divided into two sub-groups:A1 (n = 30), duct-duct reconstruction, and A2 (n = 30), duct-duodenum reconstruction. To study the influence of artery reconstruction on bile duct complication, Group B was divided into four sub-groups: B1 (n = 10), duct-duct reconstruction with hepatic artery ligation, B2 (n = 10), duct-duct reconstruction without hepatic artery ligation, B3 (n = 10), duct-duodenum reconstruction with hepatic artery ligation, and B4 (n = 10), duct-duodenum reconstruction without hepatic artery ligation. The samples were harvested 14 d after operation or at the time when significant biliary complication was found. In Group A, the anhepatic phase was 13.7 ± 1.06 min, and cold ischemia time was 50.5 ± 8.6 min. There was no significant difference between A1 and A2 in the operation duration. The time for biliary reconstruction was almost the same among all groups. The success rate for transplantation was 98.3% (59/60). Significant differences were found in the incidence of biliary complications in Groups A (41.7%), B (27.5%) and C (0%). A2 was more likely to have biliary complications than A1 (50% vs 33.3%). B3 had the highest incidence of biliary complications in Group B. Biliary complications are almost inevitable using the classical "two cuff" techniques, and duct-duodenum reconstruction is not an ideal option in rat orthotopic liver transplantation.

Role of indoleamine 2, 3-dioxygenase in diagnosing rat's acute rejection after liver transplantation

Objective To study the diagnostic value of indoleamine 2, 3-dioxygenase (IDO) gene expression in acute liver rejection in rat orthotopic liver transplantation model. Methods The rat orthotopic liver transplantation models were divided into four groups: group A, isograft transplantation group (Wistar to Wistar); group B, allograft transplantation (SD to Wistar); group C, allograft transplantation and cyclosporine; Group D, allograft transplantation and cyclosporine (the drug was withdrawn on the 3rd day after the operation). The samples (peripheral blood and liver tissue) were obtained on the operation day, 1st, 2nd, 3rd, 4th, 5th, 7th and 9th day post-operation. Luorescent quantitative polymerase chain reaction (PCR), pathological study and serum test were performed on each sample. Results In groups A and C, the IDO mRNA was expressed at a low level, aspartate aminotransferase (AST), total bilirubin (T-BIL) and alkaline phosphatase (ALP) were decreased to the normal level, and the pathological test found no acute rejection (AR). In group B, the IDO mRNA was dramatically increased on the day 2 (P<0.05), AST, T-BIL and ALP rose dramatically on the day 4 (P<0.05), and mild AR was detected on the day 5 (χ2=4.8,P<0.05). In group D, the IDO mRNA was dramatically increased on the day 4 (P<0.05), AST, T-BIL and ALP rose dramatically on the day 5 (P<0.01), and mild AR was detected on the day 7 (χ2=4.8,P<0.05). Conclusion Comparing with pathological study, detecting IDO mRNA of peripheral blood can diagnose AR of rat at earlier stage and the technique is safe and simple. Key words: Indoleamine 2,3-dioxygenase; Liver transplantation; Acute rejection

A Novel End-to-Side Anastomosis Technique for Hepatic Rearterialization in Rat Orthotopic Liver Transplantation to Accommodate Size Mismatches between Vessels

Background/Aims: We present our modification of a sutured arterial anastomosis in orthotopic rat liver transplantation as well as a literature survey and analysis of the existing techniques of rearterialization with regard to technical difficulties and potential limitations. Methods: The donor common hepatic artery (CHA) was anastomosed to the enlarged lumen of the recipient proper hepatic artery (PHA), tailored to match the size of the donor CHA, with an end-to-side interrupted suture technique. Vascular patency of hepatic rearterialization was assessed both intraoperatively and at the time the liver grafts were harvested (postoperative days 2 and 28). The effect of arterialization on hepatic morphology was confirmed by histological examination and compared to nonarterialized rat orthotopic liver transplantation. Results: The CHAs had a significantly larger diameter (up to 3-fold) compared to the PHAs, which represents a considerable size mismatch. The anastomosis procedure including the size adaptation required 15–25 min. All anastomoses were patent immediately, 5 min after rearterialization and at both harvest time points. The liver lobular architecture was intact in the rearterialized group, whereas a moderate degree of bile duct proliferation and portal/lobular lymphocytic infiltration were observed in the nonarterialized group. Conclusion: The new technique is a time-consuming and microsurgically challenging but universally applicable and robust procedure accommodating even a substantial mismatch in vessel diameter.