Jun-Jun Jia

Influence of perfusate on liver viability during hypothermic machine perfusion

AIM To optimize the perfusates used for hypothermic machine perfusion (HMP). METHODS Sprague-Dawley rats were assigned randomly to three groups (n = 12 per group) that received either saline, University of Wisconsin cold-storage solution (UW) or histidine-tryptophan-ketoglutarate solution (HTK) as the perfusate. Each group was divided into two subgroups: static cold storage (SCS) and HMP (n = 6 per subgroup). The liver graft was retrieved according to the method described by Kamada. For the SCS group, the graft was directly placed into cold perfusate (0-4 °C) for 6 h after liver isolation while the portal vein of the graft was connected to the perfusion machine for the HMP group. Then the perfusates were collected at different time points for analysis of aspartate aminotransferase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) levels. Liver tissues were obtained for evaluation of histology, dry/wet weight (D/W) ratio, and malondialdehyde (MDA) and adenosine-triphosphate (ATP) levels. The portal vein pressure and velocity were monitored in real time in all HMP subgroups. RESULTS Comparison of HMP and SCS: Regardless of the perfusate, HMP improved the architecture of donor graft in reducing the congestion around sinusoids and central vein and maintaining sinusoid lining in morphology; HMP improved liver function in terms of ALT, AST and LDH, especially during the 3-6 h period (SCS vs HMP using saline: ALT3, 225.00 ± 105.62 vs 49.50 ± 18.50, P = 0.047; LDH3, 1362.17 ± 563.30 vs 325.75 ± 147.43, P = 0.041; UW: LDH6, 2880.14 ± 948.46 vs 2135.00 ± 174.27, P = 0.049; HTK, AST6, 307.50 ± 52.95 vs 185.20 ± 20.46, P = 0.041); HMP decreased MDA level (saline, 2.79 ± 0.30 vs 1.09 ± 0.09, P = 0.008; UW, 3.01 ± 0.77 vs 1.23 ± 0.68, P = 0.005; HTK, 3.30 ± 0.52 vs 1.56 ± 0.22, P = 0.006). Comparison among HMP subgroups: HTK showed less portal vein resistance than UW and saline (vs saline, 3.41 ± 0.49 vs 5.00 ± 0.38, P < 0.001; vs UW, 3.41 ± 0.49 vs 4.52 ± 0.63, P = 0.007); UW reduced edema most efficiently (vs saline, 0.68 ± 0.02 vs 0.79 ± 0.05, P = 0.013), while HTK maintained ATP levels best (vs saline, 622.60 ± 29.11 vs 327.43 ± 44.66, P < 0.001; vs UW, 622.60 ± 29.11 vs 301.80 ± 37.68, P < 0.001). CONCLUSION HMP is superior to SCS in maintaining both architecture and function of liver grafts. Further, HTK was found to be the optimal perfusate for HMP.

Protective Effect of Remote Limb Ischemic Perconditioning on the Liver Grafts of Rats with a Novel Model

Background Remote ischemic conditioning (RIC) is a known manual conditioning to decrease ischemic reperfusion injury (IRI) but not increase ischemic time. Here we tried to establish a rat RIC model of liver transplantation (LT), optimize the applicable protocols and investigate the protective mechanism. Methods The RIC model was developed by a standard tourniquet. Sprague-Dawley rats were assigned randomly to the sham operated control (N), standard rat liver transplantation (OLT) and RIC groups. According to the different protocols, RIC group was divided into 3 subgroups （10min×3, n = 6; 5min×3, n = 6; 1min×3, n = 6）respectively. Serum transaminases (ALT, AST), creatine kinase (CK), histopathologic changes, malondialdehyde (MDA), myeloperoxidase (MPO) and expressions of p-Akt were evaluated. Results Compared with the OLT group, the grafts subjected to RIC 5min*3 algorithm showed significant reduction of morphological damage and improved the graft function. Also, production of reactive oxygen species (MDA) and neutrophil accumulation (MPO) were markedly depressed and p-Akt was upregulated. Conclusion In conclusion, we successfully established a novel model of RIC in rat LT, the optimal RIC 5min*3 algorithm seemed to be more efficient to alleviate IRI of the liver graft in both functional and morphological categories, which due to its antioxidative, anti-inflammation activities and activating PI3K Akt pathway.

A modified protocol with rituximab and intravenous immunoglobulin in emergent ABO-incompatible liver transplantation for acute liver failure

BACKGROUND The established procedure for ABO-incompatible liver transplantation (ABO-I LT) was too complicated to be used in case of emergency. We developed a protocol consisting of rituximab and intravenous immunoglobulin (IVIG) for ABO-I LT in patients with acute liver failure (ALF). METHODS The data from 101 patients who had undergone liver transplantation (LT) for ALF were retrospectively analyzed. The patients were divided into two groups: ABO-compatible liver transplantation group (ABO-C LT, n=66) and ABO-I LT group (n=35). All the patients in the ABO-I LT group received a single dose of rituximab (375 mg/m2) and IVIG (0.4 g/kg per day) at the beginning of the operation. IVIG was administered for 10 consecutive days after LT. Plasma exchange, splenectomy and graft local infusion were omitted in the protocol. Quadruple immunosuppressive therapy including basiliximab, corticosteroids, tacrolimus and mycophenolatemofetil was used to reinforce immunosuppression. RESULTS The 3-year cumulative patient survival rates in the ABO-I LT and ABO-C LT groups were 83.1% and 86.3%, respectively (P>0.05), and the graft survival rates were 80.0% and 86.3%, respectively (P>0.05). Two patients (5.7%) suffered from antibody-mediated rejection in the ABO-I LT group. Other complications such as acute cellular rejection, biliary complication and infection displayed no significant differences between the two groups. CONCLUSIONS The simplified treatment consisting of rituximab and IVIG prevented antibody-mediated rejection for LT of blood-type incompatible patients. With this treatment, the patients did not need plasma exchange, splenectomy and graft local infusion. This treatment was safe and efficient for LT of the patients with ALF.

Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and eNOS

AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning (RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation (OLT), ischemic postconditioning (IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine (Cr) and creatinine kinase-myocardial band (CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species (ROS), H2O2, mitochondrial membrane potential (ΔΨm) and total nitric oxide (NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase (eNOS) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting, and peroxynitrite was semi-quantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions (P < 0.05). ROS (P < 0.001) including H2O2 (P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC (P < 0.05) reversed this trend. The collapse of ΔΨm induced by OLT ischemia/reperfusion (I/R) injury was significantly attenuated in the RIPerC group (P < 0.001). A marked increase of NO content and phosphoserine eNOS, both in protein and mRNA levels, was observed in liver graft of the RIPerC group as compared with the OLT group (P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group (P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group (P < 0.01). CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/eNOS/NO pathway.

High neutrophil-lymphocyte ratio indicates poor prognosis for acute-on-chronic liver failure after liver transplantation

AIM To investigate the significance of pre-transplant neutrophil-lymphocyte ratio (NLR) in determining the prognosis of liver transplant (LT) recipients with acute-on-chronic liver failure (ACLF). METHODS Data were collected from the liver transplantation data bank. The NLR values and other conventional inflammatory markers were evaluated for their ability to predict the prognosis of 153 patients with ACLF after LT. The NLR cut-off value was based on a receiver operating characteristic curve analysis. A Kaplan-Meier curve analysis and univariate and multivariate Cox regression models were used to define the independent risk factors for poor outcomes. RESULTS The optimal NLR cut-off value was 4.6. Out of 153 patients, 83 (54.2%) had an NLR ≥ 4.6. The 1-, 3-, and 5-year overall survival rates were 94.3%, 92.5% and 92.5%, respectively, in the normal NLR group and 74.7%, 71.8% and 69.8%, respectively, in patients with high NLRs (P < 0.001). Furthermore, there was a significant difference in infectious complications after LT between the high and normal NLR groups. There were no significant differences for other complications. In the multivariate Cox regression model, a high NLR was defined as a significant predictor of poor outcomes for LT. CONCLUSION A high NLR is a convenient and available predictor for prognosis of LT patients and can potentially optimize the current criteria for LT in ACLF.

Mitofusin-2 mediated mitochondrial Ca2+ uptake 1/2 induced liver injury in rat remote ischemic perconditioning liver transplantation and alpha mouse liver-12 hypoxia cell line models

AIM To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS Sprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (ΔΨm), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected. RESULTS Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRT-PCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of ΔΨm and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005). CONCLUSION Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.

Optimal immunosuppressor induces stable gut microbiota after liver transplantation

AIM To study the influence of different doses of tacrolimus (FK506) on gut microbiota after liver transplantation (LT) in rats. METHODS Specific pathogen-free Brown Norway (BN) rats and Lewis rats were separated into five groups: (1) Tolerance group (BN-BN LT, n = 8); (2) rejection group (Lewis-BN LT, n = 8); (3) high dosage FK506 (FK506-H) group (Lewis-BN LT, n = 8); (4) middle dosage FK506 (FK506-M) group (Lewis-BN LT, n = 8); and (5) low dosage FK506 (FK506-L) group (Lewis-BN LT, n = 8). FK506 was administered to recipients at a dose of 1.0 mg/kg, 0.5 mg/kg, and 0.1 mg/kg body weight for 29 d after LT to the FK506-H, FK506-M, and FK506-L groups, respectively. On the 30th day after LT, all rats were sampled and euthanized. Blood samples were harvested for liver function and plasma endotoxin testing. Hepatic graft and ileocecal tissues were collected for histopathology observation. Ileocecal contents were used for DNA extraction, Real-time quantitative polymerase chain reaction (RT-PCR) and digital processing of denaturing gradient gel electrophoresis (DGGE) profiles and analysis. RESULTS Compared to the FK506-H and FK506-L groups, FK506-M was optimal for maintaining immunosuppression and inducing normal graft function; the FK506-M maintained gut barrier integrity and low plasma endotoxin levels; furthermore, DGGE results showed that FK506-M induced stable gut microbiota. Diversity analysis indicated that FK506-M increased species richness and rare species abundance, and cluster analysis confirmed the stable gut microbiota induced by FK506-M. Phylogenetic tree analysis identified crucial bacteria associated with FK506-M; seven of the nine bacteria that were decreased corresponded to Bacteroidetes, while increased bacteria were of the Bifidobacterium species. FK506-M increased Faecalibacterium prausnitzii and Bifidobacterium spp. and decreased Bacteroides-Prevotella and Enterobacteriaceae, as assessed by RT-PCR, which confirmed the crucial bacterial alterations identified through DGGE. CONCLUSION Compared to the low or high dosage of FK506, an optimal dosage of FK506 induced immunosuppression, normal graft function and stable gut microbiota following LT in rats. The stable gut microbiota presented increased probiotics and decreased potential pathogenic endotoxin-producing bacteria. These findings provide a novel strategy based on gut microbiota for immunosuppressive dosage assessment for recipients following LT.

Partial Inhibition of HO-1 Attenuates HMP-Induced Hepatic Regeneration against Liver Injury in Rats

We found better liver graft regeneration with hypothermic machine perfusion (HMP) compared with static cold storage (SCS) for the first time in our pilot study, but the underlying mechanisms are unknown. Upregulated heme oxygenase- (HO-) 1 expression has been reported to play a pivotal role in promoting hepatocyte proliferation. Here, we evaluated the novel role of HO-1 in liver graft protection by HMP. Rats with a heterozygous knockout of HO-1 (HO-1+/−) were generated and subjected to 3 h of SCS or HMP pre-half-size liver transplantation (HSLT) in vivo or 6 h of SCS or HMP in vitro; control rats were subjected to the same conditions (HO-1+/+). We found that HSLT induced significant elevation of the HO-1 protein level in the regenerated liver and that HO-1 haplodeficiency resulted in decreased proliferation post-HSLT. Compared with SCS, HMP induced significant elevation of the HO-1 protein level along with better liver recovery, both of which were reduced by HO-1 haplodeficiency. HO-1 haplodeficiency-induced decreased proliferation was responsible for the attenuated regenerative ability of HMP. Mechanistically, HO-1 haploinsufficiency resulted in suppression of hepatocyte growth factor (HGF)/Akt activity. Our results suggest that inhibition of HO-1 mitigates HMP-induced liver recovery effects related to proliferation, in part, by downregulating the HGF-Akt axis.

Optimized postconditioning algorithm protects liver graft after liver transplantation in rats

BACKGROUND Ischemia reperfusion injury (IRI) causes postoperative complications and influences the outcome of the patients undergoing liver surgery and transplantation. Postconditioning (PostC) is a known manual conditioning to decrease the hepatic IRI. Here we aimed to optimize the applicable PostC protocols and investigate the potential protective mechanism. METHODS Thirty Sprague-Dawley rats were randomly divided into 3 groups: the sham group (n = 5), standard orthotopic liver transplantation group (OLT, n = 5), PostC group (OLT followed by clamping and re-opening the portal vein for different time intervals, n = 20). PostC group was then subdivided into 4 groups according to the different time intervals: (10 s × 3, 10 s × 6, 30 s × 3, 60 s × 3, n = 5 in each subgroup). Liver function, histopathology, malondialdehyde (MDA), myeloperoxidase (MPO), expressions of p-Akt and endoplasmic reticulum stress (ERS) related genes were evaluated. RESULTS Compared to the OLT group, the grafts subjected to PostC algorithm (without significant prolonging the total ischemic time) especially with short stimulus and more cycles (10 s × 6) showed significant alleviation of morphological damage and graft function. Besides, the production of reactive oxidative agents (MDA) and neutrophil infiltration (MPO) were significantly depressed by PostC algorithm. Most of ERS related genes were down-regulated by PostC (10 s × 6), especially ATF4, Casp12, hspa4, ATF6 and ELF2, while p-Akt was up-regulated. CONCLUSIONS PostC algorithm, especially 10 s × 6 algorithm, showed to be effective against rat liver graft IRI. These protective effects may be associated with its antioxidant, inhibition of ERS and activation of p-Akt expression of reperfusion injury salvage kinase pathway.

The predictive value of blood neutrophil-lymphocyte ratio in patients with end-stage liver cirrhosis following ABO-incompatible liver transplantation

Background: The study was designed to assess the role of preoperative neutrophil, lymphocyte, and neutrophil-lymphocyte ratio (NLR) in predicting survival outcomes of ABO-incompatible liver transplantation (LT). Materials and Methods: We retrospectively collected the demographic and clinical characteristics of 71 patients with end-stage liver cirrhosis following ABO-incompatible LT in this study. Kaplan–Meier survival analysis and Cox multiple factors regression analysis were performed to determine the independent risk factors from preoperative blood parameters for poor prognosis. Results: The 1-, 3-, and 5-year overall survival were 94.9%, 80.0%, and 80.0% in the normal NLR group, respectively, and 59.4%, 55,4%, and 55.4% in patients with up-regulated NLR, respectively (P = 0.001). Furthermore, no significant difference was observed on post-LT complications between normal NLR and high-NLR groups. The high NLR was identified as the only independent prognostic risk factor for recipient survival (P = 0.015, 95% confidence interval = 3.573 [1.284–9.943]). Conclusion: The preoperative high NLR could be considered as a convenient and available indicator for selecting ABO-incompatible LT candidates.