Orthotopic Liver Transplantation Model Research Articles

Blockade of the Notch1/Jagged1 pathway in Kupffer cells aggravates ischemia-reperfusion injury of orthotopic liver transplantation in mice

Liver ischemia-reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). Kupffer cells (KCs) are the largest antigen-presenting cell (APC) group and the primary modulators of inflammation in liver tissues. The vital role of Notch1/Jagged1 pathway in mouse OLT model has been reported, however, its potential therapeutic mechanism is unknown. Here, we made use of short hairpin RNA-Jagged1 and AAV-Jagged1 to explore the effects of Notch1/Jagged1 pathway in OLT. In vitro, blockade of Notch1/Jagged1 pathway downregulated the expression of Hairy and enhancer of split-1 (Hes1) gene, which in turn increased the proinflammatory effects of KCs. Moreover, the anti-inflammatory effects of Notch1/Jagged1 pathway were induced by inhibiting Hes1/gene of phosphate and tension/protein kinase B/Toll-like receptor 4/nuclear factor kappa B (Hes1/PTEN/AKT/TLR4/NF-κB) axis in KCs. In vivo, we used a well-established mouse model of OLT to mimic clinical transplantation. Mice were stochastically divided into 6 groups: Sham group (n = 15); Normal saline (NS) group (n = 15); Adeno-associated virus–green fluorescent protein (AAV-GFP) group (n = 15); AAV-Jagged1 group (n = 15); Clodronate liposome (CL) group (n = 15); CL+AAV-Jagged1 group (n = 15) . After OLT the liver damage in AAV-Jagged1 group were significantly accentuated compared to the AAV-GFP group. While blockade of Jagged1 aftet clearence of KCs by CL would not lead to further liver injuries. Taken together, our study demonstrated that blockade of Notch1/Jagged1 pathway aggravates inflammation induced by lipopolysaccharide (LPS) via Hes1/PTEN/AKT/TLR4/NF-κB in KCs, and the blockade of Notch1/Jagged1 pathway in donor liver increased neutrophil/macrophage infiltration and hepatocellular apoptosis, which suggested the function of Notch1/Jagged1 pathway in mouse OLT and highlighted the protective function of Notch1/Jagged1 pathway in liver transplantation.

The mechanism of CD8+ CD45RClow regulatory T cell in rat liver transplantation nature tolerance model

Objective To explore the mechanism of CD8+ CD45RClow regulatory T cells and its related cytokines function during the tolerance process of orthotopic liver transplantation in rats. Methods The male Brown Norway rats were selected as the donor, the Lewis rats were the recipients, the allogenic orthotopic liver transplantation model was established by the double cuff method. Experiment animals were divided into early transplantation group (10 rats), tolerance group (10 rats), sham group. Liver function were detected. The tolerance recipients were detected by hematoxylin and eosin (HE) staining. The Luminex used to detect of cytokine and the flow cytometry were used to analyze the changes of subsets of lymphocytes in the peripheral blood/graft and draining lymph node. Results 70% of recipients in operation group could be induced natural immune tolerance. The levels of aspartate aminotransferase (AST) in tolerance group were [(456±54) U/ml] vs. those of sham group [(460±151) U/ml], (P>0.05); the levels of alanine aminotransferase (ALT) in tolerance group were [(122±35) U/ml] vs. those of sham group [(121±46) U/ml, P>0.05]. The expression of CD8+ CD45RClow T cells in tolerance group were increased than in short term group; in grafts it was up regulated in tolerance group than in sham group, and it was no significance between sham group and tolerance group in lymph node around graft. The expression of pDC in short term group are significantly increased compared with sham group. The interferon (IFN)-γ was increased in short term group, return to normal level in tolerance group. interleukin (IL)-10 in grafts milieu were dramatically increased, keep in high level in tolerance group. Conclusion In the tolerance recipients of rat orthotopic liver transplantation model, CD8+ CD45RClow regulatory T cells were significantly increased and aggregated in the peripheral blood and liver. The inhibitory cytokines IL-10 was important to maintain the long-term graft tolerance. Key words: Regulatory T cell; Liver transplantation; Immune tolerance; Interleukin-10; Dendritic cell

Effects of Panax notoginseng saponins on liver graft rejection in rats and the mechanisms

To investigate the effects of Panax notoginseng saponins (PNS) on the functional status of Kupffer cells (KCs) and immune environment after liver transplantation and explore the possible mechanisms. KCs were isolated from rats and assessed for phagocytic activity and viability using ink and Trypan blue staining. The cells were exposed to lipopolysaccharide (LPS) alone or in combination with PNS treatment at 0, 10 or 20 μmol/L. The expressions of the inflammatory factors and the oxidative stress products in the cells and the supernatant were assayed with Western blotting and ELISA; the expression of CD206 was detected using immunofluorescence assay, and the expressions of NF-κB and Keap1-Nrf2-ARE pathway proteins were detected using Western blotting. We established an orthotopic liver transplantation (LT) model in rats and assessed the effect of 200 mg/kg PNS on the graft function, inflammatory factors, pathology of the liver tissue, hepatocyte apoptosis and survival time of the rats in comparison with those in rats receiving a sham operation or PBS treatment following LT. Treatment with PNS significantly lowered the levels of inflammatory factors and oxidative stress products and increased the levels of interleukin-10 (IL-10) and SOD in a concentration-dependent manner in the KCs (P < 0.05). Immunofluorescence assay showed that PNS treatment obviously increased the expression of CD206 in the KCs. PNS treatment also significantly reduced the expressions of IRAK4, p-IKKα, p-IκBα, p-p65 and Keap1 proteins and increased the expression levels of Nrf2 and ARE proteins in the KCs (P < 0.05). In the rat models of LT, PNS treatment significantly improved the liver graft function, lowered the expression of the pro-inflammatory factors, and reduced hepatocyte apoptosis as compared with PBS treatment. PNS treatment obviously alleviated pathological changes in the liver graft and significantly prolonged the survival time of the rats following LT (P < 0.05). In addition, injection of GdCl3 to block KC function resulted in severe acute graft rejection in the rats regardless of PNS treatment (P > 0.05). PNS can reduce inflammatory response and oxidative stress in activated KCs by inhibiting NF-κB and Keap1-Nrf2-ARE pathways and promote the polarization of KCs into M2 phenotype to prolong the survival time of rats after LT.

Structural shifts in the intestinal microbiota of rats treated with cyclosporine A after orthotropic liver transplantation.

Understanding the effect of immunosuppressive agents on intestinal microbiota is important to reduce the mortality and morbidity from orthotopic liver transplantation (OLT). We investigated the relationship between the commonly used immunosuppressive agent cyclosporine A (CSA) and the intestinal microbial variation in an OLT model. The rat samples were divided as follows: (1) N group (normal control); (2) I group (isograft LT, Brown Norway [BN] rat to BN); (3) R group (allograft LT, Lewis to BN rat); and (4) CSA group (R group treated with CSA). The intestinal microbiota was assayed by denaturing gradient gel electrophoresis profiles and by using real-time polymerase chain reaction. The liver histopathology and the alanine/aspartate aminotransferase ratio after LT were both ameliorated by CSA. In the CSA group, the numbers of rDNA gene copies of Clostridium cluster I, Clostridium cluster XIV, and Enterobacteriaceae decreased, whereas those of Faecalibacterium prausnitzii increased compared with the R group. Cluster analysis indicated that the samples from the N, I, and CSA groups were clustered, whereas the other clusters contained the samples from the R group. Hence, CSA ameliorates hepatic graft injury and partially restores gut microbiota following LT, and these may benefit hepatic graft rejection.

Blockade targeting Cd47 reduces iri and improves survival in liver transplantation model

Introduction: Orthotopic liver transplantation (OLT) is currently the elective treatment for advanced liver cirrhosis and acute liver failure. Ischemia/reperfusion (IRI) injury may jeopardize graft function during the postoperative period. Treatment with anti-CD47 antibodies(CD47mAbs) has demonstrated cytoprotective properties in different experimental models of ischemia and liver injury, however, there is no evidence to demonstrate its potential use in the prevention of the ischemia/reperfusioninjury that occurs during OLT. The present study is the first report to show that the administration of CD47mAbs to donors and recipients provide benefits OLT outcomes. Method: We tested the cytoprotective effect of a CD47mAb 400, administered to the donor prior to OLT or to both the donor and to recipient during OLT in ansyngeneic experimental model in the Lewis rat. Serum transaminase levels,histopathological changes, proinflammatory cytokines,cell death parameters, oxidative and nitrosative stress, and animal survival were Hepatic histology, serum transaminase levels, pro-inflammatory cytokines,celldeath parameters, oxidative and nitrosative stress, and animal survival were analyzed. Result: Our data showed that CD47mAb antibody administration to donors reduced the level of transaminases and to both the donor and recipient not only reduced the level of transaminases, but also increased animal survival,and reduced hepatocellular injury, as well as oxidative and nitrosative stress. These beneficial effects, related to reduced caspase-3 activity and diminished IL-1b, IL-6 and TNF-a expression in vivo together with IL-6 upregulation in vivo. Conclusion: The administration of a CD47mAb to donors or to both the donor and to recipien in an experimental rat model of OLT reduced indices of ischemia/reperfusion injury. Treatment of both the donor organ and the recipient provided better protection that treating the donor organ alone and also improved survival following transsplantation.

Bone marrow mesenchymal stromal cells attenuate liver allograft rejection may via upregulation PD-L1 expression through downregulation of miR-17-5p

Studies have reported that bone marrow mesenchymal stem cells (BMSCs) play an important role in immune regulation after organ transplantation. Some of the BMSC-mediated regulatory mechanisms are related to PD-L1 expression. However, the regulatory mechanism of PD-L1 expression is not fully understood. In this experiment, we confirmed the regulatory role of BMSCs in the rejection of liver transplantation and explored the possible mechanism by which PD-L1 expression is regulated in BMSCs. An orthotopic liver transplantation model in rats was established based on the “double-cuff technique”. Third-generation BMSCs were injected into the rejection model rats via portal vein. At the same time, sera were obtained from rats in the allograft, isograft and sham-operated groups. The sera from these groups were separately added to BMSCs complete medium to partially mimic the in vivo environment in which BMSCs are exposed. After predicting and analysing microRNAs that regulate PD-L1 expression, we examined the microRNA and PD-L1 expression in BMSCs of the three groups cultured in the conditioned media. Moreover, a luciferase reporter assay was used to confirm the interaction between PD-L1 and microRNA. The study found that the liver function and liver graft histopathology of the BMSC-treated group were better than those of the allograft group. The Kaplan-Meier survival curve analysis showed that the median survival time (MST) of the BMSC-treated group was longer than that of the allograft group. Bioinformatics prediction and analysis showed that miR-17-5p is highly likely to regulate PD-L1. Compared with the other two groups of cells, BMSCs cultured with serum from allograft models showed higher PD-L1 expression, but lower miR-17-5p expression. Pearson correlation analysis showed that miR-17-5p and PD-L1 expression was negatively correlated, and further luciferase reporter assays confirmed that miR-17-5p interacted directly with the 3´-untranslated region (UTR) of PD-L1 mRNA. The results of this study demonstrate that BMSCs can attenuate liver allograft rejection and provide us with the idea that BMSCs may further upregulate PD-L1 expression by downregulating miR-17-5p expression, thereby attenuating liver allograft rejection.

Intestinal Mucosal Barrier Disruption and Microfold Cells Impairment were Required for Invasive Fungal Infection after Living Donor Liver Transplantation.

Introduction and Objective Post-transplant invasive fungal infection (IFI) is a critical problem with high mortality for patients receiving living donor liver transplantation (LDLT), however, the underlying mechanism stays unclear. Hence, identifying the risk factors and understanding the mechanisms of IFIs are essential to improve outcome of LDLT patients. Materials and Methods Clinical characteristics of patients diagnosed with opportunistic fungal infections compared with non-infected patients after living donor liver transplantation were analyzed in a retrospective cohort from 1994 to 2017. The correlation between marginal graft injury and incidence of IFIs was investigated by 1, 3-β-D-Glucan assay in rat orthotopic liver transplantation models using marginal grafts. And intestinal mucosal structural integrity as well as microfold cells of intestinal Peyer’s patches were also investigated in animal models. Results and Discussion 44 out of 584 patients were diagnosed with opportunistic fungal infections after LDLT, and they were presented with higher hospital mortality (15% vs 2.8%, p=0.002) as well as long term graft failure compared with patients without infections (Fig. 1A, HR=2.394, p<0.001). In addition, prolonged cold ischemic time and warm ischemic time were identified associated with post-transplant opportunistic infection occurrence (Fig. 1BC). In rat orthotopic liver transplantation models, incidence of IFIs and median serum β-D-Glucan value were higher in small-for-size graft, as well as in graft with prolonged ischemic time compared to whole-size graft and sham operation groups (Fig. 2AB). Furthermore, disruption of intestinal villi structure and loss of tight junctions were found associated with more severe liver graft injury (Fig. 2C). And damage to tight junctions of intestinal follicle associated epithelium covering Peyer’s patches as well as deletion of functional microfold cells (M cells) were also found in liver graft after transplantation, whilst the situation was even worse after ischemic time was extended (Fig. 2D). Conclusion Small-for-size and ischemic injury of liver graft rendered LDLT patients more vulnerable to gut derived invasive fungal infections by disrupting intestinal mucosal barrier and impairing intestinal innate immunity.

Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench-to-bedside.

This translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273).

PDL1Ig gene-loaded BMSCs Induce liver transplantation immune tolerance.

We investigated the effects of BMSCs, which was modified by programmed death ligand-1 immunoglobulin (PDL1Ig) gene, on the immunological rejection of orthotopic liver transplantation (OLT) in rats. Rat BMSCs were cultured and modified by recombinant adenovirus pAdEasy-1/PDL1Ig for 72h. The total protein was extracted, and the protein expression of PDL1Ig after transfection was detected by Western blotting. Mixed lymphocyte reaction was applied to detect the inhibitory effect of BMSCs, including pre-transfection and post-transfection, on the cell activity of T lymphocytes in peripheral blood. The male Wistar rats were used as donors, and the male Sprague-Dawley (SD) rats were used as recipients. The improved cuff method was computed in OLT to establish the rat orthotopic liver transplantation model of acute rejection. The rats were randomly divided into 4 groups, including a control group (10 pairs), BMSCs treatment group (10 pairs), BMSCs/GFP (green fluorescent protein) treatment group (10 pairs), and BMSCs/PDL1Ig treatment group (10 pairs). 5 rats in each group were randomly collected and euthanized at day 7 after the operation. The peripheral blood was gathered to detect levels of 3 types of cytokines, including interferon gamma (IFN-g), interleukin-4 (IL-4), and IL-2. In addition, the liver function of rats was checked, and the pathological changes of liver transplantation were observed under the optical microscope. The remaining five rats in every group were used for measuring the survival situation and survival time. After BMSCs were modified by a recombinant adenovirus pAdEasy-1/PDL1Ig for 72h, the expression of PDL1Ig in the BMSCs was detected. The inhibitory effects of BMSCs/PDL1Ig on the cell activity of lymphocytes were stronger than that of BMSCs/GFP. The levels of IL-4 in BMSCs/PDL1Ig group were significantly higher than those in the other 3 groups, and the levels of IFN-g and IL-2 were significantly decreased (p<0.05). The liver function of 4 groups was measured at day 7 after transplantation. Our results showed that the liver function in BMSCs/PDL1Ig group was most significantly improved, and the levels of ALT, AST, and TBil almost recovered to normal. The differences were statistically significant compared to the control group, BMSCs treatment group, and BMSCs/GFP treatment group. Results of pathological examination of liver tissue showed that the control group underwent severe rejection of liver transplantation. The BMSCs treatment group and BMSCs/GFP treatment group also rejected the liver transplantation, but the degree was lighter when compared to the control group. In addition, there was almost no rejection of liver transplantation in the BMSCs/PDL1Ig group. The recipient survival time of most rats was more than 100d, which was significantly longer than the other 3 groups (p<0.05). PDL1Ig-modified BMSCs can inhibit the rejection of liver transplantation in rats, induce the formation of the immune tolerance of liver transplantation, and the effect was more significant than that of BMSCs alone.

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

Hypoxia preconditioning protects Ca2+-ATPase activation of intestinal mucosal cells against R/I injury in a rat liver transplantation model.

AIMTo investigate the effect of ischaemia and reperfusion (I/R) injury on the Ca2+-ATPase activation in the intestinal tissue of a rat autologous orthotopic liver transplantation model and to determine if hypoxia preconditioning (HP) therapy induces HIF-1α to protect rat intestinal tissue against I/R injury.METHODSRats received non-lethal hypoxic preconditioning therapy to induce HIF-1α expression. We used an autologous orthotopic liver transplantation model to imitate the I/R injury in intestinal tissue. Then, we detected the microstructure changes in small intestinal tissues, Ca2+-ATPase activity, apoptosis, and inflammation within 48 h postoperatively.RESULTSHIF-1α expression was significantly increased in intestinal tissue at 12 h postoperatively in rats that were exposed to a hypoxic environment for 90 min compared with a non-HP group (HP vs AT, P = 0.0177). Pathological analysis was performed on the intestinal mucosa cells, and the cells in the HP group appeared healthier than the cells in the AT group. The Ca2+-ATPase activity in the small intestinal cells in the AT group was significantly lower after the operation, and the Ca2+-ATPase activity in the HP group recovered faster than that in the AT group at 6 h postoperatively (HP vs AT, P = 0.0106). BCL-2 expression in the HP group was significantly higher than that in the AT group at 12 h postoperatively (HP vs AT P = 0.0010). The expression of the inflammatory factors NO, SOD, IL-6, and TNF-α was significantly lower in the HP group than in the AT group.CONCLUSIONHypoxia-induced HIF-1α could protect intestinal mucosal cells against mitochondrial damage after I/R injury. HP could improve hypoxia tolerance in small intestinal mucosal cells and increase Ca2+-ATPase activity to reduce the apoptosis of and pathological damage to intestinal cells. HP could be a useful way to promote the earlier recovery of intestinal function after graft procedure.

Role of bone marrow mesenchymal stem cells with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation

Objective: To investigate the role of bone marrow mesenchymal stem cells (BMSCs) with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation in rats and possible mechanisms. Methods: The modified Kamada's two-cuff technique was used to establish a Lewis-BN rat model of orthotopic liver transplantation, and a total of 75 rats were randomly divided into groups A, B, C, D, and E, with 15 rats in each group. The rats in group A (control group) were given infusion of isotonic saline via the portal vein during liver transplantation, those in group B (BMSC group) were given infusion of BMSCs via the portal vein during liver transplantation, those in group C (BMSCs with CTLA4Ig gene modification) were given infusion of BMSCs carrying the CTLA4Ig gene via the portal vein during liver transplantation, those in group D (BMSCs with CD40LIg gene modification) were given infusion of BMSCs carrying the CD40LIg gene via the portal vein during liver transplantation, and those in group E (BMSCs with CTLA4Ig and CD40LIg gene modification) were given infusion of BMSCs carrying CTLA4Ig and CD40LIg gene modification via the portal vein during liver transplantation. Postoperative survival and change in liver function were observed. HE staining was used to observe the pathomorphological changes of the graft liver, and ELISA was used to measure the levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon-γ (IFN-γ) in peripheral blood. A one-way analysis of variance was used for comparison of means of multiple samples, and the Kaplan-Meier survival curve analysis was used for comparison of survival rates between multiple groups. Results: Group E had a significantly longer survival time after surgery than groups A, B, C, and D (P < 0.05), groups C and D had a significantly longer survival time than groups A and B (P < 0.05), and there was no significant difference between groups C and D (P > 0.05). On day 10 after surgery, group A had significantly higher levels of alanine aminotransferase and total bilirubin than the other four groups (P < 0.05). HE staining showed severe rejection reaction in group A, moderate rejection reaction in group B, and mild rejection reaction in groups C and D; pathological examination showed no marked rejection reaction in group E. Group A had significant increases in the levels of IL-2 and IFN-γ and significant reductions in the levels of IL-4 and IL-10 after surgery compared with the other four groups (all P < 0.05). Conclusion: Infusion of BMSCs with modification of both CTLA4Ig and CD40LIg genes can significantly inhibit acute rejection reaction after liver transplantation in rats and effectively prolong the survival time of the graft liver, with a better effect than infusion of BMSCs alone or BMSCs with modification of CTLA4Ig or CD40LIg gene.

A novel rat model of orthotopic liver transplantation after cardiac arrest and cardiopulmonary resuscitation

Objective To establish a stable rat model of orthotopic liver transplantation(OLT) after cardiac arrest and cardiopulmonary resuscitation (CPR). Methods The OLT model was established using Kammada two-cuff technique with little modification. The donor rat’s abdominal aorta was clipped for 5 minutes to simulate the cardiac arrest and CPR before transplantation. The serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) of the controlled group and the experimental group were examined before transplantation. Results Of the total 50 cases of surgery, 46 cases were successful, and the success rate of the model was 92%. The donor and recipient operation time was (49±4) min and (54±5) min, respectively. The anhepatic phase was (17±2) min. The serum levels of ALT and AST of the experimental group were significantly higher than that of the controlled group (P=0.037, 0.023). Conclusion Based on the Kammada two-cuff technique, the rat model of OLT after cardiac arrest and CPR is stable, reliable and reproducible. The model simulated the cardiac arrest and CPR of the donor before liver transplantation, providing a new experimental method of studying the liver transplantation of this kind of donors. Key words: Two-cuff technique; Rat; Liver transplantation model; Cardiac arrest and cardiopulmonary resuscitation

Glutathione Peroxidase 3 Delivered by hiPSC-MSCs Ameliorated Hepatic IR Injury via Inhibition of Hepatic Senescence.

Background and Aims: Down-regulation of GPx3 accelerated hepatic senescence, which further caused overwhelming inflammation and severe liver graft injury. MSCs derived from human induced pluripotent stem cells (hiPSC-MSCs) have been developed as more efficient delivery vehicle with the property of injury tropism. Here, we aimed to explore the suppressive role of GPx3 in hepatic IR injury using novel delivery system of hiPSC-MSCs.Methods: The mice IR injury model with partial hepatectomy was established. The engineered hiPSC-MSCs delivering GPx3 was constructed. All the mice were segregated into three groups. hiPSC-MSC-GPx3, hiPSC-MSC-pCDH (vector control) or PBS were injected via portal vein after reperfusion. Liver injury was evaluated by histological and serological test. Hepatic apoptosis was detected by Tunel staining and remnant liver regeneration was assessed by Ki67 staining. The role of hepatic senescence in liver graft injury was evaluated in rat orthotopic liver transplantation model. The suppressive effect of GPx3 on hepatic senescence was examined in mice IR injury model and confirmed in vitro. Hepatic senescence was detected by SA-β-Gal and P16/ink4a staining.Results: GPx3 can be successfully delivered by hiPSC-MSCs into liver tissues. Histological examination showed that hiPSC-MSC-GPx3 treatment significantly ameliorated hepatic IR injury post-operation. Significantly lower LDH (891.43±98.45 mU/mL, P<0.05) and AST (305.77±36.22 IU/L, P<0.01) were observed in hiPSC-MSC-GPx3 group compared with control groups. Less apoptotic hepatocytes were observed and the remnant liver regeneration was more active in hiPSC-MSC-GPx3 group. In rat orthotopic liver transplantation model, more senescent hepatocytes were observed in small-for-size liver graft, in which GPx3 expression was significantly compromised. In mice IR injury model, hiPSC-MSC-GPx3 significantly suppressed hepatic senescence. In addition, rGPx3 inhibited cellular senescence of liver cells in a dose dependent manner. Four candidate genes (CD44, Nox4, IFNG, SERPERINB2) were identified to be responsible for suppressive effect of GPx3 on hepatic senescence.Conclusion: Engineered hiPSC-MSCs delivering GPx3 ameliorated hepatic IR injury via inhibition of hepatic senescence.

Protective Effect of a Novel Technique for Liver Transplantation in the Rat

The rat orthotopic liver transplantation model with extremely short anhepatic phase was established to study its protective effect on the recipients and graft. One hundred fifty adult male Wistar rats were randomly divided into three groups: group A (n = 30), using magnetic rings for the suprahepatic vena cava reconstruction; group B (n = 30), using 7/0 Prolene sutures for suprahepatic vena cava running anastomosis as control; and a sham-operated group (n = 30) as a blank control group. The changes in liver enzyme, serum creatinine, endotoxin, and cytokine levels and histopathology were recorded. The serum creatinine, potassium, alanine transaminase, and alkaline phosphatase levels at different points in time in group A were lower than those in group B (P < .05). The level of portal vein blood endotoxin in group A was significantly lower than that in group B at each point (P < .01). At the same time, all the cytokines in group B were higher than those in group A, and the two groups were higher than those in the sham operation group. The mean levels of tumor necrosis factor-α (TNF-α), interferon-γ, (IFN-γ), and interleukin-1ß (IL-1ß) at 3 hours were higher than at 6 hours in group A. IL-10 and tissue inhibitor of metalloproteinase-1 (TIMP-1) were all higher at 3 hours in groups A and B. Levels of monocyte chemotactic protein-1, L-selectin, and TIMP-1 in group A and IL-10, monocyte chemotactic protein-1, L-selectin, and TIMP-1 in group B were higher in blood than in the liver. Levels of TNF-α, IFN-γ, IL-1, IL-10, and intracellular adhesion molecule-1 in group A and TNF-α, IFN-γ IL-1ß, and intracellular adhesion molecule-1 in group B were higher in the liver than in blood. We conclude that the extremely short anhepatic phase has protective effects on recipients and grafts in rat liver transplantation because it is related to alleviating ischemia-reperfusion injury and reducing the endotoxin release.

Heme oxygenase-1 regulates sirtuin-1-autophagy pathway in liver transplantation: From mouse to human.

Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT.

Operative Techniques, Treatment, and Precaution of Common Complications of Orthotopic Rat Liver Transplantation

<b>Objective</b> This study investigates operative techniques, treatment, and precaution for common complications of orthotopic liver transplantation (OLT) in rats. <b>Methods</b> OLT was performed in 110 rats through modified two-cuff method. Operative techniques were concluded, and causes, treatment, and precaution of common complications were analyzed. <b>Results </b>In operation phase, main causes for rat death included anesthesia, airway obstruction, pneumothorax, hemorrhagic shock, long anhepatic time, and air embolism. After surgery, main causes for rat death were as follows: stoma bleeding, infection, obstruction, and necrosis of biliary tract and poor hepatic function restoration. <b>Conclusion </b>Improving operative techniques, modifying operative methods, and familiarizing with common complications can reduce occurrence of complications, heighten operative successful rate, and establish more stable models of OLT in rats.

Surgical Techniques of Allogeneic Liver Transplantation in a Nonhuman Primate Model

Herein, we report our experience of performing allogeneic orthotopic liver transplantation (LT) in nonhuman primates. We designed an allogeneic ABO-compatible orthotopic LT model in monkeys in a manner similar to that used in humans. We applied almost the same surgical procedures used for human conventional deceased donor LT. A total of 6 monkeys underwent allogeneic LT. One cynomolgus monkey aged 45 months (3.4 kg) and 5 rhesus macaque monkeys aged 50.2 ± 14.8 months (5.40 ± 0.33 kg) were used as recipients. In the donor surgery, the liver was perfused in situ through the aorta using cold histidine-tryptophan-ketoglutarate solution. The portal vein (diameter, 5–10 mm), supra- and infra-hepatic inferior vena cava (IVC) (diameter, 12–15 mm), and common bile duct (diameter, 1.5–3.0 mm) were dissected out. The hepatic artery was kept in continuity with the celiac trunk and abdominal aorta up to the iliac bifurcation (diameter, 5–6 mm). The mean graft weight was 102.0 g (94.8–111.0 g). Recipient surgery was conducted in parallel. After recipient hepatectomy, the graft was implanted. The suprahepatic IVC and portal vein were anastomosed to those of the graft. After reperfusion, the infrahepatic IVC was anastomosed. The aorta conduit of the graft was anastomosed to the infrarenal aorta of the recipient in a retrocolic end-to-side manner. Biliary reconstruction was performed in a duct-to-duct anastomosis with cholecystectomy. Mean operative time was 107.0 minutes for donor and 198.2 minutes for recipient. There was one operative death due to unknown cause. In conclusion, for allogeneic orthotopic LT in nonhuman primate model, we can apply almost the same procedure used for human conventional deceased donor LT in a similar manner.

Orthotopic liver transplantation from cardiac death donors in the mouse: a new model and evaluation of cardiac death time.

Objective(s):The goal of this research was to develop a mouse orthotopic liver transplantation (LTx) model from donor-after-cardiac-death (DCD) grafts.Materials and Methods:Mice were randomly assigned to the experimental group or the sham group. The mice in the experimental group were divided into three groups according to the warm ischemia time (WIT) of liver graft: normal LTx, WIT 30 minute (min) +LTx and WIT 45 min +LTx. The descending aorta was clamped using a miniature aortic clamp to simulate cardiac arrest in the DCD grafts. Subsequently, the grafts were orthotopically transplanted into C57BL/6 mice. The 7-day survival rate, serum alanine aminotransferase (ALT), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) mRNA level, tumor necrosis factor-alpha (TNF-α) mRNA level, as well as hepatic pathologic alterations were observed.Results:The 7-day survival rate was markedly lower in the WIT 45 min+LTx group than that in the normal LTx group (25% versus 100%, P-value<0.05), with no significant difference between the WIT 30 min +LTx and normal LTx group (75% versus 100%, P-value>0.05). Serum ALT level of WIT 45 min+LTx group was markedly higher than that of normal LTx and WIT 30 min+LTx group (P-value<0.01). There were significant differences in necrosis and apoptosis among the three groups (P-value<0.05). The expression of iNOS, IL-6 mRNA and TNF-α mRNA in WIT 45 min +LTx group all increased significantly compared with the normal LTx and WIT 30 min+LTx group.Conclusion:The DCD LTx model is feasible in the mouse and would provide many advantages for biomedical research on LTx from DCD grafts.

A Soluble Form of P Selectin Glycoprotein Ligand 1 Requires Signaling by Nuclear Factor Erythroid 2-Related Factor 2 to Protect Liver Transplant Endothelial Cells Against Ischemia-Reperfusion Injury.

Liver endothelial cell (LEC) damage is essential in the pathogenesis of ischemia-reperfusion injury (IRI) in transplant recipients. We analyzed the mechanism of LEC resistance against IRI by using a novel recombinant soluble form of P selectin glycoprotein ligand 1, tandem P selectin glycoprotein ligand immunoglobulin (TSGL-Ig), in a mouse model of hepatic cold preservation (4°C in University of Wisconsin solution for 20 h) and syngeneic orthotopic liver transplantation (OLT). Unlike controls, TSGL-Ig protected orthotopic liver transplants against ischemia-reperfusion (IR) stress, shown by depressed serum alanine aminotransferase levels, well-preserved hepatic architecture, and improved survival (42% vs. 92%). TSGL-Ig suppressed neutrophil/macrophage sequestration and proinflammatory cytokine/chemokine programs in OLT. Treatment with TSGL-Ig mitigated LEC activation (P and E selectin, VCAM-1 and intercellular adhesion molecule 1 expression). In parallel in vitro studies, TSGL-Ig diminished cellular damage in H2 O2 -stressed LEC cultures (lactic acid dehydrogenase and alanine aminotransferase levels). Increased thioredoxin, glutamate-cysteine ligase, NAD(P)H quinone dehydrogenase 1, and hypoxia-inducible factor 1α expression, along with transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), implied that TSGL-Ig exerts antioxidant functions in IR-stressed OLT and H2 O2 -stressed LECs. Indeed, Nrf2-deficient livers suffered fulminant IRI compared with WT despite concomitant TSGL-Ig therapy. Thus, TSGL-Ig is not only acting as a competitive antagonist blocking leukocyte migration into IR-stressed liver, but it may also act directly as an agonist stimulating Nrf2-mediated cytoprotection in LECs. This study supports the role of P selectin signaling in hepatic homeostasis in OLT, with broad implications for tissue damage conditions.