Rat Orthotopic Liver Transplantation Research Articles

Bulk and single-cell RNA sequencing analysis with 101 machine learning combinations reveal neutrophil extracellular trap involvement in hepatic ischemia-reperfusion injury and early allograft dysfunction

BackgroundHepatic ischaemia–reperfusion injury (HIRI) is a major clinical concern during the perioperative period and is closely associated with early allograft dysfunction (EAD), acute rejection (AR) and long-term graft survival. Neutrophil extracellular traps (NETs) are extracellular structures formed by the release of decondensed chromatin and granular proteins following neutrophil stimulation. There is growing evidence that NETs are involved in the progression of various liver transplantation complications, including ischaemia–reperfusion injury (IRI). This study aimed to comprehensively analyse the expression patterns of NET-related genes (NRGs) in HIRI, identify HIRI subtypes with distinct characteristics, and develop a reliable EAD prediction model. MethodsMicroarray, bulk RNA-seq, and single-cell sequencing datasets were obtained from the GEO database. Initially, differentially expressed NRGs (DE-NRGs) were identified using differential gene expression analyses. We then utilised a non-negative matrix factorisation (NMF) algorithm to classify HIRI samples. Subsequently, we employed machine learning algorithms to screen the hub NRGs related to EAD and developed an EAD prediction model based on these hub NRGs. Concurrently, we assessed the expression patterns of hub NRGs at the single-cell level using the HIRI. Additionally, we validated C5AR1 expression and its effect on HIRI and NETs formation in a rat orthotopic liver transplantation (OLT) model. ResultsIn this study, we identified 11 DE-NRGs in the HIRI context. Based on these 11 DE-NRGs, HIRI samples were classified into two distinct clusters. Cluster1 exhibited a low expression of DE-NRGs, minimal neutrophil infiltration, mild inflammation, and a low incidence of EAD. Conversely, Cluster2 displayed the opposite phenotype, with an activated inflammatory subtype and a higher incidence of EAD. Furthermore, an EAD prediction model was developed using the four hub NRGs associated with EAD. Based on risk scores, HIRI samples were classified into high- and low-risk groups. The OLT model confirmed substantial upregulation of C5AR1 expression in the liver tissue, accompanied by increased formation of NETs. Treatment with a C5AR1 antagonist improved liver function, reduced tissue inflammation, and decreased NETs formation. ConclusionsThis study distinguished two apparent HIRI subtypes, established a predictive model for EAD, and validated the effect of C5AR1 on HIRI. These findings provide novel perspectives for the development of advanced clinical strategies to enhance the outcomes of liver transplant recipients.

Which incision is better for Lewis to Brown Norway rat liver transplantation, transverse or midline?

Orthotopic rat liver transplantation (OLT) is a complex microsurgical procedure extensively applied to basic science, myriad complications can occur, but incision-related self-biting has not been reported after OLT. For the project of tolerance induction through stem cells, we performed OLT from Lewis to Brown Norway (BN) rats as an acute rejection model and divided the study into the transverse incision group (n = 15) and midline incision group (n = 22), while cyclosporine A was subcutaneously injected for 10-day immunosuppression use, lidocaine cream was used for pain-relieving. The recipient survival and wound status were the primary endpoint of this study. For the transverse incision group, 30-day survival rate was 40% (6/15), self-biting occurred in 13 cases in 7–39 days, the degree 1 of biting occurred in 1 cases, the degree 2 in 2 cases. The degree 3 in 10 cases, which caused death or euthanasia, the self-biting rate was 86.7% (13/15), For the midline incision group, 30-day survival rate was 100% (22/22), the degree 1 of self-biting occurred in 3 cases, no severe self-biting occurred. There were significant differences for survival (p = 0.0003) and for self-biting rate (p < 0.01) between two groups. In conclusion, incision-related self-biting behavior occurs due to incisional injury, the transverse incision is severely pain-causing; the midline one is effective to avert occurrences.

Nanotherapeutics for solid organ transplantation: new kid onthe block.

Nanotherapeutics for solid organ transplantation: new kid onthe block.

Donor graft METTL3 gene transfer ameliorates rat liver transplantation ischemia-reperfusion injury by enhancing HO-1 expression in an m6A-dependent manner

Ischemia-reperfusion injury (IRI) is one of the most common complications in liver transplantation. METTL3 regulates inflammation and cellular stress response by modulating RNA m6A modification level. Here, the study aimed to investigate the role and mechanism of METTL3 in IRI after rat orthotopic liver transplantation. The total RNA m6A modification and METTL3 expression level was consistently down-regulated after 6 h or 24 h reperfusion in OLT, which is negatively associated with the hepatic cell apoptosis. Functionally, METTL3 pretreatment in donor significantly inhibited liver grafts apoptosis, improved liver function and depressed the proinflammatory cytokine/chemokine expression. Mechanistically, METTL3 inhibited apoptosis of grafts via upregulating HO-1. Moreover, m6A dot blot and MeRIP-qPCR assay revealed that METTL3 promoted HO-1 expression in an m6A-dependent manner. In vitro, METTL3 alleviated hepatocytes apoptosis by upregulating HO-1 under hypoxia/reoxygenation condition. Taken together, these findings demonstrate that METTL3 ameliorates rat OLT-stressed IRI by inducing HO-1 in an m6A-dependent manner, highlighting a potential target for IRI in liver transplantation.

MiR-27a-5p inhibits acute rejection of liver transplantation in rats by inducing M2 polarization of Kupffer cells through the PI3K/Akt pathway

Liver transplantation (LT), a major therapy for end-stage liver disease, is often associated with acute rejection (AR). MicroRNAs (miRNAs) have been implicated in AR-related gene regulation. In this experiment, the mechanism of miR-27a-5p in AR of LT was studied. Allotransplantation model (LEW-BN) and syngeneic transplantation model (LEW-LEW) of rat orthotopic liver transplantation (OLT) were established. miR-27a-5p was overexpressed in recipient rats 28 days before LT to detect its effects on LT pathology, liver function, and survival time. Kupffer cells (KCs) were isolated and treated with lipopolysaccharide (LPS) and miR-27a-5p overexpression. miR-27a-5p overexpression reduced lymphocyte numbers around portal areas and central veins after LT and mitigated degeneration of epithelial cells of the bile duct. Expression levels of IL-10 and TGF-β1 were increased while IL-12 was decreased. Liver function damage was alleviated and the survival time of rats with LT was prolonged. miR-27a-5p induced M2 polarization of rats with AR after LT and LPS-treated KCs in vitro and promoted activation of the PI3K/Akt pathway in KCs. Inhibition of the PI3K/Akt pathway averted induction of miR-27a-5p on M2 polarization of KCs. Taken together, miR-27a-5p inhibited AR after LT in rats by inducing M2 polarization of KCs through the PI3K/Akt pathway.

Induction of liver transplant immune tolerance in an outbred rat strain model using tacrolimus

BackgroundOrthotopic liver transplantation is the only option for patients with end-stage liver disease and hepatocellular carcinoma. Post-transplant immunosuppressive therapy is important to prevent graft failure. We investigated the effectiveness of tacrolimus (FK506) and their mechanisms for liver transplant immune tolerance in an outbred rat LT model.ResultsTo investigate the therapeutic effect of the FK506 on outbred rat LT model, FK506 and postoperative therapy were administered subcutaneously once or twice daily to transplanted rats. Histopathological and immunohistochemical analyses were conducted for all groups. The regulation of inflammatory cytokine signaling in the spleen was analyzed by flow cytometry. FK506 attenuated allograft rejection and increased survival in rat orthotopic liver transplantation models. The FK506-treated group had reduced serum levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. Furthermore, FK506 decreased the expression of inflammatory cytokines and the activation of pathogenic Th1 and Th17 cells in the liver.ConclusionsTaken together, we revealed that FK506 ameliorated strong allograft rejection in outbred liver transplantation model by anti-inflammatory effect and inhibitory peroperty of pathogenic T cells.

IL-18BP Improves Early Graft Function and Survival in Lewis-Brown Norway Rat Orthotopic Liver Transplantation Model.

Interleukin-18 (IL-18) can effectively activate natural killer (NK) cells and induce large concentrations of interferon-γ (IFN-γ). In healthy humans, IL-18 binding protein (IL-18BP) can inhibit the binding of IL-18 to IL-18R and counteract the biological action of IL-18 due to its high concentration and high affinity, thus preventing the production of IFN-γ and inhibiting NK-cell activation. Through previous studies and the phenomena observed by our group in pig-non-human primates (NHPs) liver transplantation experiments, we proposed that the imbalance in IL-18/IL-18BP expression upon transplantation encourages the activation, proliferation, and cytotoxic effects of NK cells, ultimately causing acute vascular rejection of the graft. In this research, we used Lewis-Brown Norway rat orthotopic liver transplantation (OLTx) as a model of acute vascular rejection. AAV8-Il18bp viral vectors as gene delivery vehicles were constructed for gene therapy to overexpress IL-18BP and alleviate NK-cell rejection of the graft after transplantation. The results showed that livers overexpressing IL-18BP had reduced damage and could function longer after transplantation, effectively improving the survival time of the recipients.

Stepwise Approach for Acquisition of Microsurgical Skills through Rat Orthotopic Liver Transplantation Experiments

Although rat liver transplantation (LT) is useful in training surgeons to perform microsurgery, mastering these surgical techniques remains difficult. Systematized training protocols that enable learning of the proper skills in a short period of time are needed. The present study describes an efficient five-step rat LT training protocol for surgeons designed to be mastered within 3 months through continuous training. The first step was to review all procedures by watching full videos of rat LT and to watch actual LT operations performed by a skilled surgeon, enabling recognition of the anatomy of rat abdominal organs. The second step was to perform ten donor operations, including ex vivo graft preparation, to learn the atraumatic and delicate techniques. The third step was to perform ten LTs, with the goal of achieving an anhepatic time <20 min and surviving until the next day. The fourth step was to perform ten additional LTs, with the goal of achieving 7 days of survival. The fifth step was to perform 5–10 more LTs, with the goal of achieving 7 days of survival in five consecutive LT operations. Systematizing the training was found to increase its efficiency. Furthermore, determining the specific number of operations in advance is useful to maintain motivation for training. Mastering efficient rat LT will not only enhance the success of preclinical research but will enable young surgeons to better perform vascular anastomoses under a microscope in humans.

RESULTS OF THERMAL VISION EXAMINATION BEFORE AND AFTER ORTHOTOPIC LIVER TRANSPLANTATION IN INBRED RATS

This article describes the using of thermal imaging examination in orthotopic liver transplantation in inbred rats. For the experiment, 40 inbred rats of the Vistar line were selected: 20 females, weighing 400–550 g, 20 males, weighing 600–750 g, about a year old. For comparison, we identified the control group, which underwent only an incision of the skin and muscles of the abdominal wall, and then closed the surgical wound, experimental group No. 1, in which the left lateral lobe of the liver was excluded from the bloodstream to create a model of graft ischemia, and experimental group No. 2 who underwent orthotopic transplantation. All manipulations were performed under anesthesia, in compliance with the rules of asepsis and antisepsis. Thermal imaging examination was carried out with a medical thermal imager model ULIRvision Ti120 before the operation, then on the first, third, fifth, seventh day after. When comparing the thermograms of rats from the experimental group No. 2 and the control group, differences were observed on the 3rd day after the operation, then the temperature in the graft area in the experimental group No. 2 was higher than the temperature of the same skin area in the control group. This may be due to a slight ischemia of the graft during its disconnection from the blood flow, as well as to surgical intervention. After conducting research, we came to the conclusion that on the 3rd day in the transplant area there is a local increase in temperature, which is associated with ischemia of the organ due to temporary blockage of blood vessels, which occurs from the moment of explantation of the liver until its subsequent connection to the bloodstream. Rats in which this process affects 33% of the liver die on the first day from developing fulminant sepsis.

415.1: PGC1alpha-Mediated Metabolic Reprogramming Facilitates M1 Macrophage Polarization During Steatotic Allograft Dysfunction After Liver Transplantation

Introduction: The pro-inflammatory macrophage activation is the major force to allograft dysfunction, but detailed mechanism is poorly understood. Our previous study demonstrated the pivotal role of PGC1a in mediating steatotic graft injury via mitochondrial regulation (Liu et al., 2020). This study aims to elucidate the role of PGC1a in regulating macrophage polarization regarding metabolic reprogramming during steatotic graft injury. Method: Intrahepatic PGC1a expression and macrophage subpopulation were quantified and their correlation with acute phase steatotic allograft dysfunction and long-term graft survival was analyzed in a liver transplant cohort. Macrophage subtypes with distinct PGC1a expression patterns were investigated in a rat orthotopic liver transplant model. The functional study of PGC1a in mediating macrophage metabolic reprogramming as well as its polarization were studied in a mouse ischemia and reperfusion model. Results: The cutoff value of intrahepatic PPARGC1A (PGC1a) mRNA level (relative fold change=0.385) was determined by the ROC curve with AUC of 0.811 (p<0.001) (Fig. 1A). Divided by PGC1a cutoff, lower expression recipients using steatotic grafts were shown significantly deteriorated long-term graft survival as well as acute phase elevation of ALT and AST (Fig. 1B-1C). The cytokine quantification showed that patients with early allograft dysfunction (EAD) had both cellular and humoral pro-inflammatory response, which were indicated by increased CD68 and TNFa expression in the graft and elevated IFNg but decreased IL10 concentration in the plasma (Fig. 1D-1E). Further consolidated by the rat liver transplant models, steatotic graft had a higher M1 macrophage ratio but lower M2 ratio (Fig. 2A). Especially, the subgroups of CD68+IFNg+PGC1alow macrophage and CD163+PGC1ahigh macrophage were both higher in the steatotic graft compared with the normal graft (Fig. 2B). Detailed metabolism analyses showed that steatotic graft had down-regulated oxidative phosphorylation (OXPHOS) but enhanced glycolysis, which were indicated by increased plasma lactate level and decreased OXPHOS complex II (succinate dehydrogenase, SDH) activity (Fig. 2C-2D). Functional study showed that inhibition of PGC1a decreased the expression of OXPHOS subunits Sdha and Coxii, but reactivation of PGC1a restored their expression (Fig. 2E). Consistently, inhibition of PGC1a in liver showed elevated ALT and AST, exacerbated portal triad inflammation, as well as extended steatosis and apoptosis, whereas reactivation of PGC1a attenuated these phenomena (Fig. 2F-2G). Conclusion: PGC1a is pivotal in mediating steatotic graft injury via promoting intrahepatic M1 macrophage polarization through metabolic reprogramming.Reference Liu J, Pang L, Ng KTP, et al. Compromised AMPK-PGC1alpha Axis Exacerbated Steatotic Graft Injury by Dysregulating Mitochondrial Homeostasis in Living Donor Liver Transplantation. Ann Surg. 2020.

Prolyl Hydroxylase Inhibition Mitigates Allograft Injury During Liver Transplantation.

Ischemia and reperfusion injury (IRI) determines primary allograft function after liver transplantation (LT). Primary graft dysfunction (PGD) is associated with increased morbidity and impaired graft survival and can eventually progress to graft failure requiring retransplantation. Hypoxia-inducible transcription factor-prolyl hydroxylase containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors, which control the adaptive hypoxia response through the hypoxia-inducible factor (HIF). In this study, we have investigated pharmacological activation of the HIF pathway through inhibition of PHDs as a strategy to reduce PGD after LT. Primary rat hepatocytes were isolated and the impact of the pan-PHD small-molecule inhibitor ethyl-3,4-dihydroxybenzoate (EDHB) on HIF-1 and its downstream target gene expression assessed. Subsequently, various rodent models of segmental warm liver ischemia and reperfusion and orthotopic LT were applied to study the impact of EDHB on normothermic or combined cold and warm liver IRI. Liver enzyme levels and histology were analyzed to quantify hepatic IRI. In vitro, EDHB induced HIF-1 signaling and significantly upregulated its downstream target heme-oxygenase 1 in primary rat hepatocytes. In vivo, after establishment of the optimal EDHB pretreatment conditions in a murine IRI model, EDHB pretreatment significantly mitigated hepatic IRI after warm segmental liver ischemia and reperfusion and allograft injury after orthotopic LT in rats. Mechanistically, EDHB stabilized HIF-1 in the liver and subsequently increased hepatoprotective heme-oxygenase 1 levels, which correlated with reduced hepatic IRI in these models. This proof-of-concept study establishes a strong therapeutic rationale for targeting PHDs with small-molecule inhibitors to mitigate PGD after LT.

IL-22 Protects against Biliary Ischemia-Reperfusion Injury after Liver Transplantation via Activating STAT3 and Reducing Apoptosis and Oxidative Stress Levels In Vitro and In Vivo.

Biliary complications are currently one of the leading causes of liver failure and patient death after liver transplantation and need to be solved urgently. Biliary ischemia-reperfusion injury (IRI) is one of the important causes of biliary complications. IL-22 has a protective effect on liver injury and hepatitis diseases, and its safety and efficacy in the treatment of hepatitis have also been proved in human clinical experiments. Furthermore, multiple studies have confirmed that IL-22 promotes the proliferation and repair of epithelial cells in various organs. Still, its function in the bile duct after transplantation has not been explored. This study was aimed at investigating the effects of IL-22 on cholangiocyte IRI in vitro and in vivo and exploring its underlying mechanisms. We simulated the hypoxia process of bile duct epithelial cells through in vitro experiments to investigate the protective function and molecular mechanism of IL-22 on bile duct epithelial cells. Subsequently, the function and mechanism of IL-22 in the biliary IRI model of autologous orthotopic liver transplantation in rats were assessed. This study confirmed that IL-22 could promote cholangiocyte proliferation, decrease the apoptosis rate of cholangiocytes and tissues, decrease MDA levels, and increase SOD levels by activating STAT3. In addition, IL-22 can also reduce the level of mitochondrial membrane depolarization, protect mitochondria, reduce ROS production, and play a role in protecting bile ducts. These findings provide evidence for IL-22 as a novel and effective treatment for biliary IRI after liver transplantation.

Effect of HO-1-modified BMMSCs on immune function in liver transplantation

We examined whether haem oxygenase-1 (HO-1) could enhance the immunosuppressive effects of bone marrow mesenchymal stem cells (BMMSCs) on the rejection of transplanted liver allografts in rats. The animals were divided into three groups: the normal saline (NS) group, BMMSC group and HO-1/BMMSCs group. In vitro, the extraction, culture and HO-1 transfection of BMMSCs were performed. Mixed lymphocyte response (MLR) analysis of HO-1/BMMSCs efficacy was performed. The rejection model of orthotopic liver transplantation in rats was established when BMMSCs and HO-1/BMMSCs were transfused via the portal vein. To reduce research bias, we established an isogenic Liver transplantation model of (LEW → LEW) and (BN → BN), which can achieve tolerance. Changes in histopathology and liver function in the transplanted liver and changes in regulatory T cell (Tregs), natural killer (NK) cells and cytokines after transplantation were observed in the different groups. The severe acute rejection after liver transplantation on postoperative Day 10 was observed in the NS group. The BMMSC group showed strong protective effects against rejection within the first 10 days after transplantation, while HO-1/BMMSCs showed stronger effects on rejection than BMMSCs alone. In addition, the activity of natural killer (NK) cells decreased significantly, the levels of regulatory T cells (Tregs), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) increased significantly and the levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased significantly in the HO-1/BMMSC group compared with the BMMSC group. HO-1/BMMSCs showed better immunosuppressive effects after liver transplantation than the other treatments. Our findings reveal that HO-1 can enhance the effects of BMMSCs on inhibiting acute rejection in orthotopic liver transplantation in rats.

Cytomegalovirus Latency Exacerbated Small-for-size Liver Graft Injury Through Activation of CCL19/CCR7 in Hepatic Stellate Cells.

The interplay between cytomegalovirus (CMV) latency and graft malfunction after living donor liver transplantation remains poorly defined because of the complexity of clinical confounding factors. Here, we aimed to investigate the effects of CMV latency on small-for-size graft injury and to get further insight into the pathogenic role of hepatic stellate cells (HSCs) in this process. Rat orthotopic liver transplantation with small-for-size grafts was performed in a CMV latent model developed in immunocompetent Sprague Dawley rats using Priscott strain. Posttransplant graft injury including hepatocyte damage, stellate cell activation, and fibrogenesis was evaluated. Differential gene expression of HSCs in response to CMV latency was screened by cDNA microarray. Clinical validation was further conducted in human biopsies. CMV latency aggravated hepatocyte apoptosis/necrosis in the early phase and enhanced HSC expansion and graft fibrosis during the middle-late phase in small-for-size liver grafts of the rat model. cDNA microarray mining revealed CCL19/CCR7 as one of the most noteworthy pathways bridging HSC activation and liver graft injury in the presence of CMV latency. Together with CCL19 upregulation, coherent overexpression of CCR7 in accumulated HSCs was confirmed in both rat and human CMV latent recipients. Moreover, addition of CCL19 in vitro promoted HSC migration by increasing the level of matrix metalloproteinase-2. Our data demonstrated that CMV latency aggravated early/late phase liver graft damage and fibrogenesis via CCL19/CCR7/HSCs axis. Blockade of CMV latency-related stellate cell activation may shed light on the strategy of graft protection clinically.

High Mobility Group Box 1 Contributes to the Acute Rejection of Liver Allografts by Activating Dendritic Cells.

Acute rejection induced by the recognition of donor alloantigens by recipient T cells leads to graft failure in liver transplant recipients. The role of high mobility group box 1 (HMGB1), an inflammatory mediator, in the acute allograft rejection of liver transplants is unknown. Here, rat orthotopic liver transplantation was successfully established to analyze the expression pattern of HMGB1 in liver allografts and its potential role in promoting the maturation of dendritic cells (DCs) to promote T cell proliferation and differentiation. Five and 10 days after transplantation, allografts showed a marked upregulation of HMGB1 expression accompanied by elevated levels of serum transaminase and CD3+ and CD86+ inflammatory cell infiltration. Furthermore, in vitro experiments showed HMGB1 increased the expressions of co-stimulatory molecules (CD80, CD83, and MHC class II) on bone marrow-derived DCs. HMGB1-pulsed DCs induced naive CD4+ T cells to differentiate to Th1 and Th17 subsets secreting IFN-γ and IL-17, respectively. Further in vivo experiments confirmed the administration of glycyrrhizic acid, a natural HMGB1 inhibitor, during donor liver preservation had therapeutic effects by reducing inflammation and hepatocyte damage reflected by a decline in serum transaminase and prolonged allograft survival time. These results suggest the involvement of HMBG1 in acute liver allograft rejection and that it might be a candidate therapeutic target to avoid acute rejection after liver transplantation.

Hypothermic oxygenated perfusion with defatting cocktail further improves steatotic liver grafts in a transplantation rat model.

In this study, we evaluated the restoring and defatting effect of hypothermic oxygenated perfusion (HOPE) on severe steatotic liver grafts with a defatting cocktail (DF) in a rat model. Severe (≥60%) hepatic macrosteatosis was induced by a high-fat diet (HFD) for 6weeks, after which the rats were randomly divided into four following groups: Control group, with lean livers being preserved in static cold storage (SCS) at 0°C-4°C for 45minutes; SCS group, with a steatotic liver graft (SLG) being preserved in SCS at 0°C-4°C for 4hours; HOPE group, where SLG was perfused with 3-hours HOPE followed by 1-hours SCS; and HOPE+DF group, HOPE with the addition of DF. Graft function after orthotopic liver transplantation was assessed in terms of mitochondrial function (adenosine triphosphate [ATP], Glycogen), endoplasmic reticulum stress (PPY, GRP78, CHOP, and ATF-6), cell apoptosis (Tunel assay, Caspase-3), inflammatory level (HMGB1, TLR4, IL-1β, IL-6. TNF-α, Factor V), and posttransplantation survival. HOPE protected steatotic liver grafts from microcirculation disturbance and endoplasmic reticulum stress and then promoted ATP and glycogen synthesis that improved mitochondrial function. Meanwhile, under conditions of ischemia-reperfusion injury, it prevented nuclear injury and endothelial damage by suppressing the release of an inflammatory mediator. The high efficacy of HOPE was enhanced after the addition of the DF. DF agents cannot promote the lipid decomposition of the steatotic liver graft at 0°C-4°C, but they can further improve steatotic liver and postoperative survival compared to the HOPE. The defatted steatotic liver grafts can be safely used in rat orthotopic liver transplantation.

Monocytic MDSC mobilization promotes tumor recurrence after liver transplantation via CXCL10/TLR4/MMP14 signaling

Tumor recurrence is the major obstacle for pushing the envelope of liver transplantation for hepatocellular carcinoma (HCC) patients. The inflammatory cascades activated by acute liver graft injury promote tumor recurrence. We aimed to explore the role and mechanism of myeloid-derived suppressor cell (MDSC) mobilization induced by liver graft injury on tumor recurrence. By analyzing 331 HCC patients who received liver transplantation, the patients with graft weight ratio (GWR, the weight of liver graft divided by the estimated standard liver weight of recipient) <60% had higher tumor recurrence than GWR ≥60% ones. MDSCs and CXCL10/TLR4 levels were significantly increased in patients with GWR <60% or tumor recurrence. These findings were further validated in our rat orthotopic liver transplantation model. In CXCL10−/− and TLR4−/− mice of hepatic ischemia/reperfusion injury plus major hepatectomy (IRH) model, monocytic MDSCs, instead of granulocytic MDSCs, were significantly decreased. Importantly, CXCL10 deficiency reduced the accumulation of TLR4+ monocytic MDSCs, and CXCL10 increased MDSC mobilization in the presence of TLR4. Moreover, MMP14 was identified as the key molecule bridging CXCL10/TLR4 signaling and MDSC mobilization. Knockout or inhibition of CXCL10/TLR4 signaling significantly reduced the tumor growth with decreased monocytic MDSCs and MMP14 in the mouse tumor recurrent model. Our data indicated that monocytic MDSCs were mobilized and recruited to liver graft during acute phase injury, and to promote HCC recurrence after transplantation. Targeting MDSC mobilization via CXCL10/TLR4/MMP14 signaling may represent the therapeutic potential in decreasing post-transplant liver tumor recurrence.

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

The Role of Tacrolimus Nanomicelles in Acute Rejection After Liver Transplantation in Rats.

Although there is some progress in immunosuppressive therapy of acute rejection, there is still a lack of standardized diagnosis and treatment. For the acute rejection after liver transplantation, there is still a lack of an exact treatment at this stage. Tacrolimus (TAC) side effects will also affect the survival rate and quality of life of recipients after transplantation to a large extent. Rat orthotopic liver transplantation model was established and divided into three groups. In the tolerance group, Brown Norway (BN) to Lewis liver transplantation was used; in the rejection group, Lewis to BN liver transplantation was used; in the TAC group, TAC was injected after operation on the basis of establishing rejection model. The expression of GITRL in Kupffer cells and the change of cytokines were detected 7 days after operation. In this study, the animal model of acute rejection of rat liver transplantation was established to simulate the clinical allogeneic liver transplantation, and the important role of TAC in the acute rejection of rat liver transplantation was evaluated.

Protective Effect of Retrograde Reperfusion Against Hepatic Autophagy Impairment in Rat Liver Transplantation

The retrograde reperfusion (RTR) technique was introduced in orthotopic liver transplantation (OLT) to improve initial postoperative liver function, but the related mechanisms remain unexplained. We investigated the influences of different reperfusion sequences, including initial portal reperfusion (IPR) and RTR, on hepatic ischemia/reperfusion (I/R) injury and autophagic activity in a simplified rat orthotopic liver transplantation (ROLT) model. MethodsFirst, we established an ROLT model of male Sprague-Dawley rats to simulate either the IPR or RTR technique. The operative times and survival rates until postoperative day (POD) 7 were recorded. Liver enzyme levels, histologic damage, and in situ apoptosis were assessed. Second, we evaluated differences in the autophagic flux of liver grafts at 1, 2, and 6 hours after reperfusion between the IPR and RTR techniques. All experimental procedures involving animals were approved by the Institutional Animal Ethics Committee of the 900th Hospital of PLA. ResultsIn the first experiment, all animals survived to POD 7. In contrast to the IPR sequence, the RTR technique decreased the extent of graft I/R injury. In the second experiment, reperfusion markedly impaired the autophagic flux of ischemic liver grafts, but the RTR technique could alleviate and postpone the reduction in autophagy after I/R. ConclusionsA feasible modified ROLT model with the cuff method was described and could flexibly simulate 2 reperfusion techniques: IPR and RTR. The use of the RTR sequence exhibited a protective effect against I/R injury and impairment of autophagy in liver grafts.