Rat Orthotopic Liver Transplantation Research Articles

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.

Stabilizing mast cells improves acute lung injury after orthotopic liver transplantation via promotion of apoptosis in polymorphonuclear neutrophils.

Postoperative pulmonary complications including acute lung injury (ALI) and acute respiratory distress syndrome have contributed to mortality and morbidity of orthotopic liver transplantation (OLT) with unclear mechanisms. Mast cells (MCs) and polymorphonuclear neutrophils (PMNs) are the main inflammatory cells and participants in the process of ALI. The present study was designed to investigate the role of MCs and PMNs and their potential relation to ALI following OLT. Rat orthotopic autologous liver transplantation (OALT) model was designed to determine lung injury at different time points after liver reperfusion. We also evaluated the function of MCs and the effect of tumor necrosis factor-α (TNF-α) and tryptase on ALI and PMN apoptosis in rats subjected to OALT. Histological scores and inflammatory factor levels as well as PMN apoptosis were measured. Rats suffered from ALI after OALT, which was demonstrated by a collapse of the pulmonary architecture, pulmonary edema, and infiltration of inflammatory cells in alveolar and interstitial spaces, as well as increased levels of proinflammatory cytokines. ALI maximized at 8 h after OALT. However, PMN apoptosis lagged behind the pulmonary injury and maximized at 16 h after OALT, when the acute inflammation resolution initiated. MC stabilization, and tryptase and TNF-α inhibitors could significantly decrease the lung pathophysiologic scores accompanied by an increase in PMN apoptosis. ALI after OALT was associated with MC activation and PMN apoptosis. ALI progression might be affected by delayed PMN apoptosis, which was related to MC activation. Induction of PMN apoptosis might alleviate ALI after OALT.

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

Compromised AMPK-PGCIα Axis Exacerbated Steatotic Graft Injury by Dysregulating Mitochondrial Homeostasis in Living Donor Liver Transplantation.

To investigate the association of graft steatosis with long-term outcome, and to elucidate the mechanism of steatotic graft injury in adult living donor liver transplantation. The utilization of steatotic graft expands the donor pool for living donor liver transplantation (LDLT). However, it remains controversial due to its high morbidity and mortality. Elucidating the mechanism of steatotic graft injury is crucial to develop therapeutic strategies targeting at graft injury and to further expand the donor pool. Five hundred thirty patients receiving LDLT were prospectively included for risk factor analysis and outcome comparison. Rat orthotopic liver transplantation, in vitro functional experiments and mouse hepatic ischemia/ reperfusion models were established to explore the mechanisms of steatotic graft injury. We identified that graft with >10% steatosis was an independent risk factor for long-term graft loss after LDLT (hazard ratio 2.652, P = 0.001), and was associated with shorter cancer recurrence-free survival and acute phase liver injury. Steatotic graft displayed distinct mitochondrial dysfunction, including membrane, calcium, and energy homeostasis dysregulation. Specifically, the mitochondrial biogenesis was remarkably downregulated in steatotic graft. Inhibition of AMPK-PGC1α axis impaired mitochondrial biogenesis and was lethal to fatty hepatocyte in vitro , whereas reactivation of AMPK promoted PGC1α-mediated mitochondrial biogenesis and attenuated liver injury via restoring mitochondrial function in animal model. Conclusions: We provided a new mechanism that compromised AMPK-PGC1α axis exacerbated steatotic graft injury in LDLT by dysregulating mitochondrial homeostasis through impairment of biogenesis.

Etiology of Liver Steatosis Influences the Severity of Ischemia/Reperfusion Injury and Survival After Liver Transplantation in Rats.

Liver steatosis is a leading cause of graft disposal in liver transplantation, though the degree of steatosis is often the single factor determining acceptability of the graft. We investigated how the cause of liver steatosis affects graft function in rat orthotopic liver transplantation (OLT). OLT was performed using 2 types of steatotic liver grafts: the fasting and hyperalimentation (FHA) model and the methionine- and choline-deficient diet models. The FHA and 4-week feeding of a methionine- and choline-deficient diet (MCDD4wk) groups showed similar liver triglyceride levels without signs of steatohepatitis. Therefore, the 2 groups were compared in the following experiment. With 6-hour cold storage, the 7-day survival rate after OLT was far worse in the FHA than in the MCDD4wk group (0% versus 100%, P=0.002). With 1-hour cold storage, the FHA group showed higher aspartate aminotransferase and alanine aminotransferase levels and histological injury scores in zones 1 and 2 at 24hours after reperfusion than the normal liver and MCDD4wk groups. Intrahepatic microcirculation and tissue adenosine triphosphate levels were significantly lower in the FHA group after reperfusion. Hepatocyte necrosis, sinusoidal endothelial cell injury, and abnormal swelling of the mitochondria were also found in the FHA group after reperfusion. Tissue malondialdehyde levels were higher in the MCDD4wk group before and after reperfusion. However, the grafts up-regulated several antioxidant enzymes soon after reperfusion. Even though the degree of steatosis was equivalent, the 2 liver steatosis models possessed quite unique basal characteristics and showed completely different responses against ischemia/reperfusion injury and survival after transplantation. Our results demonstrate that the degree of fat accumulation is not a single determinant for the usability of steatotic liver grafts.

WITHDRAWN: Immunomodulatory effects of sCD83 via induction of TGF-β1 and IDO: an in vitro and rat orthotopic liver transplantation study

WITHDRAWN: Immunomodulatory effects of sCD83 via induction of TGF-β1 and IDO: an in vitro and rat orthotopic liver transplantation study

Human gingiva marrow-derived stromal cells exert immunoprotection on rat liver transplantation model through regulating FAS/FASL pathway

BackgroundMarrow mesenchymal stem cells (MSCs), especially bone MSCs (BMSCs), play a vital role in immunomodulation of graft rejection but many shortcomings limit its application. In this study, we aimed to investigate the modulation effect of MSCs from gingiva (GMSCs) on the rejection of rat liver transplantation model and to explore the potential mechanisms. MethodsGMSCs were obtained from human gingival tissues and BMSCs were harvested from Brown Norway (BN) rats. The siRNA that mediated knockdown of Fas ligand (FASL) expression in GMSCs (FASL-/-GMSCs) was transfected using lentivirus plasmid. Rat orthotopic liver transplantation model was established. The rats were divided randomly into four groups: normal saline (NS) group, FASL-/-GMSCs group, BMSCs group, and GMSCs group, all of which were injected the solution via dorsal vein of penis to construct the rat rejection model Graft survival and liver function indexes were measured. The immunological reactions of recipients including immune-cytokines, T-helper type 17 (Th17) and regulatory T cells (Treg) were also evaluated by flow cytometry. ResultsThe graft survival of recipient rats in the GMSCs group was significantly prolonged in comparison with that of the NS, FASL-/-GMSCs and BMSCs group. GMSCs remarkably decreased the levels of AST, ALT and TBIL. As for immune-cytokines, serum levels of IL-2, IFN-γ and Th 17 in recipient rats from the GMSCs group reduced significantly compared with that of other three groups, while increased serum IL-10 and TGF-β expressions as well as peripheral serum Treg were also observed in GMSCs group. ConclusionGMSCs exert immunoprotection on liver transplants by regulating FAS-FASL pathway. The current study firstly provides basis for GMSCs to be used in clinical anti-rejection after liver transplantation.

Translationally controlled tumor protein exerts a proinflammatory role in acute rejection after liver transplantation

BackgroundTranslationally controlled tumor protein (TCTP), which has been verified to have a proinflammatory activity, plays an important role in allergy. However, it remains unclear whether TCTP has an impact on the acute rejection (AR) after liver transplantation. MethodsThree protocols were used to delineate the role of TCTP in AR after liver transplantation. First, in rat orthotopic liver transplantation (OLT), the expression of TCTP was measured by enzyme-linked immunosorbent assay (ELISA), real-time PCR, Western blot and immunofluorescence assays. Second, in mixed lymphocyte reaction (MLR), the role of TCTP in lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) labeling and the impact of TCTP on inflammatory factor release was detected by cytokine arrays. Third, in human OLT, the level of serum TCTP was detected by ELISA, and the relationship between TCTP and model for early allograft function (MEAF) score was assessed by Spearman's correlation. ResultsIn rat OLT, AR resulted in great harm to allografts, manifesting as deterioration of liver function, increasing inflammatory factors and infiltrating lymphocytes. Meanwhile, TCTP was overexpressed in serum and allografts. Higher level of TCTP was associated with higher rejection activity index (RAI). In an MLR protocol, TCTP knockdown inhibited the proliferation of mixed inflammatory cells and significantly suppressed the release of 15 cytokines and chemokines. In human OLT, the serum TCTP was up-regulated within a week after operation. Additionally, the increasing speed of serum TCTP positively correlated with MEAF scores (r = 0.449; P = 0.0088). ConclusionsUp-regulated TCTP positively affects AR after 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

Role of necroptosis in intestinal injury induced by autologous orthotopic liver transplantation in rats

Objective To evaluate the role of necroptosis in intestinal injury induced by autologous orthotopic liver transplantation (AOLT) in rats. Methods Twenty-four SPF adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 3 groups (n=8 each) using a random number table method: sham operation group (group S), AOLT group (group T) and necroptosis inhibitor necrostatin-1 group (group N). Necrostatin-1 1.0 mg/kg and the equal volume of dimethyl sulfoxide (DMSO) were intraperitoneally injected at 30 min before surgery in N and T groups, respectively.Blood samples were collected from the inferior vena cava at 6 h after opening the portal vein (at 6 h after the end of surgery in group S) for determination of serum diamine oxidase (DAO), D-lactic acid (D-LA) and intestinal fatty acid binding protein (I-FABP) concentrations by enzyme-linked immunosorbent assay.Rats were sacrificed after blood sampling, and the intestine was removed for examination of the pathological changes (with a light microscope) and for determination of malondialdehyde (MDA) contents and superoxide dismutase (SOD) activities (using a spectrophotometer), and the expression of receptor-interacting protein kinase-1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL) in intestinal tissues (by Western blot). Intestinal damage was assessed and scored using Chiu′s scoring system. Results Compared with group S, the serum DAO, D-LA and I-FABP concentrations, MDA content and Chiu′s score were significantly increased, SOD activity was decreased, and the expression of RIPK1, RIPK3 and MLKL was up-regulated in group T (P<0.05). Compared with group T, the serum DAO, D-LA and I-FABP concentrations, MDA content and Chiu′s score were significantly decreased, the SOD activity was increased, and the expression of RIPK1, RIPK3 and MLKL was down-regulated in group N (P<0.05). Conclusion Necroptosis is involved in the pathophysiological process of intestinal injury induced by AOLT in rats. Key words: Liver transplantation; Intestine; Necrosis

Effects of leptin on brain injury and long-term cognitive function in rats undergoing orthotopic liver transplantation

Objective To evaluate the effects of leptin on brain injury and long-term cognitive function in rats undergoing orthotopic liver transplantation. Methods Ninety clean-grade male Sprague-Dawley rats, aged 3 months, weighing 200-250 g, were divided into 3 groups by a random number table method: sham operation group(S group), liver ischemia-reperfusion(I/R) group(I/R group) and leptin group(L group), with 18 rats in each group.Orthotopic liver transplantation was performed to establish the model of liver I/R injury in I/R and L groups.Leptin 1 mg/kg was intraperitoneally injected at the onset of ischemia in L group, and the equal volume of normal saline was given instead of leptin in S and I/R groups.Twelve rats in each group were sacrificed at 3 days after operation, and brains were removed for examination of the pathological changes in hippocampal CA1 region(with a light microscope) and for determination of apoptosis in hippocampal neurons(by TUNEL assay) and expression of aquaporin 4(AQP4) and protein kinase C(PKC) in the hippocampus(by Western blot). The apoptosis rate was calculated.The remaining 6 rats in each group underwent a Morris water maze test at 30 days after surgery to evaluate long-term cognitive function. Results Compared with S group, the apoptosis rate of hippocampal neurons was significantly increased, the expression of AQP4 and PKC was up-regulated, the escape latency was prolonged, and the time of staying at the platform quadrant was shortened in I/R and L groups(P<0.05). Compared with I/R group, the apoptosis rate of hippocampal neurons was significantly decreased, the expression of AQP4 and PKC was down-regulated, the escape latency was shortened, and the time of staying at the platform quadrant was prolonged in L group(P<0.05). Conclusion Leptin can reduce the brain damage in rats undergoing orthotopic liver transplantation, the mechanism may be related to down-regulating the expression of PKC and AQP4, and leptin can also improve long-term cognitive function after orthotopic liver transplantation in rats. Key words: Leptin; Liver transplantation; Reperfusion injury; Cognition disorders

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.

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.

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.

In vivo therapeutic potential of Inula racemosa in hepatic ischemia\u2013reperfusion injury following orthotopic liver transplantation in male albino rats

Hepatic ischemia–reperfusion (I/R) injury mainly occurs following hepatic resection and liver transplantation and cause severe liver damage, organ injuries, and dysfunction. Pro-inflammatory cytokines that promote injury are released when kupffer cell activates after getting induced by I/R. Repercussions of oxidative stress and cardiac function against isoproterenol based myocardial infarction are caused by flavonol glycosides which are found in high concentrations in Inula racemosa (Ir).The root was deemed to have analgesic and anti-inflammatory effects, and no report has been published about the liver-protective activity against hepatic I/R. Therefore, the present study was aimed to understand the therapeutic impact of Ir in hepatic I/R injury. Male albino, Wistar strain rats were used and were grouped into four total phenolic content, free radical scavenging activity and serum enzymes were determined. Histopathological and immunohistochemical analysis were also carried out. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin (IL-6) and protein expression of p53, bax, and bcl-2 were determined. The administration of extracts of Ir significantly increased total phenolic and free radical scavenging activity. Altered cellular morphology, cytokines and aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were returned to near normal level. IL-6 and TNF-α levels were reduced more than 25% following treatment. Also, the protein expression of p53, bax, and bcl-2 were also returned to near normal level. Taking all these data together, it is suggested that the extracts of Ir may be a potential therapeutic agent for providing several beneficial effects in hepatic I/R injury.

WITHDRAWN: In vivo therapeutic potential of inula racemosa in hepatic ischemia-reperfusion injury following orthotopic liver transplantation in male albino rats

WITHDRAWN: In vivo therapeutic potential of inula racemosa in hepatic ischemia-reperfusion injury following orthotopic liver transplantation in male albino rats

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.

Role of programmed death ligand 1 and Kupffer cell in immune regulation after orthotopic liver transplantation in rats

IntroductionRole of programmed death ligand 1 (PD-L1) and Kupffer cells (KCs) in liver transplantation immune regulation was unclear. MethodsLewis and Brown-Norway (BN) rats were assigned to LEW-BN group (Lewis-to-BN liver transplantation) and BN-BN group (BN-to-BN). Receipts were sacrificed for histology and assessment of cytokines and PD-L1 production. Effect of PD-L1 and KCs on T cells (TCs) was monitored by co-culture of 3H-Thymidine TCs. KCs transfected with PD-L1-shRNA interference plasmids were co-cultured with TCs, PD-L1 expression and cytokines production were measured respectively. ResultsRecipients in BN-BN group survived a long time while acute rejection was found in LEW-BN group. ELISA showed plasma levels of IL-2, IFN-γ and TNF-α in BN-BN group were significantly lower and levels of IL-10 were significantly higher than that in LEW-BN group on day 7 after transplantation (P<0.05). PD-L1 expression of KCs in BN-BN group was significantly higher than that in the LEW-BN group (P<0.05). Proliferation rate of TCs in KCs+TCs group was significantly lower and its apoptosis rate was significantly higher than that in TCs group (P<0.05). IL-2, TNF-α and INF-γ levels were remarkably higher and IL-10 levels were lower in KCs+TCs group than that in TCs group (P<0.05). Levels of IL-2, IFN-γ and TNF-α in transfection group were significantly higher and that of IL-10 was notably lower than that in the un-transfected group (P<0.05). ConclusionKCs with high expression of PD-L1 could significantly suppress the proliferation and function of TCs. Silencing the expression of PD-L1 in KCs in vivo could restore the function of TCs.