Rat Orthotopic Liver Transplantation Model 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.

Nanotherapeutics for solid organ transplantation: new kid onthe block.

Nanotherapeutics for solid organ transplantation: new kid onthe block.

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.

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.

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.

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.

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.

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.

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

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.

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.

Role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation

Objective To evaluate the role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation (OLT). Methods Fifty-six pathogen-free healthy adult male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-250 g, were used in the study.Thirty-two rats were selected and assigned into 4 groups (n=8 each)using a random number table: sham operation group (group S), OLT group, hydrogen-rich saline group (group HS) and chloroquine group (group CQ). The other 24 rats severed as the donors.In group S, laparotomy was performed, and the related blood vessels were isolated.The model of OLT was established in OLT, HS and CQ groups.In group OLT, normal saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group HS, hydrogen-rich saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group CQ, autophagy inhibitor chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model, and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion, the rats were sacrificed and hippocampi were isolated for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, for pathological examination (with light microscope), and for detection of cell apoptosis (by TUNEL staining) and expression of autophagy- and apoptosis-related proteins caspase-3, cytochrome c (Cyt c), microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ), Beclin-1 and p53 in hippocampal tissues (by Western blot analysis). Apoptosis index (AI) was calculated. Results Compared with group S, the MDA content and AI were significantly increased, the SOD activity was decreased, and the expression of caspase-3, Cyt c, LC3Ⅱ, Beclin-1 and p53 was up-regulated in OLT, HS and CQ groups (P<0.05). Compared with group OLT, the MDA content and AI were significantly decreased, the SOD activity was increased, the expression of caspase-3 and Cyt c was down-regulated, and the expression of LC3Ⅱ, Beclin-1 and p53 was up-regulated in group HS (P<0.05). Compared with group HS, the MDA content and AI were significantly increased, the SOD activity was decreased, and the expression of caspase-3 and Cyt c was up-regulated, and the expression of LC3Ⅱ, Beclin-1 and p53 was down-regulated in group CQ (P<0.05). Conclusion The mechanism by which hydrogen inhibits apoptosis in hippocampal neurons is related to promotion of autophagy in a rat model of OLT. Key words: Autophagy; Hydrogen; Liver transplantation; Brain injury; Apoptosis

Establishment of liver transplantation model with improved two-cuff in rat

Objective To build rat orthotopic liver transplantation model and use this model as an experimental animal model for further research about liver transplantation. Methods Based on the classic two-cuff technique, a variety of skills were integrated,such as abdominal aorta perfusion first before ligation, suprahepatic inferior vena cava single continuous suture, preoperative and postoperative rehydration, hepatic portal vein and infrahepatic vena cava blocked by mosquito's haemostatic forceps. At last, the postoperative survival rates of 2 days and 2 weeks were recorded. Results Donors' operation time was (35±2) min, cuffing time was (7.0±1.5) min, receptors' operation time was (80±15) min, anhepatic phase was (21±3) min. The 2-day survival rate was 92 % (46/50) and 2-week survival rate was 86 % (43/50). Conclusion To some extent,the improved methods can not only reduce the difficulty of orthotopic liver transplantation operation, but also to create a uniform, stable and reliable orthotopic liver transplantation model. Key words: Liver tansplantation; Rats; Animal model